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Technical field of the invention

The present invention relates to the association between the occurrences of selected single nucleotide polymorphisms (SNPs) in genes involved in iron dependent inflammation in the Central Nervous System and the onset and development of multiple sclerosis (MS) . More particularly, the present invention relates to methods for determining a subject susceptibility to develop MS, to progress towards severe phenotypes, and/or to have prognostic information for a definite MS diagnosis and targeted treatment, based on the identification of such SNPs.

Background of the invention An association has been recently discovered between extracranial venous obstructive malformations and clinically disabling neurological disorders.

Among these stands multiple sclerosis (MS) , an inflammatory, demyelinating disease of the central nervous system of elusive origin, which leads to nerve degeneration .

The fundamental demyelinating lesions of MS are plaques, which have a vein running through their center. These veins are dilated and split the MS lesions longitudinally, as demonstrated by autopsic studies and magnetic resonance venography. Histological examination of these veins reveals unequivocally the presence of characteristic signs of impaired venous drainage, perivenous iron deposits and fibrin cuffs.

Starting from these observations and an unexpected reflux from the chest into the internal jugular vein of a patient affected by MS during a duplex scanning examination, the present inventors demonstrated (see international patent application WO-A-2009/107152) that in the jugular/vertebral veins of MS patients malformations exist (stenose) , which determine a chronic obstruction of the extracranial venous outflow, thus causing severe heamodynamic alterations. During such studies they also demonstrated that there exists a correlation between the topographic disposition of these stenosis and that of MS lesions.

Previous reports had also suggested the existence of a close relationship between the dilated cerebral veins and the inflammatory lesions in MS. It has been shown that the MS plaques arise from definite segments of large epiventricular veins and that the lesions digitating out into the cerebral hemispheres also consistently evolve in a corresponding vein relationship .

Magnetic Resonance Imaging (MRI) venography, confirms in vivo the close relationship between the main cerebral veins and the inflammatory plaques. When cortical lesions occur, they arise within the territory of the principal cortical veins. Contrast MRI allowed documentation of the break-down of the blood-brain barrier (BBB) . Such an injury preceded other MRI abnormalities and the clinical evidence of a new lesion. This supports the view that a defect in the BBB, and therefore inflammation, is an early and possible crucial event in the pathogenesis of a new MS lesion .

On these bases it is currently accepted that MS development is associated to inflammatory processes. In fact, plaque formation involves the expression of adhesion molecules in the vessel wall and in circulating cells and the migration of macrophages and T-cells across the BBB . Additionally, MS plaques are characterised by the presence of an excessive accumulation of iron. It is understood that the circulating and resident macrophages become capable of storing considerable amounts of iron intracellularly, due to the phagocytosis of the circulating senescent erythrocytes. Exceptionally, the present inventors had observed the presence of haemosiderin also in the urine of MS patients carrying an active inflammation.

The effect of manipulation of iron level had initially been investigated in Experimental Autoimmune Encephalomyelitis (EAE) , the animal model of MS. The incidence of EAE was 60-70% in mice with a normal iron level and in iron-overloaded mice, but 0 ~6 in iron deficient mice, suggesting that iron deficiency provides protection from the development of EAE. In particular, the serum concentration of soluble transferrin receptor (sTFR) was found to be significantly higher in MS patients carrying the active MS form, either in progressive, relapsing or remitting clinical form, compared to controls. Serum ferritin levels were also significantly elevated in patients affected by the active and progressive forms.

Both findings suggest that local iron overload may be among, if not the, initial signal leading to inflammation in MS. As of today, however, the evidences in support of a direct cause-effect link between iron accumulation and MS are still very limited and poorly investigated. Moreover, the true primum movens of MS has remained elusive, which, if the above hypothesis is true, makes indispensable the investigation on the exact biological mechanisms leading to iron accumulation into MS plaques and its consequences.

Based on the above evidences, the present inventors thought that in MS patients it is the altered venous flow which is fundamental to the insurgence of the inflammatory processes through favouring the accumulation of iron.

It is known that the venous drainage depends primarily on a major haemodynamic parameter called transmural pressure (TMP) , which is the differential value between two opposite pressures, the so-called external pressure (EP) that presses on the external side of the vessel wall and the internal pressure or lateral pressure (IP) that presses the internal side of the vessel wall. When the IP of the capillary is low and/or extra-capillary pressure is high, TMP is low and favourable to tissue drainage, and vice versa.

Proper venous drainage is essential to tissue life, because it contributes to eliminate catabolites which are toxic for the cells, indirectly allows the surge of arterial blood and participates to balance the liquid compartments. An excessive TMP determines venous insufficiency, lack of drainage, an increased accumulation of liquids in the interstitial and cellular compartment, cellular suffering from accumulation of toxic metabolites and ischemia due to a circulatory slowdown. Likely, the instauration of an inflammatory process follows in this district, which may results in oedema, inflammation and tissue degeneration. Nevertheless, it was apparent to the present inventors that the impairment of the venous haemodynamics itself is insufficient to explain the evolution of an inflammatory process into the complex histological and clinical manifestations of MS.

Interestingly, recent studies had demonstrated a pivotal role for tissue iron accumulation in inducing and maintaining inflammation in chronic vascular disease. The present inventors hypothesized that venous reflux and iron accumulation contribute to the instauration of the chronic inflammatory processes characteristic of MS plaques formation. More specifically, the functional impairment of the proteins involved with iron metabolism, transport, accumulation and inactivation might favour the insurgence and maintenance of the inflammatory process seen on MS patients .

Although the number of factors (likely both environmental and genetic) involved in the aetiology of chronic inflammation is extremely high, the search for candidate genes which, based on clinical and physio- pathological findings, may play a primary role in MS insurgence, is a crucial step, since they may represent the target for therapeutic strategies.

