Risk assessment for neonatal chronic lung disease

[001] The neonatal form of chronic lung disease (CLD), known as Bronchopulmonary Dysplasia (BPD), is notable for its significantly increased risk for pulmonary and neurologic impairment in the preterm and term infant persisting into adulthood. Defined by the need for oxygen supplementation or ventilator support at 28 day of life (mild BPD) or at 36 weeks postmenstrual age (moderate and severe BPD), the incidence reaches up to 77% in infants born at less than 32 weeks of gestation with a birth weight below 1 kg. Even significant improvement in perinatal care applied to all preterm infants irrespective of BPD diagnosis including administration of antenatal corticosteroids, surfactant treatment and the establishment of advanced invasive and non-invasive ventilation strategies did not alter the overall incidence of long term sequelae associated with the disease in the most immature infants.

[002] Current concepts of disease development of BPD comprise inflammation, extracellular matrix remodelling and dysregulated growth factor signalling critical for alveolo- and vasculogenesis. Clinically, BPD presents with hypoxemia leading to the need for supplemental oxygen as well as hypercapnia, reflecting impaired respiratory gas exchange and alveolar hypoventilation resulting in a mismatch of ventilation and perfusion. Long-term, increased airway hyperreactivity and decreased lung function, as well as compromised pulmonary immune response, result in a greater risk for hospital readmission due to respiratory tract infections in the first years of life.

[003] Large clinical trials have identified important risk factors for disease development, with the requirement for prolonged assisted ventilation and the need for oxygen supplementation being critical drivers of pulmonary injury in the structurally and functionally immature organ.

[004] Despite the clinical need, the diagnostic process for BPD still solely relies on clinical characteristics present at three months after birth, thereby hindering timely decision-making and initiation of individualized treatment strategies early after birth that may mitigate lung remodelling and complications later in life.

[005] With the rising number of extremely premature infants, BPD accounts for an increasing amount of pulmonary morbidity in early infancy and produces severe long-term consequences persisting into adulthood, including impaired pulmonary and neurocognitive development. Despite significant improvement in perinatal care, BPD is still diagnosed according to clinical observations near term. Thus, reliable and specific blood and imaging l markers that would allow for early detection and monitoring of BPD, as well as direct characterization of the diseased lung that could form the basis for developing personalized treatment strategies, are lacking. In order to enable early and personalized treatment strategies in preterm infants with BPD, we used advanced molecular and imaging techniques in two independent cohorts to i) identify early markers indicating disease development by proteome screening and to ii) quantify structural and functional changes in the diseased lung at the time of BPD diagnosis, i.e. 36 weeks gestational age (GA) by use of magnetic resonance imaging (M I) and infant lung function testing (ILFT).

[006] The technical problem underlying the present application is to comply with the need set out in the prior art.

[007] The solution is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.

[008] Accordingly, the present invention provides a method for assessing the risk whether a subject develops neonatal chronic lung disease comprising detecting SIGLEC-14 in a sample from said subject, wherein an increased level of SIGLEC-14 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[009] Furthermore, the present invention provides a method for assessing the risk whether a subject develops neonatal chronic lung disease comprising detecting BCAM in a sample from said subject, wherein an increased level of BCAM as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[010] Accordingly, the present invention provides a method for assessing the risk whether a subject develops neonatal chronic lung disease comprising detecting ANGPTL3in a sample from said subject, wherein a reduced level of ANGPTL3 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[011] The present inventors surprisingly found that plasma levels of SIGLEC-14, BCAM, and/or ANGPTL3, particularly in the first week of postnatal life, are highly sensitive for neonatal chronic lung disease and their elevation (SIGLEC-14, BCAM) or reduction (ANGPTL3) precedes clinical diagnosis. Structural changes in lungs of infants with neonatal chronic lung disease were detectable by MRI at the time of current clinical diagnosis at corrected 36 weeks postmenstrual age with characteristic increased T2- and decreased T1- relaxation times, mirrored by functional changes in ILFT. Accordingly, it is now possible to detect and quantify lung disease and dysfunction in infants with neonatal chronic lung disease by the use of one or more of the validated plasma markers of the invention that appear early in disease, optionally combined with imaging equivalents for interstitial remodeling and emphysematous changes, thereby providing a critical advance towards timely and accurate neonatal chronic lung disease diagnosis enabling early treatment initiation and monitoring. [012] Specifically, in a prospective study, the present inventors i) empirically identified plasma markers in the first week of life in preterm infants who went on to develop BPD and ii) measured structural and functional lung defects in BPD at the time of diagnosis by comprehensive MRI analysis complemented by pulmonary function analysis.

[013] In order to ensure unbiased, comprehensive screening to identify novel and early markers in plasma samples of preterm infants at risk for BPD development, a unique, non- hypothesis driven screening approach was employed to identify biomarkers/markers with high specificity and sensitivity. By the identification and validation of a signature protein combination comprising the inflammatory protein SIGLEC-14, the laminin receptor BCAM and the pro-angiogenic factor ANGPTL3 predicting BPD development as early as in the first week of postnatal life, this study to the best of the inventors' knowledge for the first time provides a protein panel with high potential for clinical routine diagnostics. The confirmation of the protein expression in human lung tissue obtained from preterm infants with BPD significantly underscores the importance of the findings. The present invention thereby exceeds previous investigations mostly relying on alone standing markers, lacking disease and/or organ specificity generated by hypothesis driven study approaches lacking confirmatory findings in an independent patient cohort (Rivera L et al., Front Pediatr. 2016; 4: 33). The protein pattern in infants with BPD characterized by this study reflects key processes characteristic for disease development, i.e. inflammation, extracellular matrix remodelling and dysregulated growth factor signalling critical for alveolo- and vasculogenesis (Bose et al., Arch Dis Child Fetal Neonatal Ed. 2008; 93: F455-F61 ; Bhandari et al., Semin Fetal Neonatal Med. 2010; 15: 223-9; Bland et al., Am J Physiol Lung Cell Mol Physiol. 2008; 294: L3-L14).

[014] By capturing the individual response of a structurally and biochemically immature lung to different injury mechanisms, the biomarkers/markers of the present invention can enable the identification of a "window of opportunity" to initiate monitoring and treatment measures as well as serve as potential treatment targets.

[015] The present inventors verified the protein findings in the confirmation cohort. Confirmation of the findings despite the differences in some clinical characteristics indicates the robustness of the variables predicting BPD in the first week of life.

[016] By the use of both, advanced imaging and lung function studies at term, the present inventors were furthermore able to define structural and functional changes in the BPD lung at the time of diagnosis in much more detail and with greater specificity than the clinical and imaging criteria routinely applied for BPD diagnosis. Higher T2 relaxation times in the lungs of infants with BPD may indicate an increased amount of fibrotic tissue resulting from pulmonary remodelling processes, potentially associated with pulmonary inflammation and interstitial edema. The ability of the present inventors to herewith identify infants even with mild disease underlines the detection of early processes below the resolution of conventional X-ray techniques. With increasing disease severity these changes were paralleled by shortened T1 relaxation time, potentially reflecting emphysematous changes or relative changes in pulmonary perfusion, i.e. vascular rarefication, blood redistribution or a shift towards a higher volume fraction of pulmonary connective tissue relative to blood volume. The identified image pattern thereby reflects histologic changes known to characterize BPD with alveolar and vascular hypoplasia leading to emphysematous changes accompanied by saccular wall fibrosis and extracellular matrix remodelling alongside with sustained inflammatory changes.

