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Title:
BIOMARKERS FOR LONGEVITY AND DISEASE AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2004/085996
Kind Code:
A2
Abstract:
This invention provides methods of using of the sizes and levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) particles, the -641 allele of the promoter of the gene encoding apolipoprotein C-3 (APOC-3), the 405 allele of the gene encoding cholesteryl ester transfer protein (CETP), and plasma levels of insulin-like growth factor-1 (IGF-1), adiponectin, CETP and APOC-3, for determining and increasing an individual's likelihood of longevity and of retaining cognitive function during aging, and for determining and decreasing an individual's likelihood of developing a cardiovascular-, metabolic- or age-related disease.

Inventors:
BARZILAI NIR (US)
Application Number:
PCT/US2004/008876
Publication Date:
October 07, 2004
Filing Date:
March 19, 2004
Export Citation:
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Assignee:
EINSTEIN COLL MED (US)
BARZILAI NIR (US)
International Classes:
C12Q1/48; G01N33/68; G01N33/92; G01N; (IPC1-7): G01N/
Other References:
LAMARCHE ET AL.: 'The Small Dense LDL Phenotype and the Risk of Coronary Heart Disease: Epidemiology, Path-Physiology and Therapeutic Aspects' DIABETES & METABOLISM (PARIS) vol. 25, 1999, pages 199 - 211
ARAI ET AL.: 'Deficiency of chloesteryl ester transfer protein and gene polymorphisms of lipoprotein lipase and haptic lipase are not associated with longevity' J. MOL. MED. vol. 81, 11 February 2003, pages 102 - 109
OLIVIERI ET AL.: 'ApoC-III gene polymorphisms and risk of coronary artery disease' JOURNAL OF LIPID RESEARCH vol. 43, 2002, pages 1450 - 1457
HOLMES ET AL.: 'Lifestyle Correlates of Plasma Insulin-like Growth Factor I and Insulin like Growth Factor Binding Protein 3 Concentrations' CANCER EPIDEMIOLOGY, BIOMARKERS & PREVENTION vol. 11, September 2002, pages 862 - 867
Attorney, Agent or Firm:
MILLER, Alan, D. et al. (Rothstein & Ebenstein LLP90 Park Avenu, New York NY, US)
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Claims:
What is claimed is: 1. A method of determining a subject's likelihood of longevity which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with the high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.
2. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
3. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a smaller size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease.
4. A method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.
5. A method of determining a subject's likelihood of longevity which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the 264451.1 subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.
6. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
7. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles-from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, smaller sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease.
8. A method of determining an individual's likelihood of longevity which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a larger size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.
9. The method of claim 1 or 5, which further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity.
10. The method of claim 1 or 5, which further comprises comparing the number of medium size HDL particles as a percentage of total HDL particles from the subject with the percentage of medium size HDL particles from the control population, a smaller percentage of medium size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity.
11. The method of claim 5 or 8, which further comprises comparing the number of large size LDL particles as a percentage of total LDL particles from the subject with the percentage of large size LDL particles from the control population, a larger percentage of large size LDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity.
12. The method of claim 5 or 8, which further comprises comparing the number of small size LDL particles as a percentage of total LDL particles from the subject with the percentage of small size LDL particles from the control population, a smaller percentage of small size LDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity.
13. The method of claim 2 or 6, which further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL particles from the subject than from the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
14. The method of claim 3 or 7, which further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a smaller percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease.
15. The method of claim 4, which further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL 264451. 1 particles from the subject than from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.
16. The method of claim 2,3, 6 or 7, wherein the cardiovascular related disease is selected from the group consisting of hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack.
17. A method of determining a subject's likelihood of longevity which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein (CETP), the presence of a homozygous valine/valine genotype indicating that the subject has an increased likelihood of longevity.
18. The method of claim 17, wherein the incidence of the homozygous allele is increased about 2-3 fold in a subject with longevity compared to a control population.
19. The method of any one of claims 1-8 or 17, wherein the subject is a human.
20. The method of claim 1, 5,8 or 17, wherein a subject with longevity lives to be at least 95 years of age.
21. The method of claim 1 or 5, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 2% greater than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
22. The method of claim 21, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 3% greater than the average size of the high density lipoprotein (HDL) particles from the control population.
23. The method of claim 22, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 4% greater than the average size of the high density lipoprotein (HDL) particles from the control population.
24. The method of claim 5 or 8, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 2% greater than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.
25. The method of claim 24, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 3% greater than the average size of the low density lipoprotein (LDL) particles from the control population.
26. The method of claim 2 or 6, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 2% larger than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
27. The method of claim 3 or 7, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 2% smaller than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
28. The method of claim 4, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 2% larger than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
29. The method of claim 1 or 5, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.2 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
30. The method of claim 29, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.3 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from the control population.
31. The method of claim 30, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.4 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from the control population.
264451. 1 32. The method of claim 5 or 8, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.4 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.
33. The method of claim 32, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.5 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.
34. The method of claim 33, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.6 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.
35. The method of claim 2 or 6, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.2 mn larger in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
36. The method of claim 3 or 7, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.2 nm smaller in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
37. The method of claim 6, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.3 mn larger in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.
38. The method of claim 7, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.3 nm smaller in diameter than the average size of the 264451.1 low density lipoprotein (LDL) particles from a control population of the same gender as the subject.
39. The method of claim 4, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.2 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.
40. A method of increasing a subject's likelihood of longevity which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma.
41. A method of increasing a subject's likelihood of longevity which comprises increasing the size of low density lipoprotein (LDL) particles in the subject's plasma.
42. A method of increasing a subject's likelihood of longevity which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.
43. The method of claim 40 or 42, which further comprises increasing the number of large size HDL particles as a percentage of total HDL particles.
44. The method of claim 40 or 42, which further comprises decreasing the number of medium size HDL particles as a percentage of total HDL particles.
45. The method of claim 41 or 42, which further comprises increasing the number of large size LDL particles as a percentage of total LDL particles.
46. The method of claim 41 or 42, which further comprises decreasing the number of small size LDL particles as a percentage of total LDL particles.
47. A method of decreasing a subject's likelihood of developing a cardiovascular related disease which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma.
264451.1 48. A method of decreasing a subject's likelihood of developing a cardiovascular related disease which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.
49. A method of increasing a subject's likelihood of retaining cognitive function during aging which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma.
50. The method of claim 47 or 48, wherein the cardiovascular related disease is selected from the group consisting of hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack.
51. A method of increasing a subject's likelihood of longevity which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).
52. A method of increasing a subject's likelihood of retaining cognitive function during aging which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).
53. A method of decreasing a subject's likelihood of developing a cardiovascular-related disease which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP), wherein the cardiovascular-related disease is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma.
54. The method of claim 53, wherein the cardiovascular related disease is selected from the group consisting of hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack.
55. A method of decreasing a subject's likelihood of developing a disease which is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).
264451.1 56. A method of determining a subject's likelihood of longevity which comprises determining if the subject has a mutation in a gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an increased likelihood of longevity.
57. A method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject has a mutation in a gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an increased likelihood of retaining cognitive function during aging.
58. A method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein (CETP), the presence of a homozygous valine/valine genotype indicating that the subject has an increased likelihood of retaining cognitive function during aging.
59. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
60. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a smaller size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.
61. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a larger size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
62. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a smaller size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.
63. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
64. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, smaller sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.
65. The method of claim 59 or 63, which further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of 264451.1 large size HDL particles from the subject than from the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
66. The method of claim 60 or 64, which further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a smaller percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.
67. The method of claim 61 or 63, which further comprises comparing the number of large size LDL particles as a percentage of total LDL particles from the subject with the percentage of large size LDL particles from the control population, a larger percentage of large size LDL particles from the subject than from the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
68. The method of claim 62 or 64, which further comprises comparing the number of large size LDL particles as a percentage of total LDL particles from the subject with the percentage of large size LDL particles from the control population, a smaller percentage of large size LDL particles from the subject than from the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.
69. The method of claim 59 or 63, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 3% greater than the average size of the high density lipoprotein (HDL) particles from the control population.
70. The method of claim 69, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 4% greater than the average size of the high density lipoprotein (HDL) particles from the control population.
71. The method of claim 61 or 63, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 2% greater than the average size of the low density lipoprotein (LDL) particles from the control population.
264451. 1 72. The method of claim 60 or 64, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 3% smaller than the average size of the high density lipoprotein (HDL) particles from the control population.
73. The method of claim 72, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 4% smaller than the average size of the high density lipoprotein (HDL) particles from the control population.
74. The method of claim 62 or 64, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 2% smaller than the average size of the low density lipoprotein (LDL) particles from the control population.
75. The method of claim 59 or 63, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.3 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from the control population.
76. The method of claim 75, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.4 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from the control population.
77. The method of claim 61 or 63, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.3 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from the control population.
78. The method of claim 77, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.5 mn larger in diameter than the average size of the low density lipoprotein (LDL) particles from the control population.
79. The method of claim 60 or 64, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.3 nm smaller in diameter than the average size of the high density lipoprotein (HDL) particles from the control population.
80. The method of claim 79, wherein the average size of the subject's high density lipoprotein (HDL) particles is at least 0.4 nm smaller in diameter than the average size of the high density lipoprotein (HDL) particles from the control population.
81. The method of claim 62 or 64, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.3 nm smaller in diameter than the average size of the low density lipoprotein (LDL) particles from the control population.
82. The method of claim 81, wherein the average size of the subject's low density lipoprotein (LDL) particles is at least 0.5 nm smaller in diameter than the average size of the low density lipoprotein (LDL) particles from the control population.
83. A method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma.
84. A method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the size of low density lipoprotein (LDL) particles in the subject's plasma.
85. A of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.
86. The method of claim 83 or 85, which further comprises increasing the number of large size HDL particles as a percentage of total HDL particles.
87. The method of claim 84 or 85, which further comprises increasing the number of large size LDL particles as a percentage of total LDL particles.
88. A method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).
264451.1 89. A method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity.
90. A method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.
91. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
92. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
93. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein (CETP), the presence of a homozygous valine/valine genotype indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
264451.1 94. A method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein (CETP), the presence of a homozygous valine/valine genotype indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
95. A method of determining a subject's likelihood of developing a cardiovascular related disease which comprises determining if the subject has a mutation in a gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
96. A method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject has a mutation in a gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
97. The method of claim 91,93, or 95, wherein the cardiovascular-related disease is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma.
98. A method of determining a subject's likelihood of longevity which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has an increased likelihood of longevity.
99. A method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has an increased likelihood of retaining cognitive function during aging.
264451.1 100. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
101. A method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
102. A method of determining a subject's likelihood of longevity which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has an increased likelihood of longevity.
103. A method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has an increased likelihood of retaining cognitive function during aging.
104. A method of determining a subject's likelihood of developing a cardiovascular related disease which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.
264451.1 105. A method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a metabolic syndrome.
106. The method of claim 102,103, 104 or 105, where the mutation is in a gene encoding apolipoprotein C-3 (APOC-3).
107. The method of claim 102,103, 104 or 105, where the mutation is in a promoter of a gene encoding apolipoprotein C-3 (APOC-3).
108. A method of increasing a subject's likelihood of longevity which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3).
109. A method of increasing a subject's likelihood of retaining cognitive function during aging which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3).
110. A method of decreasing a subject's likelihood of developing a cardiovascular-related disease which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC- 3).
111. A method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3).
112. The method of claim 91,93, 95,100, 104, or 110, wherein the cardiovascular related disease is selected from the group consisting of hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack.
113. The method of any one of claims 59-64, 83-85, 88, 92,94, 96,101 or 105, wherein a subject is defined as having a metabolic syndrome when the subject has three or more of the following four risk factors: 1) increased waist girth (> 94 cm for women, >102 cm for men), 264451.1 2) increased blood pressure (> 130//85 or treatment for hypertension), 3) increased fasting glucose (> 110 mg/dl or drug treatment for diabetes), and 4) elevated fasting triglycericde levels (> 150 mg/dl).
114. A method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity.
115. A method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.
116. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a cardiovascular related disease.
117. A method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF- 1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a metabolic syndrome.
118. A method of increasing a subject's likelihood of longevity, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF-1).
264451. 1 119. A method of increasing a subject's likelihood of retaining cognitive function during aging, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF-1).
120. A method of decreasing a subject's likelihood of developing a cardiovascular related disease, which comprises increasing the subject's plasma level of insulin-like growth factor- 1 (IGF-1).
121. A method of decreasing a subject's likelihood of developing a metabolic syndrome, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF- 1).
122. A method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity.
123. A method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.
124. A method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a cardiovascular related disease.
125. A method of increasing a subject's likelihood of longevity, which comprises increasing the subject's plasma level of adiponectin.
264451.1 126. A method of increasing a subject's likelihood of retaining cognitive function during aging, which comprises increasing the subject's plasma level of adiponectin.
127. A method of decreasing a subject's likelihood of developing a cardiovascular related disease, which comprises increasing the subject's plasma level of adiponectin.
128. An assay for identifying a compound that increases a subject's likelihood of longevity, increases a subject's likelihood of retaining cognitive function during aging, decreases a subject's likelihood of developing a cardiovascular-related disease, decreases a subject's likelihood of developing a metabolic syndrome, and/or decreases a subject's likelihood of developing an age-related disease, which comprises identifying a compound which: (a) increases HDL particle size in the subject's plasma, (b) increases LDL particle size in the subject's plasma, (c) increases both HDL and LDL particle size in the subject's plasma, (d) increases the percentage of large size HDL particles in the subject's plasma, (e) increases the percentage of large size LDL particles in the subject's plasma, (f) increases the percentage of both large size HDL particles and large size LDL particles in the subject's plasma, (g) increases the subject's plasma level of HDL, (h) increases the subject's plasma level of insulin-like growth factor-1 (IGF-1), (i) increases the subject's plasma level of adiponectin, (j) inhibits the activity of the subject's cholesteryl ester transfer protein (CETP), and/or, (k) inhibits the activity of the subject's apolipoprotein C-3 (APOC-3).
129. The assay of claim 128, wherein the compound increases a subject's likelihood of longevity.
130. The assay of claim 128, wherein the compound increases a subject's likelihood of retaining cognitive function during aging.
131. The assay of claim 128, wherein the compound decreases a subject's likelihood of developing a cardiovascular-related disease.
132. The assay of claim 128, wherein the compound decreases a subject's likelihood of developing a metabolic syndrome.
133. The assay of claim 128, wherein the compound decreases a subject's likelihood of developing an age-related disease.
134. The assay of any one of claims 128-133, wherein the compound increases HDL particle size in the subject's plasma.
135. The assay of claim 128,129, 131, or 132, wherein the compound increases LDL particle size in the subject's plasma.
136. The assay of claim 128,129, 131, or 132, wherein the compound increases both HDL and LDL particle size in the subject's plasma.
137. The assay of any one of claims 128-133, wherein the compound increases the percentage of large size HDL particles in the subject's plasma.
138. The assay of claim 128,129, 131, or 132, wherein the compound increases the percentage of large size LDL particles in the subject's plasma.
139. The assay of claim 128,129, 131, or 132, wherein the compound increases the percentage of both large size HDL particles and large size LDL particles in the subject's plasma.
140. The assay of any one of claims 128-133, wherein the compound increases the subject's plasma level of HDL.
141. The assay of any one of claims 128-133, wherein the compound increases the subject's plasma level of insulin-like growth factor-1 (IGF-1).
264451.1 142. The assay of any one of claims 128-133, wherein the compound increases the subject's plasma level of adiponectin.
143. The assay of any one of claims 128-133, wherein the compound inhibits the activity of the subject's cholesteryl ester transfer protein (CETP).
144. The assay of any one of claims 128-133, wherein the compound inhibits the activity of the subject's apolipoprotein C-3 (APOC-3).
145. The method of claim 116,120, 124, or 127, wherein the cardiovascular related disease is selected from the group consisting of hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack.
146. The assay of claim 128 or 131, wherein the cardiovascular related disease is selected from the group consisting of hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack.
Description:

BIOLOGICAL MARKERS FOR LONGEVITY AND DISEASES AND USES THEREOF Priority Claims [0001] This application claims the benefit of U. S. Provisional Patent Application No.