In the last years, several gene variants (SNPs, Single Nucleotide Polymorphisms) with a definite role in the etiopathogenesis , diagnosis and prognosis of diseases, as well as in the patient response to drugs, have been identified. SNPs detection has in fact become a novel important tool for improving diagnostic and prognostic methods.

The present inventors investigated the association existing between specific variants of those genes which are known to play a role in iron biology, and the onset and development of MS.

Summary of the invention

Object of the instant disclosure is to single out in advance patients at increased risk to develop MS, or to early progress towards severe clinical phenotypes, based on specific SNPs detection.

More specifically, object of the instant disclosure is the identification of specific variants in genes involved in iron biology which may play a role in the development of and/or protection against multiple sclerosis.

According to the invention, the above object is achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure .

An embodiment of the present disclosure provides for the identification of SNPs in ferroportin, transferrin, hepcidin and/or haemocromatosis genes which may play an active role in the development of multiple sclerosis.

An embodiment of the present disclosure provides for the identification of SNPs in coagulation factor XIII, and/or metalloproteinase-12 genes which may play an active role in the protection against the development of multiple sclerosis (e.g., to contrast MS progression) .

An embodiment of this disclosure provides for a method of diagnosis or prognosis of multiple sclerosis development, comprising the step of measuring in a sample of nucleic acid for the presence of at least one polymorphism in a gene selected from the group of consisting of ferroportin (FPN1) gene, transferrin (TF) gene, hepcidin (HEPC) gene, coagulation factor XIII (FXIII) gene, metalloproteinase-12 (MMP12) gene and haemocromatosis (HFE) gene, wherein the polymorphism is selected from FPN1 -8CG, FPN1 -98GC, TF P570S, HEPC - 582AG, FXIII V34L, MMP12 -82AG, HFE C282Y, HFE H63D.

Such a diagnostic method comprises:

- contacting the nucleic acid sample with at least one oligonucleotide probe being sequence-specific for the detection of at least one polymorphism, under conditions that allow binding of the nucleic acid sample with the oligonucleotide probe,

detecting the binding of the at least one oligonucleotide probe with the nucleic acid sample,

whereby the detection of i) at least one polymorphism in a gene selected from ferroprotin-1 , transferrine, hepcidin and haemocromatosis genes indicates risk of development and/or progression of multiple sclerosis, ii) at least one polymorphism in coagulation factor XIII and/or metalloproteinase-12 genes indicates a protective effect against multiple sclerosis development and/or progression.

A still further embodiment of the instant disclosure concerns a kit for determining a subject's risk for multiple sclerosis development and/or progression comprising:

one or more oligonucleotide probes each individually comprising a sequence that hybridizes under stringent conditions to detect a polymorphism in a gene selected from the group consisting of ferroportin (FPN1) gene, transferrin (TF) gene, hepcidin (HEPC) gene, coagulation factor XIII (FXIII) gene, metalloproteinase-12 (MMP12) gene and haemocromatosis (HFE) gene; and

- instructions to use the kit, wherein the polymorphism is selected from FPN1 -8CG, FPN1 -98GC, TF P570S, HEPC -582AG, FXIII V34L, MMP12 -82AG, HFE C282Y, HFE H63D,

whereby the detection of i) at least one polymorphism in a gene selected from ferroprotin-1 , transferrine, hepcidin and haemocromatosis genes indicates risk of development and/or progression of multiple sclerosis, ii) at least one polymorphism in coagulation factor XIII and/or metalloproteinase-12 genes indicates a protective effect against multiple sclerosis development and/or progression.

Brief description of the drawings The invention will now be described, by way of example only, with reference to the enclosed figures of drawing, wherein:

- Figure 1: Iron dependent inflammation. 1) stasis and increased trans-mural pressure (TMP) both facilitate red blood cells (RBC) extra-vasation and migration in the extracellular matrix. This leads to extra-vascular haemolysis and release of free iron (2), immediately inactivated and stored in ferritin- haemosiderin protein system (3) in order to avoid generation of free radicals. Increased iron deposits are potent chemo-attractants inducing adhesion molecules expression on the endothelial cell surface (4), and the consequent chain of capture rolling, adhesion and transmigration of white cells (5) , mainly by T-lymphocyte and monocyte. The latter in the matrix becomes macrophage taking up in turn iron (6) .

Figure 2: Retrospective analysis in 100 SP patients showing how long the time was they stayed in the RR-phenotype before to become progressive. Patients were stratified by HEPC genotype.

Detailed description of exemplary embodiments

In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

The etiopathogenesis of central nervous system (CNS) damage in MS is not completely understood, but likely it is the consequence of inflammation inducing autoimmune mechanisms leading in turn to demyelination, axonal injury, neuronal loss and Wallerian degeneration .

Histology focused on the veins inside the MS lesions shares findings of iron dependent inflammation consistently found in chronic venous stasis, and MRI confirms increased iron stores in MS plaques.

Whatever mechanism causing intra-plaque iron deposition, it was hypothesized that it could contribute to neurodegeneration by enhancing oxidative stress and related processes.

Iron is essential for many biological pathways; however, excess in levels can be harmful to many tissues and its role has been proven or strongly suspected in several chronic inflammatory diseases with heavy aspects of tissue injury like in MS. Several genes control iron homeostasis that must therefore be carefully regulated in all cells including those in the brain .

Thus, the present inventors reasoned that increased iron stores and interstitial protein extravasation might have been additional factors playing in favour of plaques formation, initially by acting as potent chemoattractans , subsequently representing the underlying chronic inflammatory signal, responsible for white blood-cells recruitment and migration into the matrix during MS plaques deposition .