[017] In MRI analysis, the transverse (T2) and longitudinal (T1 ) relaxation times are two of the most relevant tissue parameters for image contrast, and their quantification an important approach to obtain objective MRI parameters. Applying this technique to the preterm infant, the present invention advances the concept of imaging in BPD and significantly extends the findings from previous studies in preterm infants that had to remain inconclusive with respect to the more specific differentiation of fibrosis, edema and emphysema.

[018] Thus, the present invention concerns a method for assessing the risk whether a subject develops neonatal chronic lung disease comprising detecting SIGLEC-14 in a sample from said subject, wherein an increased level of SIGLEC-14 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[019] The present invention also relates to a method for assessing the risk whether a subject develops neonatal chronic lung disease, comprising detecting BCAM in a sample from said subject, wherein an increased level of BCAM as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[020] The present invention also concerns a method for assessing the risk whether a subject develops neonatal chronic lung disease comprising detecting ANGPTL3 in a sample from said subject, wherein a reduced level of ANGPTL3 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[021] Additionally or alternatively, the methods as described herein, especially when an increased level of SIGLEC-14 and/or a reduced level of ANGPTL3 are detected, can further comprise detecting BCAM in a sample from said subject, wherein an increased level of BCAM as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[022] Additionally or alternatively, the methods as described herein, especially when an increased level of SIGLEC-14 and/or an increased level of BCAM are detected, can further comprise detecting ANGPTL3 in a sample from said subject, wherein a reduced level of ANGPTL3 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease. [023] The term "SIGLEC-14" also known as "Sialic acid-binding Ig-like lectin 14" as used herein embraces any SIGLEC-14 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[024] As such SIGLEC-14 can, for example, comprise SIGLEC-14 of Pongo pygmaeus (Bornean orangutan) (UniProt number: Q072R5, version 1 last modified October 31 , 2006) or a fragment or variant thereof; Felis catus (Cat) (Felis silvestris catus) (UniProt number: M3WJB9; version 1 , last modified May 1 , 2013) or a fragment or variant thereof; Gorilla gorilla (western gorilla) (UniProt number: Q072R7; version 1 , last modified October 31 , 2006) or a fragment or variant thereof. The SIGLEC-14 polypeptide can comprise or have a sequence of SEQ ID NO: 1 and can also comprise a fragment or variant thereof. The SIGLEC-14 nucleic acid molecule can also comprise or have a sequence of any of SEQ ID NO: 2 or 3 and can also comprise a fragment or variant thereof. Since also polypeptides that have a sequence identity of at least 70 % or 80 % or more to the SEQ ID NO. 1 are encompassed by the present invention and as outlined herein, also SIGLEC-5 is embraced. For example, SIGLEC-5 has a sequence identity of about 84.5 % over amino acids 1 1-331 of SEQ ID NO. 1 compared to SEQ ID NO. 1 . Furthermore, methods to detect SIGLEC14 often also detect SIGLEC5 as evidenced also by the instant Examples. Accordingly, the present invention can also comprise the detection of SIGLEC-14 and/or SIGLEC-5.

[025] The term "SIGLEC-5" also known as "Sialic acid-binding Ig-like lectin 5" as used herein embraces any SIGLEC-5 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[026] As such SIGLEC-5 can, for example, comprise SIGLEC-5 of Pongo pygmaeus (Bornean orangutan) (UniProt number: Q072R6, version 1 , last modified October 31 , 2006) or a fragment or variant thereof; Gorilla gorilla (western gorilla) (UniProt number: Q072R8; version 1 , last modified October 31 , 2006) or a fragment or variant thereof. The SIGLEC-5 polypeptide can comprise or have a sequence of SEQ ID NO: 10 and can also comprise a fragment or variant thereof. The SIGLEC-5 nucleic acid molecule can also comprise or have a sequence of any of SEQ ID NO: 1 1 and can also comprise a fragment or variant thereof.

[027] "BCAM" is also termed "Basal cell adhesion molecule". The term BCAM as used herein embraces any BCAM nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[028] BCAM can for example comprise BCAM of Mus musculus (Mouse) (Uniprot number: Q9R069; version 1 , last modified May 1 , 2000) or a fragment or variant thereof; Rattus norvegicus (Rat) (Uniprot number: Q9ESS6; version 1 , last modified March 1 , 2001 ) or a fragment or variant thereof; Bos taurus (Bovine) (Uniprot number: Q9MZ08; version 2, last modified December 1 , 2001 ) or a fragment or variant thereof. The BCAM polypeptide can comprise or have a sequence of SEQ ID NO: 4 and can also comprise a fragment or variant thereof. The BCAM nucleic acid molecule can comprise or have a sequence of any of SEQ ID NO: 5 or 6 and can also comprise a fragment or variant thereof.

[029] "ANGPTL3" is also termed "Angiopoietin-related protein 3". The term ANGPTL3 as used herein embraces any ANGPTL3 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[030] ANGPTL3 can for example comprise ANGPTL3 of Rattus norvegicus (Rat) (Uniprot number: F7FHP0; version 1 , last modified July 22, 2015) or fragments or variants thereof; Mus musculus (Mouse) (Uniprot number: Q9R182; version 1 , last modified May 1 , 2000) or fragments or variants thereof. The ANGPTL3 polypeptide can also comprise or have a sequence of SEQ ID NO: 7 and can also comprise fragments or variants thereof. The ANGPTL3 nucleic acid molecule can also comprise or have a sequence of any of SEQ ID NO: 8 or 9 and can also comprise fragments or variants thereof.

[031] The term "nucleic acid molecule" when used herein encompasses any nucleic acid molecule having a nucleotide sequence of bases comprising purine- and pyrimidine bases, which are comprised by said nucleic acid molecule, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences can include DNA, cDNA, genomic DNA, RNA, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. A polynucleotide can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

[032] A variety of modifications can be made to DNA and RNA; thus, the term "nucleic acid molecules" can embrace chemically, enzymatically, or metabolically modified forms. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Modified nucleic acid molecules can for example be used in methods for detection of nucleic acid molecules described herein.

[033] The term "polypeptide" when used herein means a peptide, a protein, or a polypeptide, which is used interchangeable and which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. Also encompassed by the invention are amino acids other than the 20 gene-encoded amino acids, such as selenocysteine.

[034] The term polypeptide also refers to, and does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination; see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983), pgs. 1 -12; Seifter, Meth. Enzymol. 182 (1990); 626-646, Rattan, Ann. NY Acad. Sci. 663 (1992); 48-62.

[035] A "variant" envisions any variation of a polypeptide as described herein. For example, a variant of a polypeptide can encompass a polypeptide, wherein one or more amino acid residues are substituted. For example, the substitution can be a conservative substitution compared to said polypeptide or to a polypeptide as depicted in any of SEQ ID NO: 1 , 4 or 7 or 10. The variant can however still have the same functional properties as any of the polypeptides described herein or a polypeptide of any of SEQ ID NO: 1 , 4 or 7. Such variants can include insertions, inversions, repeats, and substitutions selected according to general rules known in the art, which have no effect on the activity of the polypeptide compared to e.g. a polypeptide of SEQ ID NO: 1 , 4 or 7 or 10.