60/456,304, filed March 20,2003, and of U. S. Provisional Patent Application No.

60/508,420, filed October 3,2003, the contents of both of which are hereby incorporated by reference in their entirety into the subject application.

Statement of Government Support [0002] The invention disclosed herein was made with U. S. Government support under grant numbers RO1-AG-18728-OlA1, MO1-RR12248-05, and DK 20541 from the National Institutes of Health, U. S. Department of Health and Human Services. Accordingly, the U. S.

Government has certain rights in this invention.

Field of the Invention [0003] The present invention relates to uses of the sizes and levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) particles, the 405 allele of the gene encoding cholesteryl ester transfer protein (CETP), the-641 allele of the promoter of the gene encoding apolipoprotein C-3 (APOC-3), and plasma levels of insulin-like growth factor-1 (IGF-1), adiponectin, CETP and APOC-3, for determining an individual's likelihood of longevity, of developing a cardiovascular related disease (e. g. , hypertension, diabetes mellitus, myocardial infarction, stroke, and/or transient ischemic attack), a metabolic syndrome and/or an age-related disease, and of retaining cognitive function with aging.

Background of the Invention [0004] Throughout this application various publications are referred to in parenthesis.

Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.

[0005] Subjects with exceptional longevity have generally been spared from major age- related diseases, such as cardiovascular disease, diabetes mellitus, Alzheimer's and cancer, which are responsible for most deaths in the elderly (1). Various studies suggest that while the effect of genetics on life expectancy is minimal across ages, this is not the case with centenarians (a rare phenotype achieved by-1/10, 000 individuals). Siblings of current centenarians have odds ratios of 8-17 of achieving 100 years of age, and parents of centenarians (born at-1870) had odds ratio of-7 of achieving ages 90-99 than an appropriate control (2-4). Furthermore, the offspring of long-lived parents had significantly lower prevalence (-50%) of hypertension, diabetes mellitus, myocardial infarctions and strokes/transient ischemic attacks compared with several age-matched control groups (4-5). hi support of the inheritance of longevity, the New England Centenarian Study reported a statistically significant linkage between a genetic locus on chromosome 4 and exceptional longevity among siblings of centenarians (3).

[0006] Since lipid profile is directly correlated to cardiovascular disease, a favorable lipid profile may play a pivotal role in longevity. The complexity of such an assumption is depicted with the example of high-density lipoprotein (HDL) levels. The Framingham (8-9) and NHANES III (10) studies have shown that cross-sectional, plasma HDL levels were comparable at different age groups both in males and females. However, looking prospectively, plasma HDL levels seemed to decrease by over 5 mg/dl per decade. These seemingly contradictory results may be explained by HDL being a'survival'factor. A decrease in HDL levels to below a certain range may result in the loss of cardiovascular protection (and possibly protection from other age-related diseases), hence in increased mortality. A study in healthy elderly and centenarians revealed a small but statistically significant reduction in the ratios of cholesterol/HDL-cholesterol and LDL- cholesterol/HDL-cholesterol, and a significant increase in HDL-cholesterol and apoAl (11).

Offspring of centenarians have significant higher plasma levels of HDL levels compared to controls (7). Lipoprotein (a) levels have been reported to be elevated in centenarians at the threshold for atherogenic risk (11-13). Favorable biological markers that are unchanged or decreased in centenarians do not rule out their role in longevity, and some of the harmful lipoprotein profile in centenarians are compatible with healthy longevity, suggesting that other characteristics may protect'down stream'of these pathways.

[0007] The metabolic syndrome (MS) of aging/syndrome of insulin resistance is most commonly associated with obesity (115), but may be inherited in lean individuals. This 264451. 1 syndrome is commonly associated with dyslipidemia, with decreased HDL cholesterol and increased LDL cholesterol levels, and with decreases in HDL and LDL particle sizes (116).

However, it is unclear if increased HDL levels have a role in preventing this syndrome. This syndrome is also associated with hypertension, the development of type 2 diabetes mellitus (117), and a markedly increased risk of developing arteriosclerosis, and is therefore linked to decreased life expectancy (118,119). Insulin resistance has been identified as a risk factor for a variety of cancers (120-122), broadening its link to shorter life span in humans and to most causes of death (123).

[0008] HDL (71) and LDL (22) constitute heterogeneous groups of particles which differ in characteristics such as density, size, electrophoretic mobility, and chemical content.

Most of the HDL particles have a globular shape, containing unesterified cholesterol distributed between the surface and the core, and proteins are found in outer parts of the lipoproteins, mainly apoAl but also apo A-II, A-IV, Cs, E, J, and sphingomyelins (79). Out of five subgroups of HDL, levels of HDL2b have been reported to be increased in a group of 16 centenarian women, while levels of HDL3a are reduced in comparison with controls (72); males were not included in this study. Levels of HDL2-C have been reported to be increased in people 65 years and older (74).

[0009] Low blood levels of HDL are strongly related with risk of atherosclerotic cardiovascular disease (57). Overexpression of the major HDL protein, apoA, markedly inhibits progression and even induces regression of atherosclerosis in animal models (40).

Decreased plasma HDL level is also a risk for stroke and transient ischemic attack (TIA), but clinical data regarding the effect of increasing HDL cholesterol on vascular events are limited, because its rise is minor and secondary to drugs that lower LDL cholesterol (58, 59).

[0010] Amongst the many effects of plasma HDL, it recently became apparent that it may protect from decreased cognitive function associated with Alzheimer's (41) and other forms of dementia (42,43). In a group of elderly (>85 years of age), the associations between low Mini Mental State Exam (MMSE) scores and low HDL was significant. This relationship was maintained even after subjects with cardiovascular disease or stroke were excluded, supporting the association between HDL and cognitive function independent of atherosclerotic disease (44). Because HDL (and not LDL) has effects that were not clearly limited to the vascular bed, it was recently hypothesized that the very old brain of centenarians, who are not characterized by Alzheimer's disease, may be protected by HDL 264451. 1 (14). Indeed, each decrease in plasma HDL tertile was associated with a significant decrease in MMSE.

[0011] Previous studies have reported that abnormalities in the LDL receptor are associated with a decreased length of life (60,61). However, LDL cholesterol has not been reported to change significantly with age in prospective or cross sectional studies (10,57), although increased age is associated with higher plasma LDL cholesterol and apoB levels in postmenopausal women (57). Studies in healthy elderly and centenarians revealed a small but statistically significant and progressive reduction with age, in total cholesterol, triglycerides (TG) and LDL concentrations, as well as a significant increase in apolipoprotein B100 and lipoprotein (a) values (11-13). Male offspring of centenarians had significant lower plasma levels of LDL-cholesterol and higher levels of HDL-cholesterol compared to controls (7).

[0012] LDL particles contain unesterified cholesterol distributes between the surface and the core, and proteins are found in outer parts of the lipoproteins (mainly apoB LDL containing particles). The distribution of mass among LDL subclasses in plasma is reflected by the particle diameter and buoyant density of the predominant LDL species. A distinct LDL subclass pattern characterized by a predominance of small, dense LDL particles (previously called LDL3) has been identified (80). The prevalence of this trait increases with aging, and the prevalence of small particle size LDL (previously called subclass pattern B) is 3-4 fold increased in older compared with young men and women (29,30).

Evidence from several studies is consistent with an autosomal dominant or codominant model for inheritance of the pattern B phenotype with varying additive and polygenic effects (66,81). One study has reported a predominance of large, buoyant LDL particles in 75% of centenarians and a predominance of small dense LDL particles in 25% of centenarians (73).

[0013] The association of plasma LDL cholesterol with a significant risk factor for a variety of cardiovascular diseases is well established (57). The oxidation of LDL is commonly considered to be a major event in the initiation and development of atherosclerosis (62). The plasma lipoprotein profile accompanying a predominance of small, dense LDL3 particles is associated with a 2-3 fold increased risk of coronary heart disease (30,63-64). More recently, nested case-control analyses in prospective studies of population cohorts have demonstrated that reduced LDL particle size is a significant predictor for the development of coronary heart disease (21,65-67).

[0014] One of the pathways that has been implicated in aging is the insulin/insulin-like growth factor (IGF-1) signaling pathway, which is involved in many functions that are necessary for metabolism, growth, and fertility in animal models as varied as flies, nematodes and mammals (147). Disruption of the insulin/IGF-1 receptor in nematodes and flies increases lifespan significantly, and several mammalian dwarf models live significantly longer, including Snell and Ames dwarf mice and heterozygous knockout mice for the IGF- 1 receptor (132,144). Low IGF-1 levels may protect humans from diseases like cancer (136-140), while normal or high IGF-1 levels may protect humans from osteoporosis (135), diabetes (143), and cardiovascular disease (134). Therefore, based on such data, an overall beneficial effect of changes in IGF-1 levels on human longevity remains uncertain.

[0015] Another factor that has been associated with disease process is adiponectin, a protein produced exclusively in adipose tissue, which occurs in serum in relatively high concentration. The plasma concentration of adiponectin is decreased in obese and in type 2 diabetic humans and in patients with coronary artery disease, and low adiponectin levels are a predictor of type 2 diabetes (reviewed in 145,146). Many clinical reports and genetic studies over the past few years demonstrate decreased circulating levels of this hormone in metabolic dysfunction, such as obesity and insulin resistance, in both humans and animal models. Pharmacologic adiponectin treatments in rodents increase insulin sensitivity, mainly by its hepatic action. This protein also suppresses the expression of adhesion molecules in vascular endothelial cells and cytokine production from macrophages, thus inhibiting the inflammatory processes that occur during the early phases of atherosclerosis.

[0016] Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that catalyzes an exchange of cholesteryl esters (CE) and TG between HDL and APOB containing lipoproteins (70). The atherogenic properties of CETP have been demonstrated by blockade of CETP in cholesterol-fed rabbits, an animal with elevated CETP activity and high atherosclerosis susceptibility (82, 83). However, studies in CETP-deficient patients did not clarify whether CETP is atherogenic (84). CETP exerts a strong and direct effect on HDL size. Expression of CETP in normolipidemic rodents has a profound effect on large sized HDL, which was suggested as a reliable index of low plasma CETP activity in vivo (48, 85). In humans, plasma levels of large HDL particles from patients homo-and hetrozygous for CETP deficiency increased two-and six-fold while levels of small HDL remained unchanged (45,46). The CETP 405 valine allele is associated with increased levels of HDL (55,86). The presence of the B2 allele at the TaqlB polymorphism in intron 1 of the CETP 264451. 1 gene has been associated with increased HDL particle size (106). Complete CETP deficiency causes a small-sized LDL population with low affinity for the LDL receptor (47).

However, because an up-regulation of the LDL receptor increases LDL clearance, CETP deficiency is characterized by lowered LDL levels (49). Conflicting observations have been reported between CETP mutations and the incidence of coronary heart disease (CHD).

Increased HDL cholesterol levels caused by mutations in CETP were associated with a slight increased risk of CHD in white Danish women (53). Similar observation of increase in CHD was observed in Japanese-American men with hetrozygous CETP D442G missense mutation (87), though their HDL levels were 10% increased. However, recently the Veterans Affairs HDL Cholesterol Intervention Trial reported that CETP TaqI B2B2 genotype is associated with higher HDL cholesterol levels and lower risk of CHD in men (54).

[0017] Other genes are also involved in lipoprotein metabolism. Of particular interest is the gene encoding apolipoprotein C-3 (APOC-3). Transgenic APOC-3 mice are hypertriglyceridemic, and'knock out'of this gene results in hypotriglyceridemic mice (124). APOC-3 is an effective inhibitor of very low-density lipoprotein (VLDL) TG hydrolysis, has a regulating role on uptake of cholesteryl esters, and may have a role as an inhibitor for lipoprotein lipase (LPL), although its exact role is not fully understood.

Polymorphisms in APOC-3 have been associated with strong effects on triglyceride levels (125-127). The APOC-3 promoter region has conferred protection against or susceptibility to severe hypertriglyceridemic. The cysteine (C) allele of the Cystein (-641) Alanine (A) polymorphism, the C allele of the C-482Threonine (T) polymorphism, and the T allele of T (- 455) C polymorphism are protective against hypertriglyceridemia (128-129). Increased incidence of the C allele in the T-455C polymorphism was noted with advanced age, indicating that this variant promoter is associated with longevity (130). Furthermore, APOC-3 has effects on lipoprotein size through displacement of apolipoprotein E (APO-E) (131).