Figure 1 shows schematically the biological mechanism underlying iron dependent inflammation. Initially, blood stasis and increased trans-mural pressure (TMP) facilitate red blood cells (RBC) extra ^¬ vasation and migration in the extracellular matrix (1) . This leads to extra-vascular haemolysis and release of free iron (2), immediately inactivated and stored in ferritin-haemosiderin protein system (3) in order to avoid generation of free radicals. Increased iron deposits play the role of potent chemo-attractants inducing adhesion molecules expression on the endothelial cell surface (4), and the consequent chain of capture rolling, adhesion and transmigration of white cells (5) , mainly by T-lymphocyte and monocyte, which become macrophage taking up in turn iron in the matrix ( 6) .

In the present disclosure it has been investigated if specific SNPs in key genes involved in iron balancing were associated to the risk of MS, its clinical phenotypes/progressions, or related clinical parameters .

SNPs will be detailed according to their accession number and reference sequence (see table 1) . When the nucleotide substitution in the gene sequence affected an aminoacid-codon responsible for aminoacid change, this latter will be preferred to indicate the SNP by using the international one-letter aminoacid-code (e.g. HFE C282Y; HFE H63D; TF P570S; FXIII V34L) . When no aminoacid change there exists, because of untranslated regions are affected, the SNP will be indicated according to the changed nucleotide position in the gene sequence (e.g. FPN1 -8CG; FPN1 FPN1 -98GC; HEPC - 582AG; MMP12 -82AG) .

Association of SNPs with clinical MS characteristics such as MS-risk, MS-onset and MS- progression will be performed.

MS-risk is defined as the probability to develop MS in subjects carrying a particular and definite SNP genotype (i.e. homozygous polymorphic condition), or combinations, compared with subjects carrying the remaining genotype conditions (i.e. wildtype homozygotes plus heterozygous carriers) . This finding is definite by the Odds-Ratio (OR) computing.

MS-onset is the age of the clinical diagnosis of

MS in a patient. The comparison of the mean age in a group of patients carrying a particular and definite SNP genotype (i.e. homozygous polymorphic condition), or combinations, with patients carrying the remaining genotype conditions (i.e. wildtype homozygotes plus heterozygous carriers) , is performed by means the t- test .

Patients suffering from multiple sclerosis are classified according to the Expanded Disability Status Score (EDSS) and the Progression Index (PI) of the d.i sease .

EDSS (developed by Kurtzke in 1983) is a rating system that is used for classifying and s andardizing the condition of people with MS. The score is based upon neurological testing and. examination of functional systems (FS) , which are areas of the central nervous system which control bodily functions . The functional systems are:

Pyramidal (ability to walk) ,

- Cerebellar (coordination) ,

- Brain stem (speech and swallowing) ,

Sensory (touch and pain) ,

- Bowel and bladder functions,

- Vis a.].,

- Mental,

Other (includes any other neurological findings due to MS) .

hese ran k i ng s are especially important i n the "less severe" lower numbers of the scale, when a patient is still ambulatory, yet experiencing some abnormal signs or disability in other areas .

The Expanded Disability Status Score (EDSS) is as follows :

0.0: Normal neurological exam.

1.0: No disability, but min i mal signs i n one functional system. (FS) are present.

1.5: No disability, but minimal signs in more than one FS are present .

2.0: Minimal disability in one FS is present, 2.5: There is mild disability in one FS or minimal disability in two FS .

3.0: There is moderate d sability in one FS or mild disability i three or four FS . However, the person is stili fully ambulatory.

3.5: The person is fully ambulatory, but has moderate disabil ity and mi .1d d isabi1 ity i one or two FS; or modera e disability in two FS; or mild disability in five FS .

4,0: The person is fully ambulatory without aid, and is up and. about most of the day (12 hours) despite relatively severe disability. Ke or she is able to walk 50 G meters without aid or rest.

4.5: The person is fully ambulatory without aid, and is up and about much of day. Ke or she is able to ork a full da , but ay otherw se have so me limitations of full activity or require minimal assistance . This is considered relatively severe disability. Able to walk 300 meters without aid.

5.0: The person is able to walk 200 meters without aid or rest. Disability impairs full daily activities, such as workrng a full day without special provisions.

5,5: The person is able to walk 100 meters without aid or rest. Disability precl u d e s f u 11 d. ally activities .

6,0: The person needs intermittent or unilateral constant assistance (cane, crutch or bra.ce) to walk 100 meters with or without resting.

6,5: The person needs constant bilateral support (cane, crutch or braces) to walk 20 meters without resting,

7.0: The person is unable to walk beyond five meters even with aid, and is essentially restricted to a wheelchair. However, he or she wheels self and transfers alone, and is active in wheelchair about 12 hours a day.

7,5: The person is unable to take more than a few steps and is restricted to wheelchair, and may need aid to transfer. He or she wheels self, but may require a motorized chair for a full day's activities .

8.0: The person is essentially restricted to bed, a chair or a wheelchair, but may be out of bed much of day. He or she retains self care functions and has generall effective use of arms. 8.5: The person is essentially restricted to bed much of day, but has some effective use of arms and retains some self care functions .

9,0: The person is confined to bed., but still able to communicate and eat.

9.5: The person is totally helpless and bedridden and is unable to communicate effectively or eat and swal low,

10.0: Death due to MS.

The progression index (PI) is defined as the ratio between EDSS and MS duration (expressed in years) .

The clinical phases of MS are classified according to the criteria of Lublin (Lublin FD. Neurol Clin 2005; 23 : 1-15) as relapsing-remitting (RR) , secondary progressive (SP) , and primary progressive (PP) .

One of the main finding of the present disclosure is the association of ferroportin (FPN1) -8GG homozygous genotype with an overall increased MS susceptibility risk of about four-folds. The risk was strongly evident among secondary progressive (SP) cases, quite reaching five-folds increasing. Similarly, stratifying the main MS clinical characteristics by FPN1 polymorphism, Kurtzke's Expanded Disability Disease Score (EDSS) and Progression Index (PI) of disease gradually increased as the number of the polymorphic -8G-allele increased in the genotype of patients. The effect was particularly evident among relapsing-remitting (RR) patients in which PI raised about 3.0-folds. It seems that MS patients carrying the -8G-allele might be at increased risk for rapid disease progression. FPN1 -98GC, is in complete linkage disequilibrium with the FPN1 -8CG. This means that all data obtained by -8CG analysis can completely be translated to the -98GC, by keeping in mind that the - 8C allele completely links with the -98G allele and the -8G allele completely links with the -98C allele.