[036] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, Science 247: (1990) 1306-1310. For example, one strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

[037] A "variant" of a nucleic acid molecule can encompass any variation of a nucleic acid molecule as described herein. For example, such a variant can encompass a nucleic acid molecule as described herein comprising a mutation. The mutation can be present with regard to any of SEQ ID NO: 2, 3, 5, 6, 8 or 9, or 1 1 or with regard to DNA sequences encoding any one of SEQ ID NO. 1 -1 1. Such mutations can include one or more point mutations, such as 1 , 2, 5, 10, 15, 20, 50 or more point mutations. A variant can also comprise insertions (addition of one or more nucleotides to the DNA/RNA), such as 1 , 2, 3, 5, 6, or more insertions. Both, point mutations and insertions can be selected according to general rules known in the art, which can have no effect on the activity of the nucleic acid molecule compared to e.g. a nucleic acid molecule of SEQ ID NO: 2, 3, 5, 6, 8 or 9. A variant may additionally be a fragment - this means that a variant may comprise mutations and may additionally comprise deletions as described for a fragment herein.

[038] Similarly, a "fragment" as used herein can be any nucleic acid molecule or polypeptide, which is the truncated form of a full length polypeptide or nucleic acid molecule as described herein. For example such a fragment may comprise a deletion of 1 , 2, 3, 4, 5, 10, 20, 30 or more amino acid residues of any of SEQ ID NO: 1 , 4 or 7 or 10 or a deletion of more than 1 , 2, 3, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or more nucleic acid bases compared to a nucleic acid molecule of any of SEQ ID NO: 2, 3, 5, 6, 8 or 9. The fragment can however still have the same functional properties as any of the polypeptides of SEQ ID NO: 1 , 4 or 7 or the nucleic acid molecules of SEQ ID NO: 2, 3, 5, 6, 8 or 9. Such fragments can be selected according to general rules known in the art which have no effect on the activity of the polypeptide as e.g. of a polypeptide of SEQ ID NO: 1 , 3 or 7 or a nucleic acid molecule of SEQ ID NO: 2, 3, 5, 6, 8 or 9, or 1 1.

[039] Given that also variants and fragments of SIGLEC-14, BCAM and/or ANGPTL-3 are encompassed by the present invention, the present invention also encompasses sequences which have a sequence identity of 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 97 %, 99 % or 100 % with any of the polypeptides/nucleic acid molecules of any of SEQ ID NO: 1 -9.

[040] In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid molecules or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 95 %, 96 %, 97 %, 98 % or 99 % identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 80 % to 95 % or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

[041] Also available to those having skill in this art are the BLAST and BLAST 2.4 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word size (W) of 28, an expect threshold of 10, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 6, and an expect threshold of 10. Furthermore, the BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) can be used. [042] For example, BLAST2.4, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments.

[043] As used herein a "control" refers to any control suitable for the methods/uses and kits of the present invention. For example, a control can be a level of expression of a biomarker/marker as described herein determined in a control sample. Alternatively or additionally, the control may also be a control value, which has been determined by means and methods known by the skilled artesian.

[044] For example, a control level expression of a biomarker/marker can be the level of expression of the marker in a healthy subject e.g. a human patient not afflicted with neonatal chronic lung disease. Thus, the control sample can e.g. be obtained from a healthy subject e.g. a subject, such as a human subject, not afflicted with neonatal chronic lung disease. Then the level of a marker as described herein is measured in this control sample to provide a control value for comparison. The subject from which the control sample can be obtained can, for example, have the same age and/or weight etc. as the subject from which the sample is obtained or which is to be tested. For example, the control or control sample can be of the same type as the sample obtained from the subject.

[045] The control for the purposes of the present invention can also comprise healthy (control) subjects, preferably subjects, who do not have neonatal chronic lung disease, or even standard controls that represent a healthy control group, or general, known in the art standards for neonatal chronic lung disease. Subjects of the control group ideally have no concurrent neonatal chronic lung disease. A control group can be a group of several healthy, for example, 3 or more, preferably 5 or more, more preferably 10, 20, 30 , 40, or 50 persons and health can be examined with known methods, some of which are also mentioned herein.

[046] For the purposes of the present invention, the risk assessment can, for example, be based of ROC curves cut-off fixed values. ROC curves (Receiver-Operating - Characteristics) provide an overview of the diagnostic accuracy of a diagnostic test. Different cut-off values (possible also each measurement point). True positive rate (or sensitivity) and false-positive rate (1- specificity =) are plotted against each other. The determination of cutoff values is governed by the Consensus Paper No. CLSI C28 -A2 the FDA. A so-determined cut-off value is then used as a reference value with which the amount/level of SIGLEC-14, BCAM and/or ANGPTL3 measured in a sample obtained from a subject (test subject) are compared.

[047] The skilled artisan can also create cut-off values e.g. according to Singh (G Singh. Determination of Cutoff Score for a Diagnostic Test. The Internet Journal of Laboratory Medicine. 2006 Volume 2 Number 1 ). Targeted is a specificity, for example, 70 %, 80 %, 90 %, 95% or 99 %. Here the group of non - patients (or the group that corresponds to the test results negative such as e.g. healthy patients) is calculated the corresponding percentile using the values (95 % percentile 95th value at 100, according to size ordered values For N < > 100 is interpolated accordingly). In MS- Excel the function "= PERCENTILE" (range of cells; 0.95) can be used - If possible, the confidence interval should be specified. This allows e.g. the software Medcalc. In the following article the calculation of the confidence interval is described using the binomial distribution (Campbell and Gardner (1988) Calculating Confidence Intervals for some non- parametric Analyses British Medical Journal, 296, 1454- 1456).

[048] As used herein the term "neonatal chronic lung disease" refers to any neonatal chronic lung disease. Neonatal chronic lung disease can have a multifactorial etiology. Risk factors may include birth at less than 30 weeks gestation, birthweight less than 1 ,000 (less than 2 pounds) to 1 ,500 grams (3 pounds 5 ounces), prematurity (the lungs, especially the air sacs, are not fully developed), infant respiratory distress, lung disease of prematurity due to lack or low amount of surfactant (a substance in the lungs that helps keep the tiny air sacs open), oxygen use/oxygen therapy (high concentrations of oxygen can damage the cells of the lungs), mechanical ventilation (e.g. the pressure of air from breathing machines, suctioning of the airways, and use of an endotracheal tube (ET tube is a tube placed in the trachea and connected to a breathing machine)), pulmonary interstitial emphysema (PIE; a problem in which air leaks out of the airways into the spaces between the small air sacs of the lungs), patent ductus arteriosus (PDA; persistence of the fetal connection (ductus arteriosus) between the aorta and pulmonary artery after birth, can result in a left-to-right shunt)), maternal womb infection (chorioamnionitis), a family history of asthma, breathing problems at birth, and/or development of an infection during or shortly after birth, and genetic predisposition.

[049] Chorioamnionitis can e.g. be defined as the presence of inflammatory alterations of the chorionic plate at histologic examination or signs of infection in both mother and infant (Franz et al., Acta Paediatr. 2001 ; 90(9): 1025-32)

[050] Postnatally, diagnosis and severity of RDS (respiratory distress syndrome) can e.g. be scored on anterior-posterior (a. -p.) chest radiographs according to Couchard et al (Couchard et al., Ann Radiol (Paris). 1974; 17(7): 669-83).