[0018] Despite the advances in knowledge discussed above, there remains a clear need for markers of longevity which may be used to decrease the risk of developing age-related diseases including dementia and metabolic-and cardiovascular-related diseases.

Summary of the Invention [0019] The present application is directed to the use of the sizes and levels of high- density lipoprotein (HDL) and low-density lipoprotein (LDL) particles, the 405 allele of a gene encoding cholesteryl ester transfer protein (CETP), the-641 allele of a promoter of the gene encoding apolipoprotein C-3 (APOC-3), and plasma levels of insulin-like growth factor-1 (IGF-1), adiponectin, CETP and APOC-3, for determining an individual's likelihood of longevity, of retaining cognitive function during aging, and of developing a cardiovascular-related disease, a metabolic syndrome or other age-related disease.

[0020] The invention provides a method of determining a subject's likelihood of longevity which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with the high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.

The invention also provides a method of determining an individual's likelihood of longevity which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a larger size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity. The invention further provides a method of determining a subject's likelihood of longevity which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.

[0021] The invention provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. The invention also provides method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing high density lipoprotein (HDL) particle size 264451.1 from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a smaller size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease.

[0022] The invention also provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, smaller sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease.

[0023] The invention provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.

[0024] The invention provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome. Also provided is a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density 264451. 1 lipoprotein (HDL) particle size from a control population, a smaller size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome. The invention provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a larger size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome. Also provided is a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a smaller size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome. The invention further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome. Also provided is method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, smaller sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.

[0025] The invention provides a method of determining a subject's likelihood of longevity which comprises determining if the subject has a mutation in the gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an increased likelihood of longevity. The invention further provides a method of determining a subject's likelihood 264451. 1 of longevity which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein, the presence of a homozygous valine/valine genotype indicating that the subject has an increased likelihood of longevity.

[0026] The invention provides a method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject has a mutation in a gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein, the presence of a homozygous valine/valine genotype indicating that the subject has an increased likelihood of retaining cognitive function during aging.

[0027] The invention provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises determining if the subject has a mutation in the gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an decreased likelihood of developing a cardiovascular related disease. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein, the presence of a homozygous valine/valine genotype indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.

[0028] The invention provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises determining if the subject has a mutation in the gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a metabolic syndrome. The invention further provides a method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein, the presence of a homozygous 264451.1 valine/valine genotype indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

[0029] The invention provides a method of increasing a subject's likelihood of longevity which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of increasing a subject's likelihood of longevity which comprises increasing the size of low density lipoprotein (LDL) particles in the subject's plasma. The invention further provides a method of increasing a subject's likelihood of longevity which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.

[0030] The invention provides a method of decreasing a subject's likelihood of developing a cardiovascular related disease which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of decreasing a subject's likelihood of developing a cardiovascular related disease which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.

[0031] The invention provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the size of high density lipoprotein (HDL) particles or of low density lipoprotein (LDL) particles in the subject's plasma. The invention also provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.

[0032] The invention provides a method of increasing a subject's likelihood of retaining cognitive function during aging which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma.

[0033] The invention provides methods of increasing a subject's likelihood of longevity, increasing a subject's likelihood of retaining cognitive function during aging, decreasing a subject's likelihood of developing a cardiovascular-related disease, decreasing a subject's likelihood of developing a metabolic syndrome, and decreasing a subject's likelihood of developing a disease which is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma, which comprise inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).

264451.1 [0034] The invention provides a method of determining a subject's likelihood of longevity, or of retaining cognitive function during aging, which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has an increased likelihood of longevity and/or of retaining cognitive function during aging. The invention also provides a method of determining a subject's likelihood of developing a cardiovascular related disease or a metabolic syndrome, which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has a decreased likelihood of developing a cardiovascular related disease and/or a metabolic syndrome.

[0035] The invention provides a method of determining a subject's likelihood of longevity or of retaining cognitive function during aging which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has an increased likelihood of longevity and/or of retaining cognitive function during aging.

The invention also provides a method of determining a subject's likelihood of developing a cardiovascular related disease or a metabolic syndrome which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a cardiovascular related disease and/or a metabolic syndrome.

[0036] The invention provides methods of increasing a subject's likelihood of longevity and/or of retaining cognitive function during aging which comprise inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3). The invention also provides methods of decreasing a subject's likelihood of developing a cardiovascular-related disease and/or a metabolic syndrome which comprise inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3).

[0037] The invention provides a method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a 264451.1 control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a cardiovascular related disease. The invention still further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a metabolic syndrome.

[0038] The invention provides methods of increasing a subject's likelihood of longevity, increasing a subject's likelihood of retaining cognitive function during aging, and/or of decreasing a subject's likelihood of developing a cardiovascular-related disease, a metabolic syndrome, and/or an age-related disease, which comprise increasing the subject's plasma level of insulin-like growth factor-1 (IGF-1).

[0039] The invention provides a method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in 264451.1 plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a cardiovascular related disease. The invention still further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a metabolic syndrome.

[0040] The invention provides methods of increasing a subject's likelihood of longevity, increasing a subject's likelihood of retaining cognitive function during aging, and/or of decreasing a subject's likelihood of developing a cardiovascular-related disease, a metabolic syndrome, and/or an age-related disease, which comprise increasing the subject's plasma level of adiponectin.

[0041] The invention provides an assay for identifying a compound that increases a subject's likelihood of longevity, increases a subject's likelihood of retaining cognitive function during aging, decreases a subject's likelihood of developing a cardiovascular- related disease, decreases a subject's likelihood of developing a metabolic syndrome, and/or decreases a subject's likelihood of developing an age-related disease, which comprises identifying a compound which: (a) increases HDL particle size in the subject's plasma, (b) increases LDL particle size in the subject's plasma, (c) increases both HDL and LDL particle size in the subject's plasma, (d) increases the percentage of large size HDL particles in the subject's plasma, (e) increases the percentage of large size LDL particles in the subject's plasma, increases the percentage of both large size HDL particles and large size LDL particles in the subject's plasma, (g) increases the subject's plasma level of HDL, (h) increases the subject's plasma level of insulin-like growth factor-1 (IGF-1), (i) increases the subject's plasma level of adiponectin, 264451.1 (j) inhibits the activity of the subject's cholesteryl ester transfer protein (CETP), and/or, (l) inhibits the activity of the subject's apolipoprotein C-3 (APOC-3).

Brief Description of the Drawings [0042] Figure 1A-1D. Percentage of large and small HDL and LDL particles in Proband, Offspring, Ashkenazi Controls, and age-matched Framingham Controls. Fig. 1A, large HDL; Fig. 1B, small HDL; Fig. 1C, large LDL; Fig. 1D, small LDL. *Significant differences (p<0.001) between Probands and Offspring versus the two Control groups.

[0043] Figure 2A-2C. Frequency distribution of lipoprotein properties in families of centenarians. The frequency distribution of plasma HDL levels (Fig. 2A), HDL particle size (Fig. 2B), and LDL particle size (Fig. 2C) in female proband (P), their offspring (O), and a control (C) population. Solid lines represent the mean and dashed lines represents 1 Standard Deviation of control. Mean plasma HDL level is similar to control in the proband, but nearly half of the offspring population has plasma HDL levels above 1 Standard Deviation of control, supporting an inherited pattern for a trait that is in decline in the proband. HDL particle size is shifted in the proband to increased size, and it is intermediate in the offspring (bi-modal pattern). Finally, LDL particle size frequency distribution is non- parametric and shifted to larger sizes in proband and offspring.

[0044] Figure 3A-3B. Odds ratio of HDL and LDL particle size to belong to proband over control. Large size particles are more likely to come from centenarians than from controls for both HDL (Fig. 3A) and LDL (Fig. 3B).

[0045] Figure 4A-4B. HDL (Fig. 4A) and LDL (Fig. 4B) particle size as a function of age. Cross-sectional data from control (closed circles) ages 60-95 (n=878 ; Ashkenazi and Framingham Study control), proband (triangles) with exceptional longevity ages 95-108 (n=191), and their offspring (open squares) ages 60-80 (n=206).

[0046] Figure 5. Plasma IGF-1 levels in offspring with (CVD+) and without (CVD-) cardiovascular-related disease.

[0047] Figure 6. Plasma IGF-1 levels and cognitive function in proband. Normal cognitive function (MMSE>25) indicated by black columns; impaired cognitive function (MMSE&lt;25) indicated by open columns.

[0048] Figure 7. Adiponectin plasma levels in proband, offspring, and control groups.

264451.1 [0049] Figure 8. Distribution of adiponectin plasma levels in proband, offspring, and control groups. Note the bimodal distribution in the offspring.

[0050] Figure 9. CETP 405 valine (V) allele in families with longevity. Frequency (%) of homozygosity for the codon 405 valine (V) allele of cholesteryl ester transfer protein (CETP) in female (F) and male (M) probands (P, n=156), offspring (O, n=163), and Ashkenazi controls (C, n=129). The frequency of the VV genotype is-2-3 fold increased in probands (P) and offspring (O) compared to Ashkenazi controls (C). **p<0.001. I, isoleucine.

[0051] Figure 10. Relationship between CETP I405V genotype and cognitive function in proband. Normal cognitive function (MMSE>25) indicated by black columns ; impaired cognitive function (MMSE&lt;25) indicated by open columns.

[0052] Figure 11. Relationship between apolipoprotein C-3 (APO-C3) genotype and adiponectin phenotype in offspring with (OAD+) and without (OAD-) high plasma levels of adiponectin.

Detailed Description of the Invention [0053] The subject invention is directed to a method of determining a subject's likelihood of longevity which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with the high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining an individual's likelihood of longevity which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a larger size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.

The invention further provides a method of determining a subject's likelihood of longevity which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of longevity.

264451.1 [0054] One embodiment of the method further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity. A further embodiment comprises comparing the number of medium size HDL particles as a percentage of total HDL particles from the subject with the percentage of medium size HDL particles from the control population, a smaller percentage of medium size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity.

One embodiment comprises comparing the number of large size LDL particles as a percentage of total LDL particles from the subject with the percentage of large size LDL particles from the control population, a larger percentage of large size LDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity. A further embodiment comprises comparing the number of small size LDL particles as a percentage of total LDL particles from the subject with the percentage of small size LDL particles from the control population, a smaller percentage of small size LDL particles from the subject than from the control population indicating that the subject has an increased likelihood of longevity.

[0055] In different embodiments of the method, the average size of the subject's high density lipoprotein (HDL) particles can be at least 2%, 3% or 4% greater than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject. The average size of the subject's low density lipoprotein (LDL) particles can be at least 2% or 3% greater than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.

[0056] In different embodiments, the average size of the subject's high density lipoprotein (HDL) particles can be at least 0.2 nm, 0.3 nm or 0.4 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject. The average size of the subject's low density lipoprotein (LDL) particles can be at least 0.4 nm, 0.5 nm or 0.6 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.

[0057] HDL and LDL particle size can be determined by a number of methods including nuclear magnetic resonance spectroscopy, gel electrophoresis, and electron microscopy measurement.

[0058] The invention provides a method of determining a subject's likelihood of longevity which comprises determining if the subject has a mutation in the gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an increased likelihood of longevity. As used herein, "decreased CETP activity"includes decreased levels of CETP and/or decreased activity of the protein itself. The CETP gene is known to be highly polymorphic, and many mutations have been described in its coding and non-coding regions (86,102-108). The presence of the B2 allele at the TaqlB polymorphism in intron 1 of the CETP gene has been reported to be associated with reduced CETP levels (103-106) and activity (107). A more recent study concluded that TaqlB polymorphism is not instrumental in determining CETP levels, but is a marker for a promoter variant of the CETP gene at position-629 relative to the transcription start, and furthermore that the-2708 and-971 polymorphisms are likely to play a role in determining CETP concentration (102).

In one embodiment, the invention provides a method of determining a subject's likelihood of longevity which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein, the presence of a homozygous valine/valine genotype indicating that the subject has an increased likelihood of longevity. The incidence of the homozygous allele can be increased about 2-3 fold in a subject with longevity compared to a control population.

[0059] The subject can be a mammalian subject or a human subject. A human subject with longevity can live to be at least 95 years of age. A subject with longevity can also be considered to be a subject in whom aging is delayed.

[0060] The invention provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. The invention also provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing sizes of both high density lipoprotein (HDL) 264451. 1 and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. One embodiment of the method further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL particles from the subject than from the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease.

[0061] In one embodiment, the average size of the subject's high density lipoprotein (HDL) particles can be at least 2% larger than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.

The average size of the subject's high density lipoprotein (HDL) particles can be at least 0.2 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject. The average size of the subject's low density lipoprotein (LDL) particles can be at least 0.3 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.

[0062] The invention also provides method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a smaller size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, smaller sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease. One embodiment of the method further comprises 264451.1 comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a smaller percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of developing a cardiovascular related disease.

[0063] In one embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 2% smaller than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject. The average size of the subject's high density lipoprotein (HDL) particles can be at least 0.2 nm smaller in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject. The average size of the subject's low density lipoprotein (LDL) particles can at least 0.3 mu smaller in diameter than the average size of the low density lipoprotein (LDL) particles from a control population of the same gender as the subject.

[0064] A subject is defined as having a"metabolic syndrome"according to the guidelines of the National Cholesterol Education Program (NCEP), Adult Treatment Panel III (ATP III) (113), if the subject has three or more of the following five risk factors: 1) increased waist girth (> 94 cm for women, >102 cm for men), 2) increased blood pressure (> 130//85 or treatment for hypertension), 3) increased fasting glucose (> 110 mg/dl or drug treatment for diabetes), 4) low plasma HDL cholesterol (< 40 mg/dl), and 5) elevated fasting triglyceride levels (> 150 mg/dl). In one embodiment of the methods described herein, the subject is defined as having a metabolic syndrome if the subject has three or more of the following four risk factors: 1) increased waist girth (> 94 cm for women, >102 cm for men), 2) increased blood pressure (> 130//85 or treatment for hypertension), 3) increased fasting glucose (> 110 mg/dl or drug treatment for diabetes), and 4) elevated fasting triglyceride levels (> 150 mg/dl).