This result can really be considered a novel and peculiar finding in the field of MS or other iron related disorders since, to date, FPN1 SNPs were never been associated to any particular disease.

The FPN1 protein is synthesized by hepatocytes, enterocytes, Kupffer cells and placenta. Recently, it has also been isolated in the CNS where it is expressed in a variety of cell types including oligodendrocytes, neurons, astrocytes, and blood brain barrier (BBB) cells. FPN1 is one of the main controllers of cellular and extra-cellular iron traffic. Its expression is regulated at several different levels. FPN1 mRNA contains in the 5'-UTR region the Iron Responsive Element (IRE) sequence which interacts with the Iron Regulatory Proteins (IRP) and it can also be regulated post-translationally by the hepatic hormon hepcidin (HEPC) . The existence of an iron-responsive element (IRE) region in the 5'-UTR of FPN1, results in increased expression of the protein under cellular iron loading leading to iron export. Conversely, under low iron availability IRPs can bind IRE, reducing in turn mRNA translation and FPN1 expression.

Knowledge on the role of mutations in the FPN1 gene is still poor. Generally, FPN1 mutations return a molecule that cannot reach the cell surface or block FPN1 internalization and degradation affecting both hepcidin interaction and iron export. Similarly, the role of SNPs in the coding region or in the promoter of FPN1 gene, are even less investigated and very few data are present in literature. The FPN1 -8CG gene variant was not found to have significant effect on ferritin or transferrin saturation levels on normal subjects or on homozygotes for the haemocromatosis (HFE) C282Y.

Nevertheless, its strong closeness to the crucial IRE region, prompted the present inventors to investigate its role in MS patients.

It has been found a significant association between the -8CG SNP and MS susceptibility and with some clinical MS characteristics that can be interpreted and summarized as follows:

i) a direct role of the FNP1 polymorphism in the CNS cells or in the macrophage membrane. Thus, the FNP1 polymorphism could hamper the efflux of iron from inside the cells strongly contributing to increase oxidative stress and cell death; and

ii) an indirect role of still unknown molecular defects in linkage with the FNP1 -8CG SNP.

Furthermore, as far as the investigated SNPs in the haemocromatosis (HFE) gene (i.e. H63D and C282Y) is concerned, they did not reveal strong associations with MS susceptibility risk or with the considered clinical parameters in our population. The only exception was the significant association between HFE 63DD homozygotes and higher PI values when compared to the remaining genotypes particularly evident in the RR patients (almost 3-fold increase) .

HFE protein is expressed on the cell surface and interacts with the transferrin receptor to limit transferrin binding necessary for iron uptake. Mutations in the HFE gene result in a molecule that fails to reach the cell membrane (C282Y) or fails to properly interact with the transferrin receptor (H63D) . Both of these variants lead to increased cellular iron load because the affinity between transferrin and transferrin-receptor is strongly hampered. Classically, HFE protein is thought to be mainly synthesized in liver and intestine, but it has been found on CNS endothelium, choroid plexus, ependymal cells, glial cells and associated to neuritic plaques and neurons in Alzheimer's disease. Thus, HFE potentially is involved in iron uptake also in the CNS as well as in other tissue districts.

Hepcidin (HPEC) is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide mainly synthesized by hepatocytes. It is encoded by the hepcidin antimicrobial peptide (HAMP or HEPC) gene and shows structural and functional properties consistent with a role in innate immunity. In mice and humans, hepcidin is a major regulator of iron balance, and controls iron levels and trafficking by binding to ferroportin. Mutations or polymorphisms in HEPC gene might have effects on iron metabolism. It has been recently described in HEPC promoter the presence of a - 582AG polymorphism associated to iron metabolism, possibly varying the transcriptional activation occurring within the promoter.

Among progressive MS patients (SP + PP) the present inventors found more than two-fold increased MS risk (OR=2.51; 1.32-4.8) associated to the HEPC GG- genotype, and the risk was drastically higher among PP cases (OR=4.8; 1.9-12.3). In addition, it was revealed an extraordinary switch-effect (HR=2.25; 1.09-4.64; log-rank P=0.019) that the G-allele has among RR- patients towards the severest secondary progressive phenotype. (see Figure 2) .

Transferrin (TF) forms a stable complex with the HFE protein facilitating iron transfer. It has been described that the effect of HFE gene on iron absorption depends on the relationships between HFE and the transferrin receptor demonstrating that the HFE H63D variant affects transferrin binding, determining a loss of HFE-repressor function for transferrin uptake, thereby increasing iron transport within the cells. A common variant in the TF gene is the P570S, with a prevalence rate of about 15% in the general population.

Considering the TF P570S SNP, the progression index (PI) of disease increased within the 570SS- homozygous carriers of about two-fold respect to the rest of cases

A joint presence of variant alleles in the HFE and TF genes is responsible for a greatest effect on iron accumulation suggesting gene-gene and gene-environment interactions .

Moreover it has been found that patients carrying a combination of at least four polymorphic alleles in at least two different iron-genes with subjects who were wild-type for all the iron-gene variants presented an increased risk of developing MS of about three-fold (OR=2.93; CI95%, 0.87-15.9; P=0.10).