[051] Systemic infections can, for example, be diagnosed according to Sherman et al. (Sherman et al., Pediatrics. 1980; 65(2): 258-63) with one or more clinical and laboratory signs of infection (C-reactive protein > 2mg/dl).

[052] Chronic lung disease can e.g. develop in premature babies, who have had mechanical ventilation (breathing machine). For example, neonatal chronic lung disease can result from lung injury to newborns, who must use a mechanical ventilator and extra oxygen for breathing. The lungs of newborn (and especially premature) babies are fragile and are easily damaged. With injury, the tissues inside the lungs become inflamed and can break down causing scarring. This scarring can result in difficulty breathing and increased oxygen needs.lt is also possible e.g. that exposure of immature lungs to high 0 ₂ concentrations and positive pressure ventilation can result in oxidative stress and ventilator induced lung injury (barotrauma/volutruma). The resulting injury and inflammation can lead to abnormal reparative processes in the lung. This process can be compounded by inflammation resulting from infections (intra-uterine/postnatal infection) that can occur in these infants. PDA can contribute further to this process by inducing pulmonary edema and vascular endothelial injury.

[053] For example, neonatal chronic lung disease can be characterized by prolonged need for ventilatory support, 0 ₂ requirements, need for home oxygen and readmission with respiratory illness in the first year of life. It is also contemplated by the present invention that neonatal chronic lung disease is defined by respiratory support (supplemental oxygen or CPAP in air) beyond 36 weeks postmenstrual age as the diagnostic criterion especially in preterm very low birth weight (VLBW) infants. Thus, neonatal chronic lung disease in an infant can mean that damaged tissue in the newborn's lungs is causing breathing and health problems. The lungs can e.g. trap air or collapse, fill with fluid, and produce extra mucus. Neonatal chronic lung disease can also describe long-term respiratory problems in preterm infants.

[054] Symptoms of neonatal chronic lung disease may include respiratory distress (rapid breathing, flaring of the nostrils, grunting, chest retractions) and/or continued need for mechanical ventilation or oxygen after a preterm infant reaches 36 weeks gestation.

[055] Neonatal chronic lung disease may be diagnosed by several factors. It is can be e.g. diagnosed when a preterm infant with respiratory problems continues to need additional oxygen after reaching 28 days of age. Chest X-rays compared with previous X-rays may show changes in the appearance of the lungs. The X-ray of lungs with CLD can have a bubbly, sponge-like appearance. X-rays are diagnostic tests that use invisible electromagnetic energy beams to produce images of internal tissues, bones, and organs onto film. Blood gas analysis (test used to determine if enough oxygen is in the blood) and echocardiography (test that use sound waves to create images of the heart to rule out defects) can also be used to confirm causes of CLD.

[056] It is also contemplated by the present invention that the neonatal chronic lung disease is bronchopulmonary dysplasia (BPD). The term "BPD" as used herein means a complication of premature birth. It can, for example, be defined by the need for oxygen supplementation or ventilator support at 28 day of life (mild BPD) or at 36 weeks postmenstrual age (moderate and severe BPD).The incidence can reach up to 77% in infants born at less than 32 weeks of gestation with a birth weight below 1 kg.

[057] As such BPD may be categorized in mild, moderate or severe BPD. Mild BPD can be diagnosed at less than 36 weeks of gestational age with breathing room air at 36 week post menstrual age or discharge, whichever comes first (assessed at 36 weeks PMA). At 36 weeks or more gestational age mild BPD can be diagnosed when breathing room air is achieved by 56 days postnatal age or discharge, whichever comes first (assessed at age 29- 55 days) (see National Institute of Child Health and Human Development Criteria for Diagnosis of Bronchopulmonary Dysplasia as described in Trembath and Laughon (2012) "Predictors of Bronchopulmonary Dysplasia" Clin Perinatol. 2012 Sep; 39(3): 585-601 ). Both of these criteria additionally require baseline requirement of > 21 % 0 ₂ for at least 28 days.

[058] Moderate BPD can be diagnosed by the need for < 30% 0 ₂ at 36 week post menstrual age or discharge, whichever comes first (assessed at 36 weeks PMA). Moderate BPG can also be diagnosed by a need for < 30% 0 ₂ at 56 days postnatal age or discharge, whichever comes first (assessed at age 29-55 days) (see National Institute of Child Health and Human Development Criteria for Diagnosis of Bronchopulmonary Dysplasia as described in Trembath and Laughon (2012) "Predictors of Bronchopulmonary Dysplasia" Clin Perinatol. 2012 Sep; 39(3): 585-601 ). Both of these criteria additionally require baseline requirement of > 21 % 0 ₂ for at least 28 days.

[059] Severe BPD max be diagnosed by need for > 30% 0 ₂ , positive pressure, or both at 35 week post menstrual age or discharge, whichever comes first (assessed at 36 weeks PMA) or by a need for > 30% 0 ₂ , positive pressure, or both at 56 days postnatal age or discharge, whichever comes first (assessed at age 29-55 days) (see National Institute of Child Health and Human Development Criteria for Diagnosis of Bronchopulmonary Dysplasia as described in Trembath and Laughon (2012) "Predictors of Bronchopulmonary Dysplasia" Clin Perinatol. 2012 Sep; 39(3): 585-601 ). Both of these criteria additionally require baseline requirement of > 21 % 0 ₂ for at least 28 days.

[060] BPD may also be defined as described in Jobe, Curr Opin Pediatr. 201 1 ; 23: 167-72, namely mild (oxygen supplementation at 28 days postnatally), moderate (oxygen supplementation below 30% or ventilator support at 36 weeks postmenstrual age), or severe (oxygen supplementation above 30% or ventilator support at 36 weeks postmenstrual age). Days with ventilator support can, for example, be recorded as endotracheal (invasive) mechanical ventilation, nasal intermittent mandatory ventilation or nasal intermittent positive pressure ventilation and/or nasal continuous positive airway pressure in days.

[061] BPD can, for example, be diagnosed when a ventilated infant is unable to wean from 0 ₂ therapy, mechanical ventilation, or both. Infants typically develop worsening hypoxemia, hypercapnia, and increasing 0 ₂ requirements. [062] For diagnosis, the patient has to have required at least 28 days of > 21 % 0 ₂ . For example, chest x-ray can initially show diffuse haziness due to accumulation of exudative fluid; appearance then can become multicystic or spongelike, with alternating areas of emphysema, pulmonary scarring, and atelectasis. Alveolar epithelium may slough, and macrophages, neutrophils, and inflammatory mediators may be found in the tracheal aspirate.

[063] BPD may also be diagnosed using magnetic resonance imaging (MRI) as described herein e.g. in the Examples and/or infant lung function testing (ILFT) as described herein e.g. in the Examples. Both techniques are also known to the skilled artesian and for example described in Walkup et al. (2015) "Quantitative Magnetic Resonance Imaging of Bronchopulmonary Dysplasia in the Neonatal Intensive Care Unit Environment" American Journal of Respiratory and Critical Care Medicine, Volume 192, Issue 10 pp. 1215-1222 and Mohtasham and Panitch (2014) "Current Approaches in Infant Pulmonary Function Testing" Current Pediatrics Reports Volume 2, Issue 1 , pp 9-17.

[064] By the use of both, advanced imaging and lung function studies at term, structural and functional changes in the BPD lung at the time of diagnosis can be analysed. In MRI analysis, the transverse (T2) and longitudinal (T1 ) relaxation times are two of the most relevant tissue parameters for image contrast, and their quantification an important approach to obtain objective MRI parameters.