[0065] The invention provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome. The invention also provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises 264451.1 comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a larger size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

The invention further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, larger sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome. One embodiment of the methods further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL particles from the subject than from the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome. One embodiment further comprises comparing the number of large size LDL particles as a percentage of total LDL particles from the subject with the percentage of large size LDL particles from the control population, a larger percentage of large size LDL particles from the subject than from the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

[0066] In one embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 3% greater than the average size of the high density lipoprotein (HDL) particles from the control population. In another embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 4% greater than the average size of the high density lipoprotein (HDL) particles from the control population. In one embodiment, the average size of the subject's low density lipoprotein (LDL) particles is at least 2% greater than the average size of the low density lipoprotein (LDL) particles from the control population.

[0067] In one embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 0.3 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from the control population. In another embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 0.4 nm 264451.1 larger in diameter than the average size of the high density lipoprotein (HDL) particles from the control population. In one embodiment, the average size of the subject's low density lipoprotein (LDL) particles is at least 0.3 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from the control population. In another embodiment, the average size of the subject's low density lipoprotein (LDL) particles is at least 0.5 nm larger in diameter than the average size of the low density lipoprotein (LDL) particles from the control population.

[0068] The invention provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a smaller size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome. The invention also provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing low density lipoprotein (LDL) particle size from the subject's plasma with low density lipoprotein (LDL) particle size from a control population, a, smaller size of the subject's low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.

The invention further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from the subject's plasma with the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles from a control population, smaller sizes of both the subject's high density lipoprotein (HDL) and low density lipoprotein (LDL) particles compared to the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome. One embodiment of the methods further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a smaller percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of developing a metabolic syndrome. One embodiment further comprises comparing the number of large size LDL particles as a percentage of total LDL particles from the subject with the percentage of large size LDL particles from the control population, a smaller percentage of large size LDL particles from the subject than from the 264451. 1 control population indicating that the subject has an increased likelihood of developing a metabolic syndrome.

[0069] In one embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 3% smaller than the average size of the high density lipoprotein (HDL) particles from the control population. In another embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 4% smaller than the average size of the high density lipoprotein (HDL) particles from the control population. In one embodiment, the average size of the subject's low density lipoprotein (LDL) particles is at least 2% smaller than the average size of the low density lipoprotein (LDL) particles from the control population.

[0070] In one embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 0.3 nm smaller in diameter than the average size of the high density lipoprotein (HDL) particles from the control population. In another embodiment, the average size of the subject's high density lipoprotein (HDL) particles is at least 0.4 nm smaller in diameter than the average size of the high density lipoprotein (HDL) particles from the control population. In one embodiment, the average size of the subject's low density lipoprotein (LDL) particles is at least 0.3 nm smaller in diameter than the average size of the low density lipoprotein (LDL) particles from the control population. In another embodiment, the average size of the subject's low density lipoprotein (LDL) particles is at least 0.5 nm smaller in diameter than the average size of the low density lipoprotein (LDL) particles from the control population.

[0071] The invention provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing high density lipoprotein (HDL) particle size from the subject's plasma with high density lipoprotein (HDL) particle size from a control population, a larger size of the subject's high density lipoprotein (HDL) particles compared to the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging. One embodiment of the method further comprises comparing the number of large size HDL particles as a percentage of total HDL particles from the subject with the percentage of large size HDL particles from the control population, a larger percentage of large size HDL particles from the subject than from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging.

264451. 1 [0072] In one embodiment of the method, the average size of the subject's high density lipoprotein (HDL) particles can at least 2% larger than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.

The average size of the subject's high density lipoprotein (HDL) particles can be at least 0.2 nm larger in diameter than the average size of the high density lipoprotein (HDL) particles from a control population of the same gender as the subject.

[0073] The invention provides a method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject has a mutation in a gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein, the presence of a homozygous valine/valine genotype indicating that the subject has an increased likelihood of retaining cognitive function during aging.

[0074] The invention provides a method of increasing a subject's likelihood of longevity which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of increasing a subject's likelihood of longevity which comprises increasing the size of low density lipoprotein (LDL) particles in the subject's plasma. The invention further provides a method of increasing a subject's likelihood of longevity which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma. One embodiment of the method further comprises increasing the number of large size HDL particles as a percentage of total HDL particles. One embodiment further comprises decreasing the number of medium size HDL particles as a percentage of total HDL particles. One embodiment further comprises increasing the number of large size LDL particles as a percentage of total LDL particles. One embodiment further comprises decreasing the number of small size LDL particles as a percentage of total LDL particles.

[0075] The invention provides a method of decreasing a subject's likelihood of developing a cardiovascular related disease which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of decreasing a subject's likelihood of developing a cardiovascular related disease 264451.1 which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.

[0076] The invention provides a method of increasing a subject's likelihood of retaining cognitive function during aging which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma.

[0077] The invention provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the size of low density lipoprotein (LDL) particles in the subject's plasma. The invention further provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises increasing the sizes of both high density lipoprotein (HDL) and low density lipoprotein (LDL) particles in the subject's plasma.

[0078] One embodiment of the methods further comprises increasing the number of large size HDL particles as a percentage of total HDL particles. One embodiment comprises increasing the number of large size LDL particles as a percentage of total LDL particles.

[0079] Several methods are known for increasing the sizes of high density lipoprotein (HDL) and low density lipoprotein (LDL) particles. For example, HDL and LDL particle size can be increased by exercise (20). Increased HDL sub-fractions with exercise training seemed dependent on CETP genotype (56). HDL particle size and the concentration of large HDL particles can be increased by administering a combination of estradiol and medroxyprogesterone (68). LDL particle size can be increased by some lipid lowering drugs, e. g. fenofibrate and atorvastatin (69).

[0080] The invention provides a method of increasing a subject's likelihood of longevity which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP). The invention also provides a method of increasing a subject's likelihood of retaining cognitive function during aging which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP). The invention further provides a method of decreasing a subject's likelihood of developing a cardiovascular-related disease which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).

The cardiovascular-related disease can be any one or more of, for example, hypertension, diabetes mellitus, myocardial infarction, stroke and transient ischemic attack. In one 264451. 1 embodiment, the cardiovascular-related disease is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma. The invention provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP).

The invention further provides a method of decreasing a subject's likelihood of developing a disease which is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma which comprises inhibiting the activity of the subject's cholesteryl ester transfer protein (CETP). The disease may be a cardiovascular-related disease.

[0081] The activity of cholesteryl ester transfer protein (CETP) can be inhibited by manipulations at any one or more of the levels of the protein itself, the protein target, or nucleic acids that encode the protein, and/or by decreasing the plasma level of CETP.

Human CETP and nucleic acid encoding it have been described (89-91,101-102). Possible methods of inhibiting CETP include use of antisense oligonucleotides, RNA aptamers (96), RNA interference (97), and antibodies to CETP (e. g. , 98-100). In addition, inhibitors to CETP and methods of making them have been described (92-94). Also, a method has been described for eliciting an immune response against CETP activity (95).

[0082] The invention provides a method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in 264451.1 plasma from the control population indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. In one embodiment, the cardiovascular-related disease is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma. The invention still further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of cholesteryl ester transfer protein (CETP) with the plasma level of cholesteryl ester transfer protein (CETP) from a control population, a lower level of cholesteryl ester transfer protein (CETP) in the subject's plasma than in plasma from the control population indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

[0083] The invention provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein (CETP), the presence of a homozygous valine/valine genotype indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. In one embodiment, the cardiovascular-related disease is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject is homozygous for a codon 405 valine allele of a gene encoding cholesteryl ester transfer protein (CETP), the presence of a homozygous valine/valine genotype indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

[0084] The invention provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises determining if the subject has a mutation in the gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. In one embodiment, the cardiovascular-related disease is characterized by a reduced size of high density lipoprotein (HDL) particles in the subject's plasma. The invention also provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises determining if the subject has a mutation in the gene encoding cholesteryl ester transfer protein (CETP) where the mutation results in decreased CETP activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

[0085] The invention provides a method of determining a subject's likelihood of longevity which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. The invention still further provides method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject is homozygous for a codon-641 cysteine allele of a promoter of a gene encoding apolipoprotein C-3 (APOC-3), the presence of a homozygous cysteine/cysteine genotype indicating that the subject has a decreased likelihood of developing a metabolic syndrome.

[0086] The invention provides a method of determining a subject's likelihood of longevity which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease 264451.1 which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a cardiovascular related disease. The invention still further provides a method of determining a subject's likelihood of developing a metabolic syndrome which comprises determining if the subject has a mutation in a gene encoding apolipoprotein C-3 (APOC-3), or a mutation in a promoter of a gene encoding apolipoprotein C-3 (APOC-3), where the mutation results in decreased APOC-3 activity, the presence of said mutation indicating that the subject has a decreased likelihood of developing a metabolic syndrome. In one embodiment, the mutation is in a gene encoding apolipoprotein C-3 (APOC-3). In one embodiment, the mutation is in a promoter of a gene encoding apolipoprotein C-3 (APOC- 3).

[0087] The invention provides a method of increasing a subject's likelihood of longevity which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3). The invention also provides a method of increasing a subject's likelihood of retaining cognitive function during aging which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3). The invention further provides a method of decreasing a subject's likelihood of developing a cardiovascular-related disease which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3). The invention still further provides a method of decreasing a subject's likelihood of developing a metabolic syndrome which comprises inhibiting the activity of the subject's apolipoprotein C-3 (APOC-3). The activity of apolipoprotein C-3 (APOC-3) can be inhibited by manipulations at any one or more of the levels of the protein itself, the protein target, or nucleic acids that encode the protein, and/or by decreasing the plasma level of apolipoprotein C-3 (APOC-3). Mammalian apolipoprotein C-3 (APOC-3) and nucleic acid encoding it have been described (150). Possible methods of inhibiting apolipoprotein C-3 (APOC-3) include use of antisense oligonucleotides, RNA aptamers (96), RNA interference (97), and antibodies to apolipoprotein C-3 (APOC-3).

[0088] The invention provides a method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's 264451. 1 plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a cardiovascular related disease. The invention still further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of insulin-like growth factor-1 (IGF-1) with the plasma level of insulin-like growth factor-1 (IGF-1) from a control population, a higher level of insulin-like growth factor-1 (IGF-1) in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a metabolic syndrome.

[0089] The invention provides a method of increasing a subject's likelihood of longevity, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF-1). The invention also provides a method of increasing a subject's likelihood of retaining cognitive function during aging, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF-1). The invention further provides a method of decreasing a subject's likelihood of developing a cardiovascular related disease, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF- 1). The invention still further provides a method of decreasing a subject's likelihood of developing a metabolic syndrome, which comprises increasing the subject's plasma level of insulin-like growth factor-1 (IGF-1). IGF-1 plasma levels could be increased by giving a subject IGF-1 or by giving a substance that increases the synthesis or release of IGF-1.

IGF-1 is commercially available.

[0090] The invention provides a method of determining a subject's likelihood of longevity, which comprises comparing the subject's plasma level of adiponectin with the 264451. 1 plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of longevity. The invention also provides a method of determining a subject's likelihood of retaining cognitive function during aging, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an increased likelihood of retaining cognitive function during aging. The invention further provides a method of determining a subject's likelihood of developing a cardiovascular related disease, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a cardiovascular related disease. The invention still further provides a method of determining a subject's likelihood of developing a metabolic syndrome, which comprises comparing the subject's plasma level of adiponectin with the plasma level of adiponectin from a control population, a higher level of adiponectin in the subject's plasma than in plasma from the control population indicating that the subject has an decreased likelihood of developing a metabolic syndrome.

[0091] The invention provides a method of increasing a subject's likelihood of longevity, which comprises increasing the subject's plasma level of adiponectin. The invention also provides a method of increasing a subject's likelihood of retaining cognitive function during aging, which comprises increasing the subject's plasma level of adiponectin. The invention further provides a method of decreasing a subject's likelihood of developing a cardiovascular related disease, which comprises increasing the subject's plasma level of adiponectin. The invention still further provides a method of decreasing a subject's likelihood of developing a metabolic syndrome, which comprises increasing the subject's plasma level of adiponectin. Adiponectin is also known as Acrp30 and AdipoQ.

The amino acid and DNA sequences for human Acrp30 have been described (151).

[0092] The invention provides an assay for identifying a compound that increases a subject's likelihood of longevity, increases a subject's likelihood of retaining cognitive function during aging, decreases a subject's likelihood of developing a cardiovascular- related disease, decreases a subject's likelihood of developing a metabolic syndrome, and/or 264451. 1 decreases a subject's likelihood of developing an age-related disease, which comprises identifying a compound which: (a) increases HDL particle size in the subject's plasma, (b) increases LDL particle size in the subject's plasma, (c) increases both HDL and LDL particle size in the subject's plasma, (d) increases the percentage of large size HDL particles in the subject's plasma, (e) increases the percentage of large size LDL particles in the subject's plasma, (f) increases the percentage of both large size HDL particles and large size LDL particles in the subject's plasma, (g) increases the subject's plasma level of HDL, (h) increases the subject's plasma level of insulin-like growth factor-1 (IGF-1), (i) increases the subject's plasma level of adiponectin, inhibits the activity of the subject's cholesteryl ester transfer protein (CETP), and/or, (k) inhibits the activity of the subject's apolipoprotein C-3 (APOC-3).

In one embodiment of the assay, the compound increases a subject's likelihood of longevity.

In one embodiment, the compound increases a subject's likelihood of retaining cognitive function during aging. In one embodiment, the compound decreases a subject's likelihood of developing a cardiovascular-related disease. In one embodiment, the compound decreases a subject's likelihood of developing a metabolic syndrome. In one embodiment, the compound decreases a subject's likelihood of developing an age-related disease. In one embodiment, the compound increases HDL particle size in the subject's plasma. In one embodiment, the compound increases LDL particle size in the subject's plasma. In one embodiment, compound increases both HDL and LDL particle size in the subject's plasma.