Coagulation factor XIII (FXIII), a circulating transglutaminase, plays its main physiologic role in final steps of blood coagulation. As other tissue transglutaminases, it catalyses the formation of γ- glutamyl-s-lysine peptide bonds between different peptide chains. Its main cross-linking activity is towards fibrin, the natural FXIII substrate. FXIII is also known having a role in wound-healing and angiogenesis , processes of particular importance in tissue repair and regeneration. It is abundantly present in platelets, deeply involved in provisional tissue repairing processes. A V34L polymorphism increases FXIII activity and was firstly associated to protection against myocardial infarction. In addition, FXIII contrasts the activity of matrix metalloproteinases (MMPs) in monolayered fibroblast cells yielding in turn o more robust extracellular matrix favouring in turn cell proliferation. The present study demonstrated that patients homozygous for FXIII-LL34 had an overall risk reduction of developing MS of about 2.3-fold.

Metalloproteinase-12 (MMP12) digests several ECM components and enables macrophages to penetrate injured tissues. MMP12 may block angiogenesis by converting plasminogen to angiostatin a potent angiogenesis inhibitor. The -82AG polymorphism, influence the binding of the transcription factor activator protein-1 (AP-1) influencing MMP12 promoter activity. Association of the polymorphism with coronary artery disease, cancer and endometriosis, have been recently published. Patients carrying the -82GG genotype had a slower progression of the disease if compared to the remaining genotypes (AA+AG) , thus yielding a protective effect of this SNP on MS progression.

The SNPs-evaluation by DNA-array techniques, or by means of any other sequencing tool (e.g. Pyro- Sequencing technique) , it could be added to the clinical assessment of MS. By analyzing simultaneously selected SNPs it could be possible to have precious information in predicting MS progression and/or in treatment response on the basis of pharmacogenetics aims .

Materials and Methods

Patients and controls

400 consecutive and unrelated patients (female/male = 240/160) affected by clinically definite MS according to the revised criteria of McDonald (Polman CH, et al . Ann Neurol 2005; 58, 840-4640) and classified according to the criteria of Lublin (Lublin FD. Neurol Clin 2005; 23: 1-15) as having relapsing- remitting (RR=270), secondary progressive (SP=100), and primary progressive (PP=30) courses, entered the study. They were consecutively selected from the patient population referring at two multiple sclerosis Centers, both in Northern Italy. Disease severity was scored in all patients using Kurtzke' s Expanded Disability Status Scale (EDSS) . The duration of the disease was expressed in years from the time of official neurological 5 diagnosis. Moreover, the progression index (PI) defined as the ratio between EDSS/MS duration was assessed in the entire MS group.

As controls 400 healthy volunteers were used matched for age, gender and geographic origin with the

10 MS patients without any sign or familial history for neurological diseases.

The study was approved by the local Ethical Committee (Comitato Etico della Provincia di Ferrara) and all the recruited subjects signed an informed

15 consent to participate to the study.

Genes and their polymorphisms .

Table 1. Main characteristics of the SNPs investigated

*According to data-bank provided by NCBI (The National Center for

Biotechnology Information) DNA extraction and PCR conditions

DNA was isolated from peripheral frozen whole blood coming from the different Centres participating to the study by using automated DNA extraction and purification robot (BioRobot EZ1 system from QIAGEN; Hilden, Germany) , which offers fully automated extraction and purification of nucleic acids using magnetic bead technology. The PCR-amplification protocol for the simultaneously detection of the gene regions containing the C282Y and H63D substitutions in the HFE gene and the -8CG substitution in the FPN1 gene promoter was as follows: an initial 10 min at 94°C followed by 35 cycles of 95°C for 30 s, 56°C for 20 s, and 72°C for 90 s. All PCR cycles were performed in a Peltier Thermal Cycler apparatus (PTC-200; M. J. Research, Inc., Watertown, MA, USA) and were completed with a final extension step of 5 min at 72°C.

Table 2 shows the sequences of the eight different groups of oligonucleotides needed for PCR amplification and sequencing (i.e. Pyro-Sequencing technique) and restriction details related to the eight different SNPs in study and the specific characteristics.

Table 2.

PCR Restric .

Restric . SEQ ID

Oligo name Oligo sequence size Products

enzyme No. bp (bp)

FNPl -8CG

Fw R/P 5 ' CCAGTTCCTTGCACTCCTG-3 ' BstUI 85+44 1

129

Rv R/P 5 ' CATCCTCTCTGGCGGTTG-3 ' [B] (60°C) (mut) 2 Sq P 5 ' AGAGCCAGCGGGGTC-3 ' 3

FNPl -98GC

Fw R 5 ' CTGCTGAGCCTCCCAAACCGCT-3 ' 4

Rv R 5 ' TCAACGACACTCACCACAGCAT-3 ' 171+157 5

328 Ava II

Fw P 5 ' TGCCAAAGTCGTCGTTGTAGT-3 ' (mut) 6

Rv P 5 ' AGGAGTGCAAGGAACTGGAGATA-3 ' [B] 7

Sq 5' GGCTGTTGTGTTTTTAGAG-3 ' 8

HFE C282Y

Fw R 5 ' GGCAAGGG AAACAGA CC-3 ' 9

Rv R 5 ' CTCAGGCACTCCTCTCAACC-3 ' Rsal 247+140 10

Fw P 5 ' CGAACCTAAAGACGTATTGCC-3 ' 387 (37°C) (wt) 11

Rv P 5 ' CCCAATAGATTTTCTCAGCTCCT-3 ' [B] 12

Sq 5 ' GGAAGAGCAGAGATATACG-3 ' 13 PCR Restric .