[065] Higher T2 relaxation times in the lungs of infants with BPD may be obtained when compared to results from infants with no or mild BPD. Additionally or alternatively, diagnosis of moderate or severe BPD can be associated with decreased T1 relaxation times.

[066] Furthermore, with increasing disease severity these changes can be paralleled by shortened T1 relaxation times.

[067] In severe BPD increased T2 relaxation times can be paralleled by shortening of T1 relaxation times, which can indicate emphysematous changes.

[068] Clinically, BPD can present with hypoxemia leading to the need for supplemental oxygen as well as hypercapnia, reflecting impaired respiratory gas exchange and alveolar hypoventilation resulting in a mismatch of ventilation and perfusion. Long-term, increased airway hyperreactivity and decreased lung function, as well as compromised pulmonary immune response, can result in a greater risk for hospital readmission due to respiratory tract infections in the first years of life.

[069] The "risk assessment" in the methods, uses and kits of the present invention can comprise every suitable method for assessing the risk of future development of a neonatal chronic lung disease. Assessing the risk can, for example, include assigning a likelihood of future development of neonatal chronic lung disease to the subject. [070] It is also envisioned by the present invention that such risk assessment can comprise correlating assay result(s) obtained for any of the biomarker(s)/marker(s) as described herein to a likelihood of development of neonatal chronic lung disease. For example, the measured concentration(s) of marker(s) may each be compared to an appropriate threshold value such as a control (and/or a control value).

[071] In principle, the risk assessment can take place at any time point. The present invention also encompasses that the risk assessment takes place within the first 30 days or less of life of the subject. Particularly, risk assessment can take place within 30, 27, 25, 23, 20, 17, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days of life of the subject. For example, the risk assessment takes place within the first two weeks (14 days) of life of said subject. For example, the risk assessment takes place within the first week (7 days) of life of said subject.

[072] The methods of the present invention can be performed on a sample obtained from any subject. For example, a subject at risk of developing neonatal chronic lung disease can be born prematurely (e.g., about 10 weeks before the due date), have breathing problems, low birth weight, prolonged 0 ₂ administration, use of a ventilator, and/or have an infection before, during, or shortly after birth. All of these factors can place a neonate at risk for BPD.

[073] A subject at risk of developing a neonatal chronic lung disease can be genetically predisposed to the disease, e.g., have a family history or have a mutation in a gene that causes the disease, or show early signs or symptoms of the disease.

[074] A subject currently afflicted with neonatal chronic lung disease can have one or more than one symptom of the disease and may have been diagnosed with the disease.

[075] The term "subject" as used herein can thus relate to any subject. For example, a subject can be human or an animal. The subject can be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. Preferably, a mammal is as a human, dog, cat, horse, cow, pig, mouse, rat etc. The mammal can also be a human being. Thus, the subject can be a vertebrate, preferably a human being.

[076] Additionally or alternatively, the subject can also be an infant.

[077] An infant as used herein can also be a neonate. As used herein, the terms "neonate" and "newborn" are used interchangeably and refer to subjects, who have recently been born. In some embodiments, the neonate is a human within the first three months of being born. In some embodiments, the neonate is a human within the first two months of being born. In some embodiments, the neonate is a human within the first month of being born. The neonate can also be born after 39, 40, 41 , 42 or more weeks of gestational age.

[078] The present invention also contemplates that the subject can be a preterm infant. A preterm infant means that the neonate is prematurely born. For example, the premature neonate is a human neonate born between 23 and 37 weeks of gestational age. For example the premature human neonate can be born after 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30,

31 , 32, 33, 34, 35, 36, 37 weeks of gestational age.

[079] The methods of the present invention also contemplate that the infant can be born at less than 32 weeks of gestational age. For example, the infant can be born at 32 or less than

32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20 or less weeks of gestational age.

[080] It is further envisioned that the subject can be a preterm infant, which has a weight of less than 2 kg, less than 1 .9 kg, less than 1 .8 kg, less than 1.7 kg, less than 1 .,6 kg, less than 1 .5 kg, less than 1 .4 kg, less than 1 .3 kg, less than 1.2 kg, less than 1 .1 kg, less than 1 kg, less than 900 g, less than 800 g, less than 700 g, less than 600 g or less than 500 g. For example, the preterm infant can have a weight between 450-750 g, or between 750-1250 g, or between 1250-1500 g. The preterm infant can also have a weight of less than 1 kg.

[081] As used herein, the term "gestational age" refers to age of an embryo, fetus, or neonate as calculated from the first day of the mother's last menstrual period. In humans, the gestational age may count the period of time from about two weeks before fertilization takes place. In other words, the gestational age (completed weeks) can be the time that has elapsed between the first day of the last menstrual period and the day of delivery. If pregnancy was achieved using assisted reproductive technology, gestational age can be calculated by adding two weeks (14 days) to the conceptional age.

[082] The "postmenstrual age" (weeks) as used herein means the gestational age plus the chronological age, wherein the chronological age (days, weeks, months, or years) is the time elapsed from birth.

[083] As used herein a "sample" means any probe, which has been obtained from the subject. The sample may also comprise a probe/sample obtained from the mother of the subject. Exemplary samples include body fluid, a biopsy, cell material or tissue material. Body fluid samples can e.g. include blood, airway aspirate, tracheal aspirate and/or urine. Cell material or tissue material may comprise airway scrapping, bronchoalveolar lavage (BAL) and/or lung tissue. The sample can, for example, be a blood sample, such as a plasma sample.

[084] Methods to detect SIGLEC-14, BCAM and/or ANGPTL3 in a sample are known to the skilled artesian. Methods to detect SIGLEC-14, BCAM and/or ANGPTL3 are also apparent for the skilled artesian, especially when considering the sequences as depicted in Table 1. In principle, the detection of SIGLEC-14, BCAM and/or ANGPTL3 can be performed by DNA, RNA or polypeptide analysis.

[085] When analyzing DNA, such as DNA of SIGLEC-14 encoding any of SEQ ID NO: 1 , 2, and/or 3, DNA of BCAM encoding any of SEQ ID NO. 4, 5, and/or 6 and/or DNA of ANGPTL3 encoding any of SEQ ID NO. 7, 8 or 9, it can, for example, be determined if the DNA is transcribed/expressed by analyzing e.g. DNA methylation or histon modifications. Methods to detect DNA and also to detect if DNA is expressed are known to the skilled artesian and for example described in Wagner et al. (2014) "The relationship between DNA methylation, genetic and expression inter-individual variation in untransformed human fibroblasts." Genome Biology 2014, 15:R37 or Karlica et al. (2010) Histone modification levels are predictive for gene expression. PNAS vol. 107 no. 7, 2926-2931.

[086] The detection of DNA (or methylation pattern or histone modifications of the DNA), can be performed by any method. Such methods are known to the skilled artesian and for example described in Ghosh et al. (2006) "Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics." Mol. BioSyst; 2, 551 -560. Exemplary methods for the detection of DNA (or methylation pattern or histone modifications of DNA) include PCR, southern blot, in situ hybridization or transcription-mediated amplification. Methylation pattern and histon modification patterns may also be analyzed with immunohistochemistry or immunocytology as described herein.