In one embodiment, the compound increases the percentage of large size HDL particles in the subject's plasma. Large HDL particles range in size from 8.8-13 nm. In one embodiment, the compound increases the percentage of large size LDL particles in the subject's plasma. Large LDL particles range in size from 21.3-23 nm. In one embodiment, the compound increases the percentage of both large size HDL particles and large size LDL particles in the subject's plasma. In one embodiment, the compound increases the subject's plasma level of HDL. In one embodiment, the compound increases the subject's plasma level of insulin-like growth factor-1 (IGF-1). In one embodiment, the compound increases the subject's plasma level of adiponectin. In one embodiment, the compound inhibits the 264451. 1 activity of the subject's cholesteryl ester transfer protein (CETP). In one embodiment, the compound inhibits the activity of the subject's apolipoprotein C-3 (APOC-3).

[0093] In any of the methods or assays described herein, the cardiovascular-related disease can be hypertension, diabetes mellitus, myocardial infarction, stroke and/or transient ischemic attack.

[0094] Since an increased likelihood of longevity also correlates with a decreased likelihood of developing an age related disease, the present invention also provides methods of determining a subject's likelihood of developing such age related diseases, as well as methods of decreasing a subject's likelihood of developing such diseases. Age related diseases include, but are not limited to, forms of cancer and Parkinson's disease.

[0095] In any of the methods described herein, the subject can be a human subject or a mammalian subject. The subject can be a male subject or a female subject. In one embodiment, the subjects are male subject. In another embodiment, the subjects are female subjects.

[0096] In any of the methods described herein, the control population can be a normal healthy population or a patient population, as appropriate for the particular method being carried out. Differences between the control population and the test group or subject can be assessed using any of the appropriate statistical measures known to those skilled in the art.

[0097] This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details 1. Methods and Materials [0098] Study desig7Z : The study was designed to characterize the phenotype and genotype of exceptional longevity, as well as certain disease states. Because subjects with exceptional longevity do not have a control group, important in the design was the recruitment of the offspring generation and appropriate control group. A complete description of the design of the study has been published (1,4, 14). Informed written consent was obtained in accordance with the policy of the Committee on Clinical Investigation of the Albert Einstein College of Medicine. Subjects were recruited by word of mouth and through advertisement in Jewish aging centers and homes. Informed written 264451.1 consent was obtained in accordance with the policy of the Committee on Clinical Investigation of the Albert Einstein College of Medicine.

[0099] Study Subjects : Two hundred and thirteen subjects with exceptional longevity (proband) 95-107 years of age (157 females and 56 males, average age 98.2 i 5.3 (S. D.) years) were recruited to participate in the study. The participant's age was defined by their birth certificate or date of birth as stated in their passport. Proband had to be living independently at 95 years of age, to reflect relatively good health, although at the time of recruitments they could be at any institution or dependency. A population of Ashkenazi Jews, which originate from relatively few founders, was chosen for study. Ashkenazi Jewish founders lived in the 16th-17th centuries in the'Pale of Settlement'of eastern and central Europe. They were subjected to isolation, inbreeding and then rapid expansion.

There is much evidence to support the use of this unique population as a resource to discover important genes, the breast cancer (BRCA) gene being a prominent example (15).

This population is also considered homogeneous from socio-economical and educational perspectives; however, this population is comparable to non-Jewish populations in regard to longevity, prevalence of atherosclerotic cardiovascular diseases, and dementia (4). In addition, proband had to have at least one offspring who was willing to be recruited to the study (122 females and 94 males, average age 68. 3 6.7 years, range 51-89 years). A case- control study was designed to control for the offspring group. Two different control groups were used. The first control group consisted of the spouses of the offspring (n=75, mean age 70.2 10.2 years, 53% female). Fewer spouses of offspring were recruited than offspring of centenarians because 22 spouses had died, 18 were divorced/separated, 10 were not Ashkenazi Jews, and 55 spouses elected not to participate, leaving the total number of participating spouses at 75. The first control group also consisted of age-matched Ashkenazi Jewish control from the Einstein Aging Study (16) (n=183 ; 732 years of age, 57% female), for a total of 258 subjects. The second control group was an age-matched Caucasian population from the Framingham study (n=589 ; 67.8 3.5 years, 48% female), to confirm the differences with a non-related population.

[00100] Procedures : A single nurse practitioner was sent to all probands in the morning to draw venous blood sample, conduct a physical examination, and obtain a medical history report, including review of the questionnaire, determining weight and body mass index, and administrating the Mini Mental Standard Exam (MMSE) test (17). At that visit the offspring and the participating spouses underwent similar procedures. The control group from the 264451.1 Einstein Aging Study underwent these procedures at their clinical site in the University, and all blood samples were assigned and handled by the General Clinical Research Center at Albert Einstein College of Medicine.

[00101] The guidelines of the National Cholesterol Education Program (NCEP), Adult Treatment Panel III (ATP HI) (113) were followed in defining"metabolic syndrome"as the presence of three or more of the following five risk factors: 1) increased waist girth (> 94 cm for women, >102 cm for men), 2) increased blood pressure (> 130//85 or treatment for hypertension), 3) increased fasting glucose (> 110 mg/dl or drug treatment for diabetes), 4) low plasma HDL cholesterol (< 40 mg/dl), and 5) elevated fasting triglyceride levels (> 150 mg/dl).

[00102] Lipids and Lipoproteins : Total plasma cholesterol, triglycerides, HDL, LDL, very low-density lipoprotein (VLDL), apolipoprotein AI (apo-AI) and apolipoprotein B (apo-B) concentrations were performed at the laboratories of Montefiore Medical Center- Moses division, where the intra and interassay variations ranged between 2.5 and 3.5% in all these determinations. These measurements were performed for the Framingham study in the central Framingham laboratory as previously described (109). Lipoprotein profiling by nuclear magnetic resonance spectroscopy was analyzed by LipoScience, Raleigh, North Carolina, as previously described (18,19). Each NMR measurement produces the concentrations of intermediate density lipoprotein (IDL), three LDL subclasses, and five HDL subclasses of varying size, given in mg/dL cholesterol concentration units. From the LDL and HDL subclass levels are calculated weighted-average LDL and HDL particle sizes (nm diameter) and LDL particle concentrations (nmol/L). After grouping the 5 individual HDL subclasses into 3 size categories (large, intermediate, small), the following lipoprotein subclasses were considered in the analyses: IDL (23-27 nm), large LDL (21.3-23 nm), intermediate LDL (19.8-21. 2 mou), small LDL (18.3-19. 7 nm), large HDL (8. 8-13 nm), intermediate HDL (8.2-8. 8 nm), and small HDL (7.3-8. 2 nm). LDL and HDL subclass distributions and particle sizes determined by NMR are highly correlated with those measured by gradient gel electrophoresis (67,110) and density gradient ultracentrifugation (20). LDL subclass diameters, which are consistent with those measured by electron microscopy, are uniformly-5 mu smaller than those estimated by gradient gel electrophoresis (111).

[00103] As described by LipoScience (76,78), LipoScience's clinical research service employs proton nuclear magnetic resonance (NMR) spectroscopy to simultaneously 264451.1 quantify subclasses of LDL and HDL. The NMR method exploits the fact that each lipoprotein particle in plasma of a given size broadcasts its own characteristic lipid NMR signal. The signal intensity is directly proportional to the lipoprotein's bulk lipid mass and particle concentration. Larger particles give rise to signals at a different point in a resonance spectrum compared to those from smaller particles, and the shape of each signal is distinct.

Measured signal intensities give the particle numbers, since there is a direct proportionality between signal size and the number of particles giving rise to the signal. The increased efficiency of the NMR method is apparent because each lipoprotein subclass in plasma broadcasts its own characteristic lipid NMR signal, analogous to the different sounds produced by bells of different size. Using proprietary spectral software, the lipid methyl group signals are decomposed to give the spectral contributions made by separate sub- populations of lipoprotein particles. A detailed description of the NMR analytical process employed by LipoScience is given in the Handbook of Lipoprotein Testing (77).

[00104] IGF-1 and adiponectin assays. IGF-I was measured using commercial enzyme- linked immunosorbent assay (ELISA): 2-site immunoassays based on paired mouse monoclonal antibodies (DSL, Inc.) after acid/ethanol extraction of the samples (148). These assays do not detect mouse IGF-1. Sensitivity for both is estimated at 0.03 ng/mL at a 1: 100 sample dilution post-extraction (149). IGF binding protein assays were carried out using commercial ELISA kits from DSL (133). These assays are based on mouse monoclonal antibodies to the human proteins and display no cross-reactivity with the mouse counterparts. Plasma adiponectin levels were determined using a radioimmunoassay kit (Linco Research, St. Charles, MO).

[00105] Genotypiflg assay for Cholesterol Esther Transfer Protein (CETP), Hepatic Lipase (HL), and 4polipoprotein C-3 (APOC-3) : DNA was extracted from whole blood from all patients. A total of 50 jj. l PCR amplifications were performed using 100 ng of DNA, 10 pmol of primers and 50 mM dNTPs. The promoter region for HL and CETP gene regions containing polymorphic sites, including nucleotides 250 and 514 for HL and 405 and 442 for CETP were sequenced and analyzed. A 667 bp fragment was amplified using the following primers: HL- (Forward) 5'CAGTCCTCTACACAGCTGGAAC3' (SEQ ID NO : 1) and (Reverse) 5'CGGGGTCCAGGCTTTCTTGG3' (SEQ ID NO : 2), CETP- (Forward) 5'AGCGGTGAT CATTGACTGCAGGAAGCTCTGGC3' (SEQ ID NO : 3) and (Reverse) 5'TATTTTTTT CACGGATGGGCA3' (SEQ ID NO : 4). PCR conditions were: 3' at 94°C for one cycle, 45s at 94°C, 1'at 66°C, 1'at 72°C, for 30 cycles conclude with 10'at 264451. 1 72°C, for one cycle. The product than was sent to sequencing using ABI3700 capillary sequencers, and a basic sequencing process consisting of template preparation, reaction, clean up, electrophoresis, and sequence trimming was carried out. Analysis of the sequence outputs was done both manually on the print out and electronically comparing sequences to standard sequences using BLAST software. In addition, several other known CETP multiallelic polymorphic markers (CETP AC (-631) (-629), CEPT D442G, CEPT G (+1), CEPT I405V) were studied, using a multilocus PCR-based genotyping assay (75). Briefly, DNA was extracted from whole blood and amplified using two multiplex'cocktails'of biotinylated primer pairs to target genomic fragments ranging from 75 to 375 base pairs in size. Amplified fragments within each PCR product pool were then detected colorimetrically with sequence-specific oligonucleotide probes immobilized in a linear array on nylon membranes. Probe specificities have previously been confirmed by sequencing and through use of DNA genotyped independently through other methods such as restriction length polymorphism analysis (75). CETP concentrations in human serum were measured by Elisa (Wako chemicals USA, Inc. Richmond, VA). Ill addition, several multiallelic polymorphic markers were analyzed for apolipoprotein C-3 (APOC-3) and its promoter, in particular APOC-3 C (-641) A.

[00106] Statistical analyses : Statistical analysis of the data was performed using PROC MIXED in SAS System Version 6.12 (SAS Institute, Cary, NC) and Stata version 8. 0SE (Stata Corp., College Station, TX). Since the distribution of lipoproteins and sizes were non-normal in many cases, the non-parametric Mann-Whitney test was used for comparisons. Additionally, a multinomial logistic regression was performed to determine which of the incommensurable predictor variables most strongly predicts the phenotype of longevity. Each variable's contribution to predicting longevity was represented by its t- statistic in the multinomial logit model output. Pearson's Correlation Coefficient was also used to express the correlation between variables. Narrow sense heritability (h2) was estimated from the slope of the linear regression of the traits of each parent on the mean value of offspring (112). For a comparison of the difference in allele frequency between the groups, Hardy-Weinberg equilibrium was tested, and the chi-square test was performed. A p-value less than 0.05 was considered to indicate a significant difference.

2. Results [00107] Lipoprotein properties in fa77zilies with exceptional longevity (Tables 1 and 2, Figure 1A-1D) : Four groups of subject are represented: centenarians, offspring of centenarians, and 2 controls (Ashkenazi Jews and age-matched Framingham Offspring study). The first group consists entirely of people endowed with a propensity to extreme longevity. The second group should be a mixture of those with and without a propensity to extreme longevity. The two control groups should contain few, if any, people destined to survive in good health to age 95. There were no significant differences between the groups for routine blood chemistries including electrolytes, liver function, and kidney function tests. Body mass index (BMI) was similar in offspring and control groups, which were both significantly greater than in probands. Total cholesterol levels are determined mainly by levels of HDL, LDL, and very low-density lipoprotein (VLDL), which are presented in the Table 1. Particles sizes were directly measured, enabling their assessment independently of the measurements of their plasma apoA, and apoB levels. While probands had lower levels of HDL cholesterol and apo-AI than either their offspring or control groups, their HDL particle size was markedly increased. The number of LDL particles and their size were also increased in probands, and were associated with decreased plasma LDL and apo-B levels.

[00108] Differences among the probands, offspring, and controls in the proportions of total LDL and HDL contributed to by the large and small subclasses of these lipoproteins are shown in Figure 1A-1D. In both probands and offspring, the large HDL subclass accounted for a much greater proportion of total HDL than in the controls, whereas the relative amounts of small HDL were much less than in controls. Similarly, the large LDL subclass was relatively much more abundant in probands and offspring than in controls, while the opposite was true for the small LDL subclass.

[00109] Because it was predicted that strong biological markers may be inherited in families with exceptional longevity, studies were conducted of the offspring (likely to inherit biological markers) and control groups, matching their age, total cholesterol, LDL cholesterol, and body mass index (BMI). When offspring are compared with control, the offspring are also remarkable for the large sizes of their LDL and HDL particles. In this case however, HDL and apo-AI levels were increased, in addition to the decreased plasma LDL and apo-B levels. Because offspring and Jewish control were matched for BMI and VLDL, differences in these variables did not seem to be required for the effects of particle size on longevity. The age-matched Framingham group had higher BMI and cholesterol levels and their lipoprotein profile seems to be worse than the Ashkenazi Jewish control.

These differences decreased when adjusted for BMI. Thus, both proband and offspring lipoprotein particle sizes were significantly different than control groups. Females, at any of the lipoprotein characteristic presented, have better profile than males.