Restric . SEQ ID

Oligo name Oligo sequence size Products

enzyme No. bp (bp)

HFE H63D

Fw R 5 ' ACA GG AAGGCC G GC-3 ' 14

Rv R 5' GCCACATCTGGCTTGAAATT-3 ' Bell 137+70 15

207

Fw P 5 ' CCACATCTGGCTTGAAATTCT-3 ' (50°C) (wt) 16

Rv P 5 ' GTTTGAAGCTTTGGGCTACG-3 ' [B] 17

Sq 5 ' GGGCTCCACACGGCG-3 ' 18

TF P570S

Fw R 5 ' GCTGTGCCTTGATGGTACCAGGTAA-3 ' 19

Rv R 5' GGACGCAAGCTTCCTTATCT-3 ' BstEII 89+21 20

110

Fw P 5 ' GAAAAAGAC A GAG GC G GC-3 ' (60°C) (wt) 21

Rv P 5 ' CTGTGACCACAGCGTGATTC-3 ' [B] 22

Sq 5 ' TGATGGTACCAGGAA-3 ' 23

HEPC -582AG

Fw R 5 ' ACCCTCCTGCCTTGGCCTC-3 ' 24

Rv R 5' CCATTGCTTTAAGCTCTCACC-3 ' HpyCH4IV 226+26 25

252

Fw P 5 ' ACATC CAAGGG C GACAC GG-3 ' (37°C) (mut) 26

Rv P 5 ' GAGCAGGGCAAGCA CAGC-3 ' [B] 27

Sq 5 ' C GACAC GGGAAAAC-3 ' 28

FXIII V34L

Fw R 5 ' CATGCC C G GTC C-3 ' 29

Rv R 5 ' TACCTTGCAGGTTGACGCCCCGGGGCACTA-3 ' Ddel 164+28 30

192

Fw P 5 ' AA GCAGCGGAAGA GACC-3 ' (37°C) (mut) 31

Rv P 5 ' GCTCATACCTTGCAGGTTGAC-3 ' [B] 32

Sq 5 ' CACAGTGGAGCTTCAG-3 ' 33

MMP12 -82AG

Fw R 5 ' AG CAAGGGA GATA CAGC -3 ' 34

Rv R 5' GCCTAAGTTCCTGAACTGTTCCT-3 ' PvuII 80+20 35

100

Fw P 5 ' TCCGGGTTCTGTGAATATGAAT-3 ' [B] (37°C) (mut) 36

Rv P 5' CTTTTGTTTGCATGTTTTTGAGA-3 ' 37

Sq 5 ' CAAGGGA GATA CAAC -3 ' 38

Fw and Rv, indicate respectively the forward and reverse sequence primer; P and R, indicate Pyrosequencing an Restriction detection technique utilized respectively. [B] , indicate the byotinilated primer. Sq, indicates the oligonucleotide utilized for sequence by Pyrosequencing technique. Oligonucleotide sequence, amplicon size, restriction enzyme, temperature and products (expressed in base pairs) are showed. Mut and Wt indicate respectively the polymorphic (rare) and ancestral (more common) allele . The multiplex PCR-reaction was performed in 50 μΐ final volume containing 50 ng of genomic DNA, 20 μΜ dNTPs, 1.5 mM MgC12 , 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 15 pmol of each primer, and 1.25 U of recombinant DNA polymerase (AmpliTaq Gold, Applied Biosystems, Branchburg, NJ, USA) .

Genotyping on Nanogen System

Amplicons were genotyped by Nanogen microchip system (Nanochip™ Molecular Biology Workstation, Nanogen Corporate, San Diego, CA, USA) . A volume of 5 to 10 μΐ containing the HFE C282Y, H63D and FPN1 -8CG fragments was mixed with histidine buffer to 50 mmol/L final concentration in 60 μΐ final volume. About 150- 200 ng of each amplicon were addressed onto the Nanochip® Cartridge (H2-type) by means of Nanochip® Loader using default parameters. A chemical denaturation of amplicons, by means of NaOH 0. IN for 5 min of treatment, ended the addressing protocol. Each reporter mix contained probes, stabilizers and reporter oligonucleotides (detailed published on DNA-array of gene variants in venous leg ulcers: detection of prognostic indicators . Gemmati D. et al . J Vase Surg. 2009 Dec; 50 ( 6) : 1444-51 ) specific for each SNP, and hybridization step and fluorescence scan of the cartridge were carried out by the Nanochip® Reader. An optimized hybridization touch down protocol was determined for the analysis of each polymorphism. Details of the instrumentation and general protocols have been followed by previous reports: i) Ferrari M, et al . Single-nucleotide polymorphism and mutation identification by the Nanogen microelectronic chip technology. Methods Mol Med 2005; 114: 93-106 and ii) Sosnowski R, et al . Active microelectronic array system for DNA hybridization, genotyping and pharmacogenomic applications. Psychiatr Genet 2002; 12: 181-92. About 20% of samples were loaded and analysed in duplicate and heterozygous controls were always included in each assay. Quantitative analysis and genotype designation were realized by using dedicated softwares supplied by the Nanogen® Company and both were set at a diagnostic level.

Genotyping on Pyrosequencing technique

Amplicons were purified by Pyromark procedure according to standard Supplier's protocols (PyroMark protocols by Qiagen) . The automated sequencing procedure on PyroMark instrument was the default method utilized by the software according to the SNP detection step. All the oligonucleotides utilized (SEQ ID No . : 1 to 38) were built by the PyroMark software choosing oligonucleotide triplets having no less than a detection score of 95.

Genotype confirming procedure

Haplotypes were confirmed by re-genotyping about 20% of randomly samples selected among each different genotype group for each specific polymorphism by means of enzymatic restriction of the same amplicon utilized for Nanochip® procedure. Table 2 reports the restriction enzymes used (New England Biolabs Inc., Hitchin, UK) , the digestion fragments obtained and the temperature specific for each different restricted amplicon. All the digestion reactions were carried out according to the Supplier's instructions. There were no discrepancies between genotypes determined in duplicate and/or by different methods.