[087] Alternatively or additionally, SIGLEC-14, BCAM and/or ANGPTL-3 can also be detected by RNA analysis. Also these are standard techniques known to the skilled artesian. Exemplary methods for the detection of RNA are in situ hybridization, northern blot, RT-PCR or transcription-mediated amplification. The RT-PCR can also be a quantitative RT-PCR.

[088] In general, for the detection of DNA or RNA such as mRNA it may be useful to utilize one, two, three or more oligonucleotides (also called primers), which specifically hybridize to SIGLEC-14, BCAM and/or ANGPTL-3 nucleic acid molecules or fragments or variants thereof as also described herein. Such oligonucleotides can have a length of 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40 or more nucleic acid bases. Knowing the nucleic acid sequence of SIGLEC-14, BCAM and/or ANGPTL-3 (e.g. SEQ ID NO: 2, 3, 5, 6, 8, or 9) various oligonucleotide primers spanning the SIGLEC-14, BCAM and/or ANGPTL-3 RNA may be designed e.g. in order to amplify the genetic material by Polymerase Chain Reaction (PCR).

[089] Conventional methods for designing, synthesizing, producing said oligonucleotides (primers) and performing PCR amplification may be found in standard textbooks, see, for example Agrawal (Ed.), "Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Methods in Molecular Biology, 20)", Humana Press, 1993; Innis et al. (Ed.), "PCR Applications: Protocols for Functional Genomics", Academic Press, 1999; Chen and Janes (Ed.), "PCR Cloning Protocols: From Molecular Cloning to Genetic", 2nd edition, Humana Press, 2002.

[090] Once a nucleic acid molecule has been amplified, nucleotide structure can be analyzed by sequencing methods and compared to e.g. SIGLEC-14, BCAM and/or ANGPTL- 3 nucleic acid molecules such as of SEQ ID NO: 2, 3, 5, 6, 8, or 9 or fragments or variants thereof. Sequencing may be performed manually by any molecular biologist of ordinary skills or by an automated sequencing apparatus. These procedures are common in the art, see, for example, Adams et al. (Ed.), "Automated DNA Sequencing and Analysis", Academic Press, 1994; Alphey, "DNA Sequencing: From Experimental Methods to Bioinformatics", Springer Verlag Publishing, 1997.

[091] Thus, suitable oligonucleotide can hybridize to the nucleic acid sequence as described herein. Suitable oligonucleotides can be at least 65 %, 70 %, 80 %, 90 %, 95 %,

99 % or 100 % complementary to the nucleic acid sequence as described herein.

[092] "Complementary" as used herein refers to nucleic acid sequences also including oligonucleotides that base-pair according to standard Watson-Crick complementary rules, or that are capable of hybridizing to a particular nucleic acid sequence or fragment or variant thereof.

[093] The term "hybridizes" as used herein preferably relates to hybridizations under stringent conditions. The hybridization reaction and washing step(s), if any, may be carried out under any of a variety of experimental conditions. Numerous hybridization and wash protocols have been described and are well-known in the art (see, for example, Sambrook et al. (1989), Innis (Ed.) (1995), and Anderson (Ed.) (1999) as cited in the reference list). The methods of the invention may be carried out by following known hybridization protocols, by using modified or optimized versions of known hybridization protocols or newly developed hybridization protocols as long as these protocols allow specific hybridization to take place.

[094] It is also envisaged that the oligonucleotide (primer) or pair oligonucleotides (primers) is labeled. The label may, for example, be a radioactive label, such as ³² P, ³³ P or ³⁵ S. The label can also be a non-radioactive label, for example, digoxigenin, biotin and fluorescence dye or a dye.

[095] It is further envisioned that SIGLEC-14, BCAM and/or ANGPTL3 can be detected at the level of polypeptide.

[096] Alternatively or additionally to the detection on the nucleic acid level, SIGLEC-14, BCAM and/or ANGPTL3 can also be detected by the detection of the respective polypeptides, but also variants or fragments thereof. The polypeptide to be detected can also be a polypeptide of SEQ ID NO: 1 , 4 or 7 or a polypeptide having a sequence identity of 80 %, 85 %, 90 %, 95 %, 97 %, 99 % to a sequence of any of SEQ ID NO: 1 , 4 or 7.

[097] Exemplary means to detect SIGLEC-14, BCAM and/or ANGPTL3 polypeptide can include any means/technique suitable for the detection of such polypeptides. Such means/techniques are well known to the person skilled in the art and, for example, described by Arasaradnam et al. (2014) "Review article: next generation diagnostic modalities in gastroenterology— gas phase volatile compound biomarker detection." Aliment Pharmacol Ther. 39(8):780-9. [098] In general, methods of detection of polypeptides are known in the art. Exemplary methods to detect the level of polypeptides used in the methods, uses or kits of the present invention include an assay selected from the group consisting of a Western blot, an enzyme- linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), an immunohistochemistry (IHC) assay, a protein array, mass spectrometry (MS), MS/GC, antibody-enriched MS. Further exemplary methods to detect SIGLEC-14, BCAM and/or ANGPTL3 polypeptide are immunohistochemistry, immunocytology, chromatographic methods or western blot. The method to detect the level of polypeptides described herein can be an ELISA. Kits for performing such ELISAs are commercially available. For example, the Siglec 5/Siglec 14 (DY1072 from R&D systems), BCAM (EHBCAM from Thermofischer Scientific), ANGPTL3 (ELH-ANGPTL3 from Raybiotech).

[099] With immunohistochemistry immunohistochemical samples such as sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the tissue can be analyzed. For immunocytology extracellular matrix and other stromal components can be removed, leaving only whole cells to stain. Therefore, immunocytology can include the analysis of cells obtained in a sample. Both, immunohistochemistry and immunocytology can comprise the use of antibodies to detect the SIGLEC-14, BCAM and/or ANGPTL3 polypeptide. Thus, both immunohistochemistry and immunocytology can also comprise fluorescent or non-fluorescent immunohistochemistry and/or immunocytology.

[100] Further exemplary but non-limiting techniques also include chromatographic separation techniques, and/or mass spectrometry.

[101] "Mass spectrometry (MS)" as used herein encompasses all techniques, which allow for the determination of the molecular weight (i.e. the mass) or a mass variable corresponding to a polypetide to be determined/analyzed in accordance with the present invention. Mass spectrometry in general and also of peptides and proteins is a technique well known to the skilled artesian and for example described in Wysocki et al. (2005) "Mass spectrometry of peptides and proteins" Methods. 2005 Mar;35(3):21 1-22. In particular, protein/polypeptide mass spectrometry can comprise two main ways to identify proteins. For example, so called top-down sequencing involves fragmenting intact proteins directly, while the bottom-up sequencing fragments peptides in the gas phase after protein digestion. Ion activation and dissociation can, for example, be performed by gas-phase collision-activated dissociation (CAD), infrared multiphoton dissociation (IRMPD) or electron capture dissociation (ECD).

[102] Mass spectrometry can also be coupled to different chromatographic techniques. Thus, mass spectrometry as used herein can relate to LC-MS and/or GC-MS, i.e. to mass spectrometry being operatively linked to a prior chromatographic separation step. Mass spectrometry as used herein can also encompass quadrupole MS. The method to detect the level of polypeptides described herein can also be protein/polypeptide mass spectrometry. Mass spectrometry may further be coupled to chromatography.