[00110] A relationship exists between HDL particle sizes to high HDL levels, high apo-A levels, low LDL and VLDL levels and low BMI, and between LDL particle sizes to low LDL and VLDL levels, low apo-B levels, high HDL levels and low BMI. A multinomial logistic regression was performed to determine which predictor variables contribute most strongly to distinguish proband, offspring, and control (Table 2). The predictor variables studied were HDL size, LDL size, HDL, LDL Apo-Al, Apo-B, and BMI. The relative importance of these predictors cannot be assessed by comparing regression coefficients because the variables are not measured in common units or dimensions. Instead, the t- statistics were compared for each variable's contribution to the multinomial logi model.

LDL-size and HDL-size were found to consistently exhibit the strongest contribution to this model across all three levels of the subject grouping. In fact, each other variable was non- significant in one or two of the other groups.

[00111] HDL and LDL particle sizes as a function of age (Figures 2A-2C and 3A-3B).

When HDL and LDL particle sizes of all the population are drawn by age, sizes are relatively unchanged until age 85, and then the particles sizes of HDL and LDL increase (p<0.001 between centenarians and their offspring compared to either control group, Table 1). The offspring as a group have increased particle size for both LDL and HDL. The same relationship is demonstrated when male and female are considered separately (not shown).

Largest HDL particle sizes are greater than 20-fold more likely to belong to proband than to control (Figure 3A). Similarly largest LDL particle sizes are more than 10-fold more likely to belong to proband than to control (Figure 3B).

[00112] The percent of large, medium and small and LDL and HDL particles (Table 3).

The levels of small, medium and large LDL (L1-L3) and HDL (H1-H2, H3, H4-H5) particles were determined (not shown), but since study sub-groups had different total levels of lipoprotein (Table 1), the data represent these particles as a percentage of the total levels of LDL and HDL. Proband HDL particle size increased significantly (p&lt;0. 001), mostly due to a-40% increase in the subfraction of large size and a-50% decrease in the subfraction of medium size HDL particles (p<0.001 for both). In parallel, proband LDL particle size was also significantly increased primarily due to a-20% increase in the fraction of large 264451.1 size LDL particles and a-50% decrease in the fraction of small size LDL particles (p<0.001 for both). Offspring also had increased large HDL and decreased medium HDL particle sizes and increased large LDL and decreased small LDL particle sizes.

[00113] The percentage of large HDL was also compared in people aged 85 years and older who had died in a seven year period following collection of the blood samples versus survivors. People who were alive at the end of the seven year period had a higher percentage of large HDL than did those who died during the seven year period (average % large HDL: alive, 67.5%, n = 60; died, 60.5%, n = 16; p 0.01 ; large HDL (mg/dL): alive, 32.7, died 25, p 0.002).

[00114] Frequency distribution of lipoproteins properties in families of centenarians (Figure 4A-4B). The frequency distribution reveals whether the variable is parametric (important for the appropriate statistical test), and also as revealed here, important to suggest patterns of inheritance. The plasma HDL frequency distribution is parametric in female (Figure 4A) and male (not shown) control with mean values of-50-60 mg/dl for female and 40-50 mg/dl for male. Offspring have a peak near the mean but other peaks are seen on the right, demonstrating that 46% of the female offspring and 42% of the males (not shown) have plasma HDL levels that are above 1 standard deviation of normal. This pattern is consistent with inheritance of this trait. However, while many probands have unusually high plasma levels of HDL, their distribution is non-parametric, and the mean is approximately averaged. Thus, while plasma HDL levels may prove to be a good marker for longevity in the offspring, proband often do not have, or maintain this characteristic. As with the example for HDL levels above, offspring have a bi-modal frequency distribution of HDL particles size, shifting the distribution to the right, suggesting inheritance of large size HDL particles, as described for plasma HDL levels. However, the most striking feature is that most proband have large particle size. Lastly, the frequency distribution of LDL particle size is non-parametric, and demonstrates that LDL particle size is greater in probands and their offspring than in controls (Figure 4B). Data for males are similar, although average HDL and LDL particle sizes are lower than in females. When the offspring who had the upper tertile for HDL or LDL particle size were compared with their parents, each of the individual proband had smaller particle sizes than the respective offspring (p<0.001). This may indicate that although particle size is higher on average in proband, it may be on the decrease, and their real size and effects may be underestimated here.

264451. 1 [00115] Since proband variance was equal between genders while the offspring variance was not equal between genders, heritability of lipoprotein traits was performed in the two offspring genders separately. The heritability (h2) of HDL size is 0.32 (0.16-SD) in female and 0.70 (0.22) in male offspring. Similarly, the heritability (h2) of LDL size is 0.46 (0.20) in females and 0.6 (0.26) in males. All were statistically significant (p<0.01), supporting a genetic linkage with lipoprotein sizes.

[00116] LDL and HDL particles size and cardiovascular disease (CVD) and metabolic syndrome (Tables 4-6). LDL and HDL particle size, and the percent of large particles comprising total levels of the lipoproteins, in the control and offspring with and without hypertension, diabetes mellitus, myocardial infarctions, and strokes/transient ischemic attack (TIA), are presented in Table 4. Subjects without these risks had significantly increased LDL and HDL particle size and higher percent of large particles comprising total levels of the lipoproteins. Similar observation was noted when analyzed only for the offspring with and without CVD risks. Increased HDL levels and decreased LDL levels were also associated with CVD protection, whereas VLDL level was not. The same trend was noted for each individual disease risk, but the numbers were too small to reach statistical significance. This suggests a link between the size of lipoprotein particles and age-related CVD.

[00117] Similar results were obtained upon analyzing the relationship between lipoprotein particle size, hypertension, and history of CVD (defined as myocardial infarction, stroke, or transient ischemic attack) in the combined group of offspring and control (Table 5). Significantly higher percentage of large HDL particles, HDL particle size, percentage of large LDL, and LDL particle size where observed in healthy subjects compared to those with hypertension. Moreover, significantly higher percentage of large HDL particles (32%), HDL particle size, percentage of large LDL (54%), and LDL particle size where observed in healthy subjects compared to those with a history of CVD.

Interestingly, the lower LDL levels in the hypertension and CVD groups are probably accounted for by use of cholesterol-lowering drugs (18% in the healthy group, 38% in the hypertension group and 60% in the CVD group). Significantly higher levels of HDL in the hypertension and CVD groups were observed compared to the healthy group, but VLDL levels were not significantly different. This observation is also striking when healthy offspring only are analyzed vs. those with hypertension and history of CVD (data not 264451. 1 shown). In total, these findings suggest a link between the size of lipoprotein particles and age-related hypertension and CVD.

[00118] The metabolic syndrome (a. k. a. insulin resistance syndrome, syndrome X, dysmetabolic syndrome X) (113) is a risk factor for many causes of death (114). Thus, the frequency of metabolic syndrome according to the NECP III guidelines was determined.

Since the frequency of the metabolic syndrome increases with age, probands would be expected to have a much higher frequency of the metabolic syndrome than the younger control group. However, the frequency of the metabolic syndrome in probands was 44%, similar to a frequency of 39% in the much younger control group. Most important, offspring had a significantly lower frequency of the metabolic syndrome (26%; p<0.03 vs. control), although these groups were well matched for BMI and age. Subjects with and without the metabolic syndrome were tested to determine their lipoprotein sizes (Table 6). Indeed, larger HDL and LDL particle sizes were apparent in healthy subject than in age-matched subjects with the metabolic syndrome. This effect was not noted for LDL levels, perhaps because of the widespread use of statin therapy. Because reduced HDL level is one of the criteria for having the metabolic syndrome, and is associated with HDL particle size, the analysis was repeated with subjects whose metabolic syndrome was re-defined by 3 criteria other than plasma HDL levels. Lipoprotein particle sizes were still significantly larger in those without the metabolic syndrome. These findings suggest a lower frequency of metabolic syndrome-related traits in subjects genetically predisposed to longevity.

[00119] Offspring of centenarians are healthier (Table 7). The prevalence of selected chronic age-related diseases was evaluated in probands and their offspring (n=180, 55% female), compared to three control groups. The first control group consisted of their spouses. The second control group were age group-matched Whites from the National Health and Nutrition Examination Survey III (NHANES III) (n= 6728, sample mean 64.7 i 4.6 years of age). The third control group included all Ashkenazi Jews living in Israel and insured by the largest HMO-type health insurer in Israel (Clalit Health Services; n= 219,042, mean age 64. 4i2. 8 years). Each participant in the offspring case control study completed a questionnaire including questions (worded similarly to the ones used in the Israeli data set, and in NHANES III) regarding a personal history of hypertension, type 2 diabetes mellitus, heart attack and stroke. Because increased body mass index (BMI) is a major determinant of death from cardiovascular, cancer, and all causes of death (88), all subjects underwent a physical examination, which included measurements for BMI, and 264451. 1 body fat mass (BFM, using a RJL System for bioelectrical impedance analysis). Offspring of centenarians had significantly better health in all disease categories than did controls (Table 7).

[00120] HDL particle size and cognitive function (Table 8). Subjects with exceptional longevity usually escape forms of dementia; however, at the end of life many of these subjects have decreases in their cognitive function. LDL and HDL particle sizes and percentage of large particles comprising total levels of the lipoproteins were assessed in the proband with (MMSE<25) and without (MMSE>25) cognitive dysfunction. While LDL particle size had no relationship with MMSE scoring, HDL particle size and HDL levels demonstrated a significant relationship. Coupled with the apparent protective effects of higher HDL levels and large HDL-particle size on CVD described above, these findings suggest pleiotropic effects of large HDL-particle size. Because the offspring of this population almost always score maximally in this test, an assessment of he effect of their lipoprotein sizes on their cognitive function could not be done. <BR> <BR> <BR> <BR> <P>[001211 IGF-I levels correlate positively with protection froni cognitive decline and cardiovascular disease (Table 9, Figures 5 and 6). As demonstrated in Table 9, offspring and control, matched by gender, reported similar maximal heights. Proband maximal height was compared to a white population height of U. S. men and women who were 55-64 years of age in 1960-1962, as reported in the National Health Examination Survey of 1960-62 ("General Control"in Table 9, on average less than-10 years younger than the present cohort). As a group, proband are not shorter than the NHANES control. Because IGF-1 levels decrease with age, it is difficult to assess the meaning of low IGF-1 levels in proband compared with-30 years younger control. However, female offspring have higher IGF-1 levels than controls (P<0.01), while male offspring have IGF-1 levels similar to controls (Table 9). Strikingly, offspring without cardiovascular disease (CVD-) have significantly higher IGF-1 levels than offspring with cardiovascular disease (CVD+) (Figure 5).

Similarly, male probands with normal cognitive function (MMSE>25) have significantly higher IGF-1 levels than male probands with impaired cognitive function (MMSE<25) (Figure 6).

[00122] High levels of adipoizectiii in fainilies with exceptional longevity (Table 10, Figures 7 and 8). Adiponectin levels were measured in 100 proband, 100 offspring, and 100 controls. Adiponectin levels were twice as high in proband as in controls (Figure 7); however, proband had a lower BMI (-22). Although offspring and controls had similar BMI 264451. 1 (-26), adiponectin levels in offspring as a group were significantly higher than in controls (Figure 7). Figure 8 illustrates the distribution of adiponectin levels in these three groups.

Clearly, there is a bimodal distribution for adiponectin in the offspring. Table 10 shows data obtained from two subgroups of offspring: those with adiponectin levels over 13 pg/ml (which is over 1 SD for control) (OAD+) and those with lower levels of adiponectin (OAD- ). There was no correlation between BMI (not shown) and body fat (by bioimpedance) for OAD+ offspring, while this correlation was maintained in the OAD-offspring. The group of OAD+ offspring had significantly fewer subjects with metabolic syndrome than the OAD-group, consistent with the role of adiponectin as an insulin sensitizer. HDL levels, and HDL and LDL particle sizes were all dramatically elevated in the OAD+ group.

[00123] Genes regulating lipoproteijz sizes iya farzilies with longevity (Table 11, Figures 9-11). The enzymes cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) have been shown to modulate HDL and LDL levels and sizes. CETP mediates the transfer of cholesteryl ester from HDL in exchange for triglycerides in apolipoprotein B-containing lipoproteins. Analysis was carried out of several of the common polymorphic alleles of CETP (CETP AC (-631) (-629), CEPT asp442, CEPT G (+1)), along with sequencing of most of the HL promoter in these cohorts (negative results not shown), as well as polymorphic sites in apolipoprotein C-3 (APOC-3) and its promoter.

[00124] Allele frequencies of the Isoleucine (1) 405 Valine (V) allele of CETP were 0.46, 0.43 and 0.29 in probands, offspring and Ashkenazi controls, respectively (p< 0.01).

Strikingly, the frequency of homozygosity for the codon 405 valine allele (VV genotype) was 24.8% in female and male probands compared to only 8.6% in Ashkenazi controls.

These differences were statistically significant (p < 0.0003) in both males and females (Figure 9). The offspring of probands had a VV genotype frequency of 20.7%, intermediate between probands and controls and also significantly greater than in controls (p < 0.004).

The frequency of II, IV and VV genotypes of the CETP Ile405Val mutation in the control is similar to that in other reports (53,55). The relationships were assessed between the CETP I405V genotype and lipoprotein particle sizes and CETP activity in the control, offspring and proband, respectively. The VV phenotype was associated with significantly larger LDL and HDL particle sizes (Table 11). Furthermore, subjects with the VV genotype had 17% lower CETP concentrations compared to those with II or IV genotype, and HDL level and CETP levels were negatively correlated (r=-0. 29, p=0.03 ; Spearman's rho). These findings suggest a survival advantage for individuals with the VV genotype, perhaps 264451.1 mediated through decreased levels of CETP and its effects on lipoproteins and their particle sizes. No other alleles in CETP or HL had changes in frequency compared to control.