Statistical analysis

Statistical differences among groups were assessed by the Student's t-test and the Chi-squared test respectively for mean value and genotype distribution comparisons. When appropriate, Yates' correction or Fisher's exact test was applied. Adjusted Odds Ratios (OR) and 95% confidence intervals (95% CI), calculated by logistic regression models, were used to estimate the risk of developing different types of MS associated to the presence of the polymorphic homozygous (if present) or heterozygous (HFE 282CY) condition compared to the rest of genotype counterpart. The model accounted for sex and age distribution among cases and healthy controls. P-values lower than or equal to 0.05 were considered statistically significant. Survival analysis for HEPC -582AG SNP was performed by Kaplan- Meier curve. All analyses were performed by using Systat V.5.0 (Systat Inc., Evanston, IL, USA) and SPSS Statistical Package (SPSS Inc., Chicago, IL, USA).

Results

MS patient characteristics

Table 3 shows clinical and demographic characteristics in the whole group of MS patient and in the subgroups .

Table 3.

As expected, PP and SP subtypes showed significantly higher EDSS values when compared with the RR course. Similarly, disease duration was longer in SP and in PP courses, whereas the other clinical findings, with the exception of the onset age among PP cases, were not significantly different among subgroups.

SNPs genotype analysis and associated MS susceptibility Table 4 shows the prevalence of all the iron-gene polymorphisms investigated in the patient populations and the associated OR-values computed in the total MS patients and clinical subtypes compared to healthy controls .

Globally, the prevalence of FPN1 -8GG homozygotes was 7.0% in MS cases and 1.75% in healthy controls. This yielded an overall OR-value of 4.23 (95%CI, 1.82- 9.8; P<0.0001) when compared with the rest of genotypes (reference group) . In RR cases the assessed risk was the same of the entire MS population, whereas in SP patients the risk was further higher (OR=4.9; 95%CI, 1.73-13.8; P=0.003). In contrast, statistical analysis among the PP cases (n=30; CC=20; CG=9; GG=1) did not reveal significant associations, probably for the shortage of PP cases in our survey.

As far as HFE gene polymorphisms are concerned, H63D yielded appreciable greater than one OR-values in all the subgroups considered though far from statistical significance. Conversely, C282Y yielded non-significant OR values lower than or quite equal to the one-unit value.

Considering the HEPC -582AG variant, a quite two- fold increased MS-risk was observed among SP-subset, and the risk strongly increased among PP patients with values quite close to five-fold. The rate of GG- homozygotes was about 8% among the whole MS population considered, but it showed a clear stepwise trend increasing from RR (5.5%), to SP (11%) to PP (23.3%). MS patients carrying the GG-genotype are at increased risk for rapid disease progression.

Table 4.

FPN1 -8CG HFE H63D HFE C282Y HEPC -582AG TF P570S

Cases CC CG GG HH HD DD CC CY YY AA AG GG PP PS SS

Total 239 133 28 278 109 13 388 12 0 198 169 33 265 121 14

(n=400) (59.8) (33.25) (7.0) (69.5) (27.25) (3.25) (97) (3) (0) (49.5) (42.25) (8.25) (66.25) (30.25) (3.5)

OR 4.23 (1.82-9 .8) ; 1.65 (0.67-4 01) 0.7 (0.33-1. 48) 1.41 (0.82-2.43) 1.28 (0.58-2 86)

(95%CI) P<0.0001 (NS) (NS) P=0.074 (NS)

RR 161 90 19 191 73 6 263 1 0 143 112 15 185 76 9

(n=270) (59.6) (33.4) (7.0) (70.7) (27) (2.3) (97. 4) (2.6) (0) (53) (41.5) (5.5) (68.5) (28.1) (3.3)

OR 4.25 (1.76-10 25) ; 1.11 (0.38-3 25) 0. 5 (0.25-1 .5) 0.92 (0.48-1.80) 1.22 (0.5-2. 98)

(95%CI) P=0.001 (NS) (NS) (NS) (NS)

SP 58 34 8 65 30 5 96 4 0 43 46 11 59 37 4

(n=100) (58) (34) (8) (65) (30) (5) (96) (4) (0) (43) (46) (11) (59) (37) (4)

OR 4.9 (1.73-13 .8) ; 2.58 (0.83-8 .1) 0.94 (0.31-2 .86) 1.94 (1.12-4.1) 1.47 (0.46-4 73)

(95%CI) P=0.003 P=0.104 (NS) P=0.02 (NS)

PP 20 9 1 22 6 2 29 1 0 12 11 7 21 8 1

(n=30) (66.7) (30) (3.3) (73.3) (20) (6.7) (96. 7) (3.3) (0) (40.0) (36.7) (23.3) (70) (26.7) (3.3)

OR 1.94 (0.23-16.3) 3.5 (0.71-17 .3) 0.78 (0.1-6. 04) 4.8 (1.9-12.3) 1.22 (0.15-9 .8)

(95%CI) (NS) (NS) (NS) P=0.001 (NS)

Controls 268 125 7 294 98 8 383 17 0 229 147 24 270 119 11

(n=400) (67) (31.25) (1.75) (73.5) (24.5) (2) (95. 75) (4.25) (0) (57.25) (36.75) (6) (67.5) (29.75) (2.75)

OR-values have been calculated comparing the wildtypes vs the rest of genotypes.

(NS) means non significant level.

Relationships between genotype and MS clinical characteristics (Single analyses)

Table 5 shows the clinical characteristics (age of onset, disease duration, EDSS, and PI) in the whole group of MS patients stratified by the different polymorphism genotypes.

Table 5.

Considering the EDSS, homozygous carriers of FPN1 -8GG genotype had a slight higher disability score when compared with the -8CC wild-type genotype (3.2312.32 vs 2.5312.36) . Although a trend among the three different genotypes was observed it did not reach significance (P-trend= 0.09). Slightly significant differences were instead observed in the RR subgroup (P-trend= 0.03) . Similarly, considering PI, the homozygous carriers for FPN1 -8GG genotype had a significant higher index when compared with the -8CC wild-type genotype (1.3612.18 vs 0.43+0.54; P=0.03) and a significant trend among the different genotypes was maintained. The significance further increased for the same comparison among the RR subgroup (P-trend=0.015) . A significant trend for PI among the different genotypes was also confirmed among RR subgroups (P=0.01). In relation to disease duration, no significant differences or trends were instead observed in either the whole or the subset groups.