[103] "Chromatographic separation techniques" as described herein can, for example, be selected from the group consisting of liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, size exclusion or affinity chromatography, ion exchange chromatography, expanded bed adsorption (EBA) chromatographic separation, reversed-phase chromatography, two- dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography or countercurrent chromatography. The chromatographic separation technique can furthermore be coupled to mass spectrometry. Also these methods are all known to the person skilled in the art and, for example, described in Gowda and Djukovic (2014) Overview of Mass Spectrometry-Based Metabolomics: Opportunities and Challenges" Methods Mol Biol. 1 198: 3-12.

[104] How to apply the techniques/methods as described herein is also well known to the person skilled in the art. Moreover, suitable devices are commercially available. The techniques described herein can be assisted by automation, for example, sample processing or pre-treatment can be automated by robotics. Data processing and comparison can be assisted by suitable computer programs and databases. Automation as described herein allows using the method/uses of the present invention in high-throughput approaches.

[105] Further exemplary means to detect SIGLEC-14, BCAM and/or ANGPTL3 polypeptide can include utilization of suitable binding molecules directed e.g. against one of these molecules. The binding molecules can be selected from the group consisting of an antibody, or a proteinaceous binding molecule with antibody-like binding properties.

[106] Such an "antibody" can be a full length antibody, a recombinant antibody molecule, or a fully human antibody molecule. Additionally or alternatively, the antibody may be a divalent antibody fragment, a monovalent antibody fragment. A full length antibody can be any naturally occurring antibody. The term "antibody" can also include immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as lgG1 , lgG2 etc.). Such full length antibodies can be isolated from different animals such as e.g. different mammalian species.

[107] A "recombinant antibody molecule" refers to a antibody molecule the genes of which have been cloned, and is produced recombinantly in a host cell or organism, using well- known methodologies of genetic engineering. Typically, a recombinant antibody molecule has been genetically altered to comprise an amino acid sequence, which is not found in nature. Thus, a recombinant antibody molecule can be a chimeric antibody molecule or a humanized antibody molecule. Exemplary antibodies that can be used in the methods of the present invention include an anti-SIGLEC-14 monoclonal antibody (abeam; clone MM0550- 4G4), anti-SIGLEC-14 antibody (1 :50, #MAB10721 ; R&D systems), a polyclonal anti-BCAM antibody (Sigma-Aldrich; product number: HPA005654), anti-human BCAM antibody (1 :200, #sc-99188; Santa Cruz Biotech), a polyclonal anti-ANGPTL3 antibody (Merk Millipore; product number: ABC83), and anti-human ANGPTL3 antibody (1 :50,#600-401-Y15; Rockland antibodies and assays).

[108] The antibody can also be an "antibody fragment". Such antibody fragments comprise any part of an antibody, which comprises a binding site. Illustrative examples of such an antibody fragment are single chain variable fragments (scFv), Fv fragments, single domain antibodies, such as e.g. VHH (camelid) antibodies, di-scFvs, fragment antigen binding regions (Fab), F(ab')2 fragments, Fab' fragments, diabodies or domain antibodies, to name only a few (Holt et al (2003) "Domain antibodies: proteins for therapy." Trends Biotechnol. 2003 Nov; 21 (1 1 ):484-90).

[109] The binding molecule may also only have a single binding site, i.e., may be monovalent. Examples of monovalent binding molecules include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties. Examples of monovalent antibody fragments include, but are not limited to a Fab fragment, a Fv fragment, a single-chain Fv fragment (scFv) or an scFv-Fc fragment. Furthermore, the antibody or antibody fragment may be monoclonal or polyclonal.

[110] The binding molecule that can be used in this invention can be a monoclonal antibody or antibody fragment. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kohler and Milstein Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., US Patent 4,946,778) can be adapted to produce single chain antibodies to SIGLEC-14, BCAM and/or ANGPTL3 polypeptides as described herein.

[111] The binding molecule can also be a proteinaceous binding molecule with antibodylike binding properties. Exemplary but non-limiting proteinaceous binding molecules include an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, an avimer or a (recombinant) receptor protein.

[112] Further illustrative examples of proteinaceous binding molecules with antibody-like binding properties that can be used as inhibitor include, but are not limited to, a EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a G1 a domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin- like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP- type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, "Kappabodies" (III CR, Gonzales JN, Houtz EK, Ludwig JR, Melcher ED, Hale JE, Pourmand R, Keivens VM, Myers L, Beidler K, Stuart P, Cheng S, Radhakrishnan R. Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions. Protein Eng. 1997 Aug; 10(8):949-57) "Minibodies" (Martin F, Toniatti C, Salvati AL, Venturini S, Ciliberto G, Cortese R, Sollazzo M. The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6. EMBO J. 1994 Nov 15;13(22):5303-9), "Janusins" (Traunecker A, Lanzavecchia A, Karjalainen K. Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells. EMBO J. 1991 Dec;10(12):3655-9 and Traunecker A, Lanzavecchia A, Karjalainen K. Janusin: new molecular design for bispecific reagents. Int J Cancer Suppl. 1992;7:51-2), a nanobody, a tetranectin, a microbody, an affilin, an affibody or an ankyrin, a crystallin, a knottin, ubiquitin, a zinc-finger protein, an autofluorescent protein, an ankyrin or ankyrin repeat protein or a leucine-rich repeat protein, an avimer (Silverman J, Liu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer WP. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol. 2005 Dec;23(12): 1556-61 ); as well as multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains as also described in Silverman et al. (2005) cited herein). In some embodiments, the inhibitor used in the present invention is a proteinaceous binding molecule with antibody-like binding properties, which is selected from the group of an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, and an avimer.

[113] Alternatively, a binding molecule used in the present invention can also be an aptamer. Such an aptamer is an oligonucleic acid that binds to a specific target molecule. These aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist. More specifically, aptamers can be classified as: DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides.

[114] A proteinaceous aptamer as described herein may also include an oligonucleotide portion in addition to a protein portion. This is e.g. the case for aptamers that can be obtained from Somalogic (SOMAmers). SOMAmers are short, single-stranded deoxyoligonucleotides selected in vitro from large random libraries for their ability to bind to discrete molecular targets endowed with protein-like properties by adding functional groups that mimic amino acid side chains, thereby expanding their chemical diversity. Such aptamers are engineered with dU residues functionalized at the 5-position with different protein-like moieties (e.g., benzyl, 2-napthyl or 3-indolyl-carboxamide).

[115] It is within skill of the art that the polypeptide is not detected as whole. Rather, certain epitopes specific for SIGLEC-14, BCAM and/or ANGPTL3 are detected by e.g. binding molecules as described herein. These binding molecules specifically bind to one of these biomarkers/markers.

[116] The term "specifically binds" is not intended to indicate that a binding molecule binds exclusively to its intended target since, as noted above, a binding molecule binds to any polypeptide displaying the epitope(s) to which the binding molecule binds. Rather, a binding molecule "specifically binds" if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the binding molecule will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. For example, the binding molecule can bind with affinities of at least about 10 ⁷ M ^~ , and preferably between about 10 ⁸ M ^"1 to about 10 ⁹ M ^"1 , about 10 ⁹ M ^"1 to about 10 ¹⁰ M ^"1 , or about 10 ¹⁰ M ^"1 to about 10 ¹² M ^~1 .