[00125] In regard to cognitive function, probands with the CETP VV genotype had an average MMSE score of 27 (95% CI 28.1, 29.4 ; normal MMSE score being >25). Probands with the II genotype had an average MMSE score of 22 (95% CI 19.4, 25.5 ; P&lt;0. 0004), and probands with the IV genotype had an average MMSE that was intermediate between those with the VV and II genotypes (average for IV = 24,95% CI 22.5, 26.4 ; p<0.01 vs. VV). As Figure 10 demonstrates, proband who scored >25/30 (consistent with good cognitive function) had lower frequency of the II genotype (20 vs. 36 %) and increased frequency in the VV genotype (33 vs. 15%), with no influence by the IV genotype. Indeed proband with MMSE<25 are more likely to have the I allele as oppose to subjects with MMSE>25 who are more likely to have V allele (58% and 55% respectively, p<0.05). Thus, proband with CETP VV had significantly better cognitive function than these without. In fact, they were the only proband subgroup with mini mental scores above 25, which is the threshold for cognitive dysfunction.

[00126] In regard to cardiovascular disease, subjects with the CETP VV genotype also show a trend toward having a lower incidence of cardiovascular-related disease.

[00127] In regard to apolipoprotein C-3 (APOC-3), there is a striking increase in proband and their offspring, compared to controls,. in the frequency of the homozygous cysteine/cysteine (CC) genotype at the C (-641) A promoter site (Frequency = 25% proband, 22% offspring, 10% control; p value = 5*10-4 control vs. offspring, 1*10-4 control vs. proband). The favorable CC genotype was associated with significant higher LDL size (21.39 vs. 21.21 nm ; p=0.01). In particular, female offspring with the CC genotype had larger LDL particle size than controls (21.66 vs. 20.97 ; p = 0.0001). Furthermore, HDL levels (72.7 vs. 62.0 ; p=0.009), but not HDL size, were increased in subjects with the CC genotype. The incidence of the CC genotype was also higher in offspring with high levels of adiponectin (OAD+) than in offspring without high adiponectin levels (OAD-) (Figure 11).

In contrast, CETP did not show any relationship to adiponectin in the OAD+ group. Only 5% of subjects with CETP VV and APOC-3 CC have overlapping genotypes.

3. Discussion [00128] The present application discloses that families with exceptional longevity have a biological marker in the form of increased particle sizes of HDL and LDL, which is largely 264451.1 independent of the levels of their lipoprotein and apolipoproteins. This particular phenotype is associated with a lower prevalence of risk of hypertension, cardiovascular related disease, and metabolic syndrome in their offspring and improved cognitive function in the probands, supporting a functional role in their longevity. The pattern of distribution in the offspring and the markedly increased frequency of homozygosity for the codon 405 valine CETP allele and for the codon-641 cysteine APOC-3 allele support the inheritability of this phenotype and exceptional longevity.

[00129] Because subjects with exceptional longevity escape many other age-related diseases, such as several forms of dementia, infections, and forms of cancer, one may ask whether large HDL and LDL particles are only markers for exceptional longevity or do they have a causative role? Current knowledge and the data disclosed herein support the possibility of causation between lipoprotein particle sizes and exceptional longevity, for several reasons which are discussed below.

[00130] In population studies there is usually a strong, mechanistically linked, inverse correlation between plasma levels of LDL and VLDL to HDL levels and HDL and LDL particle size. However, there is compelling evidence implicating LDL lipoprotein particle size as a stronger predictor of cardiovascular disease than LDL levels (21-23). Small LDL particles were shown to penetrate more readily into arterial tissue (24), bind more tightly to arterial proteoglycans (25), and oxidize more rapidly than larger LDL particles (26,27), and are associated with endothelial dysfunction (28), all mechanisms involved in the development of cardiovascular related diseases. Therefore, large LDL particle size may be important in protecting the vascular bed, and ensuring cardiovascular longevity in the proband. Considering that the prevalence of small particle size LDL (previously called subclass pattern B) is 3-4 fold increased in older compared with young men and women (29, 30), aging effects seem to be reversed in the proband and their offspring. In regard to HDL particle size, small HDL particle size has been demonstrated in patients with cardiovascular related disease (32). In addition, some lipid lowering drugs may shift the HDL particles to' bigger sizes in patients with cardiovascular disease, to sizes similar to those seen in patients without cardiovascular disease (31). However, causality between HDL particle size and cardiovascular disease has been debated because of the association of cardiovascular disease with small LDL particles and increased triglycerides (32).

[00131] HDL has been thought to exert its effects through reverse cholesterol transport.

This ability to clear cholesterol from the endothelial and other peripheral cells may lead to 264451. 1 improved organ function. Thus, HDL may have protective effects from'lipotoxicity'similar to that obtained in caloric restricted rodents, whose life span is dramatically prolonged (33).

In addition, other biological properties of HDL have been described including anti- inflammatory (34), anti-oxidative (36), anti-aggregatory (35), anti-coagulant (37), and pro- fibrinolytic activities (38), which are exerted by HDL particles and other components, including interactions with apolipoproteins, enzymes, and even specific phospholipids (39- 40). This complexity emphasizes that changes in the functionality of HDL, which could occur through changes in mass or size, may determine the anti-aging effects of HDL.

Indeed, the effects of exercise imposed on an elderly population increase lipoprotein size, in the absence of change in lipoprotein levels (20). Such data support the notion that lipoprotein sizes may also be mediating protection from a variety of age-related diseases, down stream to environmental factors such as exercise.

[00132] The strongest support for a clinical role for lipoprotein size in exceptional longevity is derived from the data presented herein. The elderly subjects in this study who escaped hypertension, type 2 diabetes mellitus, myocardial infarction, or stroke/transient ischemic attack (TIA) have significantly larger LDL and HDL particle sizes. These data lead to the hypothesis that cardiovascular protection achieved by increased particle size of HDL and LDL is important for exceptional longevity. While centenarians'brain are chronologically old, post-mortem analysis suggests they are free from the typical pathology of Alzheimer's or vascular dementia. Surprisingly, upon testing the cognitive function of proband with exceptional longevity, large HDL, but not LDL, particle size seemed protective from cognitive decline. This may be due to the fact that the large particle size of HDL lipoprotein may play an additional or different role than LDL particle size, and that such a role is unique in the brain. Indeed, increased plasma levels of HDL (which is correlated with HDL particle size in-70 year old subjects) may offer protection from Alzheimer's (41) and other forms of dementia (42,43). Furthermore, in a group of elderly, low cognitive function was significantly associated with low HDL levels even after subjects with cardiovascular disease or stroke were excluded, supporting the association between HDL and cognitive function independent of cardiovascular disease (44). As is the case for HDL levels, HDL and LDL particle sizes are significantly larger in women than in men, and may explain why out of 100 people reaching the age of 100 years, 85 are women. It is possible that in order for men to get to 100 years old, VLDL levels also need to be very low.

264451.1 [00133] Because lipoprotein particle sizes correlate with their levels and with their respective apolipoprotein, it is possible that particle size is a marker for another variable that may exert the actual effect. However, the normal plasma levels of HDL and apo-Al, in the face of the largest HDL particle size in proband, strongly challenge this possibility.

Moreover, a careful analysis of the data demonstrates that in each of the groups studied (proband, offspring and control), LDL and HDL sizes were markedly and strongly correlated with each other in all groups; however, the other variables assessed by regression had weaker effect and significantly contributed to the phenotype in only one or two of the groups. Furthermore, adjusting for lipoprotein levels, apolipoprotein and BMI, still left lipoprotein particle sizes as the most prominent finding in this study.

[00134] The implication of CETP in the genetic analysis also supports the role of HDL particle size rather than its levels in exceptional longevity. In plasma from patients homo- and heterozygous with CETP deficiency, levels of large HDL particles increase two-and six fold, while levels of small HDL remain unchanged with apoE-containing and CE-rich HDL (45,46). Complete CETP deficiency causes a small-sized LDL population with low affinity for the LDL receptor (47). However, because an upregulation of the LDL receptor increases LDL clearance, CETP deficiency is characterized by lowered LDL levels (49).

Interestingly, because cholesteryl ester is not transferred off the HDL particle when CETP activity is low, it may indicate that reverse cholesterol transport may not be the main benefit exerted by HDL and its large particles, and this may have implications for relevant drug development. The support for CEPT involvement in HDL size and its association with exceptional longevity derives from the markedly increased frequency of homozygosity for the codon 405 valine in families with longevity. While this mutation was observed in only ~30% of the proband, it implicates that pathway in the phenotype of longevity. In fact, it represents a 2-3 fold increase in the frequency of this polymorphic allele compared with several control groups. Female offspring of proband had intermediate frequency, and those with the valine allele had the highest HDL and HDL sizes. Interestingly, increased HDL cholesterol levels caused by mutations in CETP were associated with a slight increased risk of ischemic heart disease in white Danish women (53); however, recently the Veterans Affairs HDL Cholesterol Intervention Trial reported that CETP TaqI B2B2 genotype is associated with higher HDL cholesterol levels and lower risk of coronary heart disease in men (54). It has been suggested that this polymorphic allele in codon 405 valine, which on its own may not be functional, is a marker for anther functional mutation in the TaqI B2B2 264451.1 (55). Nevertheless, there is no other example for polymorphic allele whose frequency has so dramatically increased in centenarians. As mentioned above, exercise training increases HDL and LDL particle size (20), an effect that seems dependent on CETP genotype (56).

Delipidation of HDL by hepatic lipase leads to the generation of smaller HDL subclasses in most subjects (50-52), but increased rates of known or new mutations in the promoter of hepatic lipase were not observed in the present study.

[00135] Finally, there is strong evidence for the inheritance of exceptional longevity (2- 4). While the increased frequency of the homozygous valine allele in the CETP gene in proband and their offspring support inheritance, so does the pattern of distribution of HDL and HDL particle size in the offspring. While the distribution of this trait is near-normal in controls, offspring have bi-modal distribution for HDL levels and HDL particle size. For plasma HDL levels, 46% of the offspring have levels above 1 standard deviation of the normal, and this proportion is also increased for HDL particle size, supporting the inheritance of a very marked effecter. Plasma HDL levels was shown to decrease with aging, a fact that may explain why levels are slightly low or normal in the proband.

Furthermore, the parents of the offspring with the largest HDL and LDL particle sizes had significantly lower particle sizes than their offspring, suggesting that the life-time size of these particles may be underestimated. Following longitudinally the offspring with increased HDL and LDL particle sizes may provide the best evidence for their importance.

While these results were obtained in Ashkenazi Jews, the similarity between the control and the Framingham Offspring confirms that this population is not different than other Caucasian populations in the United States. Furthermore, the rates of age-related diseases and life expectancy of the Ashkenazi population of Israel is similar to that of the United States (4), suggesting that the longevity of the subjects of this population is not unique.

However, even if the mechanism for exceptional longevity of this population is unique, its applicability may be universal.

[00136] In recent years genetic experiments have extended life-span in lower species by mechanisms that sometimes seem unlikely to be relevant for humans (1). Because humans aging is long and is associated with specific diseases, the relevant mechanisms for longevity should be universal. It is interesting that from all mammalians, CETP is one enzyme that is lacking in rodents, and may have been evolutionary dropped to ensure the longevity of rodents. While the proband have nearly doubled their life expectancy at birth, they are unique in staying healthy until very late age, certainly at the age where their friends got sick 264451.1 and died. The present application indicates that larger HDL and LDL particle sizes are important for longevity, maybe by ensuring protection of the cardiovascular system and the aging brain. It is still to be determined if these particle sizes are sufficient to ensure exceptional longevity, protecting from all causes of death. The CETP 405 valine genotype results in inactivation of the CETP enzyme and larger HDL particle size. Thus, manipulations at the level of DNA, RNA and/or protein that inhibit CETP and/or increase HDL and LDL particle size would be expected to promote longevity and protect from cognitive dysfunction and cardiovascular and age-related diseases.