Considering the HFE polymorphisms among the selected clinical parameters, PI was significantly related with the H63D gene variant. Accordingly, by comparing homozygous 63DD genotypes with the remaining cases (63HH and 63HD) , PI significantly increased in the 63DD cases either in the whole or in the RR subgroup (1.5+2.62 vs 0.47+0.96; P=0.0015 and 1.08+1.45 vs 0.38+0.63; P=0.04 respectively).

In relation to the HFE C282Y polymorphism no clinical characteristic significantly related with a particular genotype, very likely because no 282YY homozygotes and very few heterozygous carriers were found, not supporting significant statistics. Interestingly, an unexpected significant mean later onset (about 7-yy) was observed among HFE 282Y-carriers respect to wildtypes (P=0.014), and this behaviour was quite similar among all the iron-gene variants investigated (i.e. homozygotes had a lower mean age onset respect to the rest of genotypes) .

Considering HEPC variants, it was found a significant higher EDSS among -582GG homozygous patients of about 1.4-fold respect to the rest of cases (P=0.025) .

Similarly, considering the TF P570S variant, PI increased within the 570SS-homozygous carriers of about 2.1-folds respect to the rest of cases (P=0.08).

Relationships between genotype and MS clinical characteristics (Combined analyses)

a) Case-Control analysis

In an attempt to calculate MS risks associated to the coexistence of polymorphic genotypes in the iron- genes, the present inventors compared the whole groups of cases and controls carrying a combination of at least four polymorphic alleles in at least two different genes with subjects who were wild-type for all the gene variants. Combined homozygotes at least in two iron-genes, single homozygotes in one iron-gene and combined carrier in at least two, or carrying at least a quadruple heterozygous condition, were 11.37 ~6 in patients and 5.0% in controls. The wild-type condition in all the iron-gene variants was 11.5% in cases and 17% in controls. Although appreciable risk-values were obtained, no significant association yielded from these kind of comparisons (OR=2.93; CI95%, 0.87-15.9; P=0.10) .

b) Intra-case analysis

In addition, by means of an intra-patients analysis, relationships between EDSS score or PI and genotypes, the present inventors demonstrated that the same combined carrier patients (i.e. those carrying a combination of at least four polymorphic alleles in at least two different genes) had elevated EDSS (3.512.4 vs 2.4+2.6; P=0.09) or PI (1.2+2.55 vs 0.34+0.39; P=0.005) when compared with fully wild-type patients. Thus, in the combined carriers EDSS increased about 1.6-fold and PI 3.0-fold.

This means that additive effects of different genes modulate the risk/progression of such a complex disease .

A second important finding of the present study was the recognition of a progression-switch within the HEPC gene. HEPC -582AG showed an extraordinary output, ascribing to the G-allele the role of earlier progression-switch .

In detail, during a retrospective observational period of ten years, those patients carrying the -582G- allele had a chance to progress in the SP-phenotype of more than two-folds (HR=2.25; 1.09-4.64; log-rank P=0.019) if compared to the -582AA counterpart genotype (Figure 2) . This sub-analysis was performed in a sub- group of 100 RR-patients (see Figure 2) .

In the risk computation obtained in a subgroup of 373 MS patients, FXIII-LL34 homozygotes had an overall significative MS risk of 0.43 (CI95%, 0.19-0.91), yielding a risk reduction of about 2.4-fold (Table 6) .

Table 6

When the computation was carried out in the sub ^¬ group of RR patients, the risk reduction was even higher (OR=0.25; CI95%, 0.07-0.82) yielding a 4.0-fold decreasing. This, according to the higher transglutaminase activity found in FXIII LL34 compared to the counterpart isoform. This may have effects on extracellular matrix (ECM) and improve angiogenesis and cell proliferation. Although no data are present in scientific literature as regard this particular SNP and MS, it was found to interact with other neurodegenerative disorders such as Alzheimer disease.

As regards MMP12 -82AG polymorphism, it was not reached a statistically different genotype distribution comparing cases vs controls (Table 7), but considering EDSS and PI in a subgroup of 384 MS patients, the present inventors found noteworthy results.

Table 7

Although EDSS non-significantly changed among genotypes, PI was significantly higher within -82AA cases. Accordingly, those carrying the -82GG genotype had a slower progression of the disease if compared with the remaining genotypes (AA+AG) (PI: 0.23 ± 0.17 vs 0.50 ± 1.1; P=0.0092) (Table 8 - PI stratification according to MMP12 genotype) ) . This agrees with the less aggressive MMP12 activity present in MMP12 -82GG compared to the counterpart isoform. Such MMP12 characteristic may have less detrimental effects on nervous system and give back more viable cells and tissues improving in turn a more feasible regeneration. Table 8

On the basis of the significant lower PI observed in -82GG carriers, and supported by the smaller lesion size observed in CVD patients carrying the same gene variant, the present inventors ascribe protective effects against MS to MMP12 polymorphism. Similarly, the present inventors ascribe to FXIII V34L SNP protective effects against MS establishment. Detailed data are shown in table 9, wherein clinical characteristics in the whole MS group stratified by different polymorphism genotypes (FXIII V34L and MMP12 -82AG) are reported.

Table 9

A simplified overview of findings related to protective and risk SNPs is summarized in table 10.

Naturally, while the principle of the invention remains the same, the details of construction and the embodiments may widely vary with respect to what has been described and illustrated purely by way of example, without departing from the scope of the present invention.

Table 10