Table 1. Lipoproteins properties in families with exceptional longevity. The Table presents the levels of total, LDL, VLDL and HDL cholesterol, levels of apolipoprotein B (apo B), apolipoprotein AI (apo AI), sizes of LDL and HDL particles, number of LDL particles, and body mass index (BMI) of male and female probands, their offspring, controls, and subjects from the Framingham Study (F). *These measurements of the Framingham Study were not obtained using the same laboratory as the other groups. Data are expressed as means and 95% confidence intervals. Significant differences between Probands and Offspring: *p&lt;0. 05, **p<0.01, ***p<0.001. NS, not significant. Female Proband N=157 Offspring N=122 Control N=147 Framingliam Pvs. O C vs. P, O F vs P, O N=276 Trait Avr CI Avr CI Avr CI Avr CI P< P< p< Cholesterol 204*** (198, 211) 227 (220, 233) 222 (211, 232) 0. 001 0. 006, N. S (m dL) LDL 117* (111, 123) 128 (121, 134) 130. 2 (120, 140) 0. 02 0. 02, N. S m dL 96* (92, 100) 104 (99, 109) 100 (91, 108) 0. 01 N. S, N. S (made 96* (92, 100) 104 (99, 109) 100 (91, 108) * 0. 001 0. 001, 0. 001 LDL (1118, (1012 (1525 LDL * (1118, (1012, (1525, Particles 1079* (1022, 1136) 1190 1262) 1135 1257) 1576 1627) 0. 01 N. S, N. S 0. 001, 0. 001 (nmol/L) HDL 56. 3 (53. 6, 59) 69. 9 (67, 73) 60. 7 (56. 7, 64. 6) 0. 001 0. 08, 0. 001 m dL *** HDL Size 9. 546 (9. 469, (9. 296, (9. 100, (9. 299, 00010001 NS (nom) 9. 622)9. 469) 9. 267 9. 4Q2 AprtAi APO Al 151*** (146, 157) 186 (179, 191) 165 (156, 174) 0. 001 0. 01, 0. 001 m dL VLDL 72. 8 (66. 5, 79. 1) 71. 8 (63. 3, 80. 4) 77. 2 (70. 7, 83. 7) 78. 5 (73. 4, N. S. N. S., N. S. N. S., N. S. BMI (kg/m2) 7 (22. 1, 23. 3) 24. 8 (24. 1, 25. 5) 24. 7 (23. 8, 25. 7) 0. 001 0. 001, N. S % Fat 26. 8 (24. 6, 29) 32. 1 (30. 6, 33. 6) 32. 2 (29. 8, 34. 7) 0. 001 0. 001, N. S Male Proband N=56 Offspring N=94 Control N=111 Framingham P vs. O C vs. P, O F vs P, O Il=309 Trait Avr CI Avr CI Avr CI Avr CI P< P< P< Cholesterol 183* (174, 192) 196 (188, 203) 184 (173, 195) 0. 03 N. S, 0. 06 m dL LDL 105 (97, 113) 112 (105, 118) 102. 8 (93, 112) * N. S N. S, 0. 1 APO B 91 (84, 97) 95 (90, 99) 86. 5 (80, 93) * N. S N. S, 0. 04 e T 21. 11 20. 91 20. 7 06'0. 001, 0. 03 0. 001, 0. 001 nu 21. 53) 21. 27 21. 02 20. 74) LDL (1007, (860, (1546, Particles 1019 (946, 1092) 1077 1147) 4 1038) 1589 1632) NS N. S, 0. 05 0. 001, 0. 001 (nmol/L HDL 50 (45, 55) 53 (49. 6, 55. 9) 48. 8 (45, 52. 5) * N. S N. S, 0. 1 HDL Size 9. 407 (9. 243, 9, 195 t8. 998, 8, 9 (8. 903, 9, 04 8. 996 p, 001 0. 002, 0. 06 0. 001, N. S (nm)*** 9. 572) 9. 192) 9. 055),9. 08,) APOA1 APO Al 131** (123, 140) 148 (141, 155) 136. 1 (124, 148) 0. 002 N. S, 0. 05 m dL VLDL 71 (60. 1, 82) 75. 1 (64. 8, 85. 5) 93. 1 (87. 3, 98. 8) 74. 6''N. S. N. S., N. S. 0. 005, 0. 004 BMI (kg/m2) *** (22. 4, 24) 26. 6 (26, 27. 3) 25. 9 (24. 8, 27) 0. 001 0. 001, N. S % Fat 21 (19. 3, 22. 7) 24. 2 (22. 9, 25. 4) 23. 3 (21, 25. 7) 0. 006 0. 1, N. S Table 2. Multiple logistic regression analysis for HDL and LDL particle size and the level of HDL, LDL, apolipoprotein B (apo B), apolipoprotein AI (apo AI), and body mass index (BMI) of male and female probands (P), their offspring (O), and control (C). A multinomial logistic regression was performed, and t-statistics (t value) and its P value are presented for each variable's contribution to the multinomial logit model. LDL-size and HDL-size consistently exhibited the strongest contribution to this model across all three levels of the subject grouping. Proband Offspring Control HDL size t value P value t value P value t value P value HDL-C 4. 48 <. 0001 8. 28 <. 0001 0. 01 0.99 APOA1-1. 1 0. 27-4. 44 <. 0001 1. 7 0.09 LDL-C-2. 37 0. 02-4. 88 <. 0001-1. 88 0.06 LDL Size 6.4 <. 0001 9. 19 <. 0001 4.72 <. 0001 APO B -0. 53 0. 6 0.97 0. 3347 -0. 84 0.4 BMI -1. 75 0. 08-1. 02 0. 3111-2. 34 0.02 LDL size LDL-C 1.37 0.1739 3. 52 0.0006 2. 81 0.0072 APOB 1.02 0. 3099 -1. 98 0.0499-0. 98 0.3307 HDL-C-0. 68 0. 4955 -2. 53 0.0123 1.71 0.0938 HDL Size | 6.4 <. 0001 9. 19 <. 0001 4.72 <. 0001 APO A1 l 0.53 0.5985 3. 37 0. 001-1. 04 0.303 BMI 0.34 0. 7353 0.48 0. 6285 -0. 17 0.87 Table 3. The percent of large, medium and small LDL and HDL particles as a function of the total levels of LDL and HDL. Probands and offspring had increased large LDL and decreased small LDL particle sizes. Probands and offspring also had increased large HDL and decreased medium HDL particle sizes. Data are expressed as means and 95% confidence intervals. Significant differences between Probands and Offspring: *p<0.05, **p&lt;0. 01, ***p&lt;0. 001.

Proband vs. Offspring vs. <BR> <BR> <P> Proband Offspring Control Framingham Control, Control,<BR> <BR> <BR> (N=143) (N=114) (N=147) (N=276) Framingham Framingham LargeLDL (%) 72. 3 (68-76. 5) 76.8 (72-81. 6) 59. 6 (55-64.2) 56. 1 (53.4-58. 7) 0. 001, 0.001 0. 001, 0.001 MediumLDL (%) 21. 6 (17.7-25. 3) * 15.3 (11.4-19. 1) 17. 1 (13.7-20. 4) 29. 6 (27. 6-31. 6) 0.08, N. S 0.001, 0.001 Small LDL (%) 6. 18 (3.92-8. 43) 7.87 (4. 64-11. 1) 23.3 (19. 3-27. 2) 14.3 (12.6-15. 9) 0.001, 0.001 0.001, 0.001 Large HDL (%) 61.6 (59.2-63. 9) * 57.4 (54. 7-60. 1) 45.5 (42.8-48. 1) 46.7 (44.5-49) 0.001, 0.001 0.001, 0.001 Medium HDL (%) 10.4 (9. 01-11. 9) * 13.5 (11. 5-15. 4) 17. 1 (13.7-20. 4) 21.7 (20.1-23. 2) 0. 08, N. S 0.001, 0. 001 Small HDL (%) 28 (26.1-29. 8) 29.1 (26.9-31. 2) 23. 3 (19.3-27. 2) 31. 6 (30-33.1) 0.001, 0.001 0.004, 0.001 Male Proband Offspring Control Framingham (N=48) (N=92) (N=tH) (N=309) Large LDL (%) 66. 7 (58.7-74. 6) 58.8 (52. 4-65. 1) 55.7 (51.3-60. 2) 44. 6 (41.9-47. 2) 0.01, N. S 0.001, 0.001 MediumLDL (%) 23. 6 (16.8-30. 4) 22.2 (17.7-26. 7) 18.5 (14.4-22. 6) 31.9 (29.7-34) N. S, N. S 0.007, 0.001 Small LDL (%) 9.74 (4.13-15. 3) * 19.0 (13. 5-24.5) 25.7 (21.7-29. 7) 23.5 (21.5-25. 6) 0.001, 0.001 0.001, 0. 06 Large HDL (%) 56.9 (52. 5-61. 4) *** 46. 9 (43. 3-50. 4) 39.8 (37.5-42. 1) 33 (30.9-35. 1) 0.01, 0.001 0.001, 0.001 MediumHDL (%) 9.8 (7. 3-12. 3) *** 15.8 (13.4-18. 1) 18.5 (14.4-22. 6) 25.1 (23.7-26. 4) N. S, N. S 0.001, 0.001 Small HDL (%) 33.3 (29.2-37. 3) 37.3 (34.5-40. 1) 25.7 (21.7-29. 7) 41.9 (40.1-43. 6) 0.001, 0.04 0.001, 0. 06 Table 4. LDL and HDL particles size and cardiovascular disease (CVD). LDL and HDL particle size, and the percent of large particles comprising total levels of the lipoproteins, in the control and offspring with and without CVD risk factors (hypertension, type 2 diabetes mellitus, myocardial infarctions, or strokes/TIA). Subjects without these risks had significantly increased LDL and HDL particle size and higher percent of large particles comprising total levels of the lipoproteins. Similar observation were noted when analyzed for offspring with and without CVD risks. This suggests a link between the size of lipoprotein particles and age-related CVD. Data are expressed as mean standard deviation. Healthy CVD P Variable (N=160) (N=82) value HDL mg/dL 55.9 +/- 1.2 47.8 +/- 1.7 0.001 Large HDL mg/dL (% Total HDL) 31.9 +/- 1.17 958%) 23.6 +/- 1.49 (49%) 0.001 HDL Size (nm) 9.33 +/- 0.04 9.12 +/- 0.05 0.001 LDL mg/dL 114.2 +/- 2.5 101.9 +/- 3.6 0.005 Large LDL mg/dL (% Total LDL) 79.5 +/- 3.01 (70%) 57.8 +/- 4.43 (56%) 0.001 LDL Size (nm) 21.4 +/- 0.05 21.1 +/- 0.09 0.002 VLDL mg/dL 70.1 +/- 3.3 72.3 +/- 5.5 NS Table 5. LDL and HDL and their particles size in healthy subjects, subjects with hypertension (HTN), and subjects with a history of cardiovascular disease. Cardiovascular disease (CVD) is defined here as myocardial infarction, stroke or transient ischemic attack.

Subjects are pooled Offspring and Control. Data expressed as mean S. D. NS, not significant. Healthy vs. Healthy HTN Variable CVD HTN, CVD (N=209) (N=64) P value (N=20) HDL (mg/dL) 64. 5 ~ 19. 4 57. 1 ~ 17. 2 50. 8 ~ 18. 4 0.004, 0.003 Large HDL (% Total) 54. 5 ~ 16 46. 8 ~ 17. 1 41. 3 ~ 18. 1 0.002, 0.001 HDL Size (nm) 9. 32 ~ 0. 49 9. 07 0. 42 8. 96 i 0. 55 0.001, 0.001 LDL (mg/dL) 120.3+/-34. 9 116. 9 ~ 38. 2 104. 1 ~ 35. 7 N. S, 0.03 Large LDL (% Total) 66. 5 27. 5 57. 6 A 30. 7 43. 3 28. 1 0.02, 0.001 LDL Size (nm) 21. 4 A 0. 6 21. 1~ 0. 8 20. 8 ~ 0. 8 0.008, 0.001 VLDL (mg/dL) 80. 2 ~ 77. 9 93. 7 ~ 84. 6 114 ~ 90. 86 NS, NS Table 6. LDL and HDL and their particles size in healthy subjects and subjects with Metabolic Syndrome (MS). Metabolic Syndrome defined by NCEP III criteria (113) and by NCEP III criteria excluding HDL (HDL-). Data expressed as mean i S. E.

Healthy MS MS (HDL-) Healthy vs.<BR> <BR> <BR> <BR> <BR> <BR> <P>Variable (N=221) (N=47) (N=25) MS,MS (HDL-)<BR> <BR> <BR> P Value HDL (mg/dL) 63.2 (1. 14) 46.6 (1. 75) 46.8 (4.98) 0.0001, 0.002 Large HDL (% Total) 66.9 (1. 58) 42 (3.94) 40.89 (2.9) 0.0001, 0.0001 HDL Size (nm) 9.28 (0.03) 8.88 (0.05) 8.95 (0.07) 0.0001, 0.001 LDL (mg/dL) 122 (2.14) 112.9 (5.4) 114.9 (6.43) 0.09, 0. 32 Large LDL (% Total) 56.5 (0.9) 39.7 (2.06) 51.3 (2.23) 0.0001, 0.0004 LDL Size (nm) 21.33 (0.04) 20.76 (0.11) 20.97 (0.13) 0.0001, 0.01 VLDL mg/dL 66. 8 (2.5) 106.4 (6.3) 108 (7.2) 0.0001, 0.0001 Table 7. Health of centenarians and their offspring compared to controls. Abbreviations: BMI, body mass index; DM, type 2 diabetes mellitus; HTN, hypertension; IDB, Israeli data base; MI, myocardial infarction ; PBF, percent body fat. (A) (B) (C) (D) (E) P value P value P value P value Proband Offspring Control NHANES IDB A vs. D B vs. D B vs. C B vs. E Disease n=145 n=180 n=75 n=6728 n=219042 HTN (%) 36 33 38 43 45 0.22 0.03 0.57 0.08 DM (%) 6 7 18 14 18 0.006 0.01 0.02 0.001 MI (%) 13 4 15 10 * 0.19 0.007 0.001 Stroke (%) 2 0 2 6 4 0.07 0.01 0.001 0.001 BMI 22.7 25.2 24.8 0.3 (kg/m2) # 0.3 # 0.3 # 0.4 PBF (%) 23.3 27.0 28.1 0.15 # 0.7 # 0.5 # 1.2 Table 8. HDL level and particle size and cognitive function. LDL and HDL particle sizes and percentage of large particles comprising total levels of the lipoproteins in the proband with (MMSE<25) and without (MMSE&gt;25) cognitive dysfunction. While LDL particle size had no relationship with Mini Mental scoring, HDL particle size demonstrated a significant relationship. Data are expressed as mean 1 S. D. MMSE, Mini Mental Standard Exam. MMSE>25 MMSE<25 Variable P Value (N=68) (N=71) HDL mg/dL 60.4 +/- 2.5 48.3 +/- 1.7 0.0001 Large HDL mg/dL (% Total HDL) 39.9 +/- 1.17 29.6 +/- 1.49 0.0001 HDL Size (nm) 9.51 +/- 0.04 9.28 +/- 0.05 0.0001 LDL mg/dL 113.5 +/- 4.21 116.8 +/- 4.41 NS Large LDL mg/dL (% Total LDL) 69.5 +/- 3.01 61.8 +/- 4.43 NS LDL Size (nm) 21.0 +/- 0.05 21.1 +/- 0.09 NS VLDL mg/dL 72.8 +/- 4.8 80.2 +/- 4.7 NS Table 9. Height, and IGF-1 and IGF binding protein-3 (BP-3) plasma levels in Proband, Offspring and their Control, and in a General Control group.

General Offspring Control Proband Control Female Male Female Male Female Male Female Male <BR> <BR> <BR> <BR> <BR> N=height 143 134 65 39 136 43 443 418<BR> <BR> Maximal<BR> <BR> <BR> 63.4 69.5 64.4 69.4 62.5 66.8 60.5 66.9<BR> Height<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> (2.26) (2.57) (2.56) (2.24) (2.33) (2.87)<BR> (inches) N=IGF-1 65 45 21 14 78 26 <BR> <BR> <BR> <BR> IGF-1 130* 163 115 163 111* 87.5*<BR> <BR> <BR> (ng/ml) (57) (51.4) (52. 6) (61.2) (51.8) (39. 1) BP-3 1356 948 1389 860 934 531 (ng/ml) (1857) (1498) (1815) (1393) (1416) (984) *p<0.01 vs. control. Data presented as Mean and (S. D.).

Table 10. Body fat, metabolic syndrome, HDL levels, and HDL and LDL particle sizes in offspring with (OAD+) and without (OAD-) elevated levels of adiponectin. OAD+, adiponectin levels over 13 ilg/ml (which is above 1 SD for control).

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