[117] Affinity can e.g. be calculated as K _d =k _off /k _on (k _off is the dissociation rate constant, K _on is the association rate constant and K _d is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c=K(n-r): where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n=number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991 ; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

[118] The term "epitope" refers to an antigenic determinant capable of specific binding to a binding molecule. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. [119] The methods, uses and kits of the present invention can include that SIGLEC-1 , BCAM and/or ANGPTL3 is/are detected in a sample. These markers can all be detected in the same sample or in different samples. Furthermore, these markers can be sequentially or simultaneously detected in the sample(s). Thus, any further marker can be detected in the same sample as SIGLEC-14, BCAM or ANGPTL3 or in a different (second or even third) sample. The sample can be any sample, which is suitable for the methods of the present invention.

[120] As used herein the expression "detecting" means any detection method suitable to analyze biomarkers/markers used in the methods, uses and kits of the present invention. For example, the detection can be performed on the mRNA or polypeptide level. Furthermore, the detection can include determining the level such as the level of expression of one or more markers as described herein. Detecting can also mean measuring a physiologically relevant concentration of a marker as described herein. Detection can e.g. be performed using aptamers, which can be obtained from SomaLogic. SomaLogic e.g. provides protein- capture SOMAMER® (Slow Off-rate Modified Aptamer(s)), which can also be used for detection of a marker as described herein. Detection can also be performed with an antibody e.g. with an antibody as described herein in the Examples. Detection can also include detection of any of SEQ ID NO. 1 -9 or a variant or fragment thereof in a sample.

[121] The "level" may refer to any level suitable for the purposes of the methods, uses and kits of the present invention. For example, the level can mean the polypeptide expression level or the mRNA expression level.

[122] The term "increased level" or "upregulated level" when used herein means any increase in level, which may be suitable for the methods, uses and kits of the present invention. Some of these techniques are also described herein. For example, it can mean that a certain marker (such as SIGLEC-14 and/or BCAM) is expressed at a higher level compared to a control or a control level as described herein.

[123] In principle both, polypeptide expression level or mRNA expression level can be upregulated when compared to a control. For example, polypeptide or mRNA expression level of marker of interest (such as SIGLEC-14 and/or BCAM) in a sample may be upregulated by 3 %, 5 % 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % or more when compared to the polypeptide or mRNA expression level of a control as described herein.

[124] The term "decreased level" or "reduced level" means any decrease in level, which may be suitable for the methods, uses and kits of the present invention. For example, it can mean that a certain marker (such as ANGPTL3) is expressed at a lower level compared to a control or a control level as described herein. [125] In principle both, polypeptide expression level or mRNA expression level can be reduced when compared to a control sample. For example, polypeptide or mRNA expression level of marker of interest (such as ANGPTL3) a sample may be reduced by 3 %, 5 % 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % or more when compared to the polypeptide or mRNA expression level of a control as described herein.

[126] The "control" can, for example, be a sample from a healthy subject, or a subject not afflicted with a neonatal chronic lung disease as described herein.

[127] However, in the present invention such a comparison to a control can be dispensable, because the presence of both, SIGLEC-14 and BCAM and the absence of ANGPTL3 in a sample already provides for a valuable diagnosis and/or prognosis and/or assessing the risk whether a subject develops neonatal chronic lung disease.

[128] The present invention further relates to a method for preventing or treating neonatal chronic lung disease in a subject, the method comprising:

(a) determining whether the level of SIGLEC-14 is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control; and

(b) subjecting the subject to a treatment regime.

[129] The present invention additionally concerns a method for preventing or treating neonatal chronic lung disease in a subject, the method comprising:

(a) determining whether the level of BCAM is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control; and

(b) subjecting the subject to a treatment regime.

[130] The present invention further relates to a method for preventing or treating neonatal chronic lung disease in a subject, the method comprising:

(a) determining whether the level of ANGPTL3 is reduced in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control; and

(b) subjecting the subject to a treatment regime.

[131] As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, namely neonatal chronic lung disease. Also meant by "prophylaxis" is the reduction or inhibition of the recurrence of neonatal chronic lung disease in a subject.

[132] It is further envisioned that the methods and uses of the present invention can further comprise step (a1 ) which is determining whether the level of BCAM is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control (especially in methods where an increased level of SIGLEC-14 and/or a reduced level of ANGPTL3 is detected in step (a)). The methods can further comprise a step (a1 ) which is determining whether the level of SIGLEC-14 is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control (especially in methods where increased levels of BCAM and/or ANGPTL3 are detected in step (a)). The methods can further comprise a step (a1 ) which is determining whether the level of ANGPTL3 is reduced in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control (especially in methods where an increased level of BCAM and/or an increased level of SIGLEC-14 is detected in step (a))

[133] It is further envisioned that the methods of the present invention can comprise a step (a2), which is determining whether the level of ANGPTL3 is reduced in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control (especially in methods where increased levels of BCAM and/or SIGLEC-14 are detected in step (a) and (a1 ), respectively, or especially in methods where increased levels of BCAM and/or SIGLEC-14 are detected in step (a1 ) and (a), respectively).

[134] It is further envisioned that the methods of the present invention can comprise a step (a2), which is determining whether the level of SIGLEC-14 is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control (especially in methods where increased levels of BCAM and/or reduced levels of ANGPTL3 are detected in step (a) and (a1 ), respectively, or especially in methods where increased levels of BCAM and/or reduced levels of ANGPTL3 are detected in step (a1 ) and (a), respectively).

[135] It is further envisioned that the methods of the present invention can comprise a step (a2), which is determining whether the level of BCAM is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control (especially in methods where increased levels of SIGLEC-14 and/or reduced levels of ANGPTL3 are detected in step (a) and (a1 ), respectively, or especially in methods where increased levels of SIGLEC-14 and/or reduced levels of ANGPTL3 are detected in step (a1 ) and (a), respectively).

[136] Thus, also methods for preventing or treating neonatal chronic lung disease in a subject, the method comprising:

(a) determining whether the level of SIGLEC-14 is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control; and (a1 ) determining whether the level of BCAM is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control;

(a2) determining whether the level of ANGPTL3 is reduced in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control; and

(b) subjecting the subject to a treatment regime are envisioned by the present invention.

[137] The present invention also relates to a method for determining the effectiveness of a treatment regime for neonatal chronic lung disease in a subject, the method comprising: (a) identifying a first level of SIGLEC-14 in a first sample from the subject before administration of a treatment regime to the subject;

(b) identifying a second level of SIGLEC-14 in a second sample from the subject after administration of a treatment regime to the subject;

(c) comparing the first and second level of SIGLEC-14; and

(d) adjusting the treatment regime if the second level of SIGLEC-14 is the same or higher than the first level.

[138] The present invention also relates to a method for determining the effectiveness of a treatment regime for neonatal chronic lung disease in a subject, the method comprising:

(a) identifying a first level of BCAM in a first sample from the subject before administration of a treatment regime to the subject;

(b) identifying a second level of BCAM in a second sample from the subject after administration of a treatment regime to the subject;

(c) comparing the first and second level of BCAM; and

(d) adjusting the treatment regime if the second level of BCAM is the same or higher than the first level.

[139] The present invention also relates to a method for determining the effectiveness of a treatment regime for neonatal chronic lung disease in a subject, the method comprising:

(a) identifying a first level of ANGPTL3 in a first sample from the subject before administration of a treatment regime to the subject;

(b) identifying a second level of ANGPTL3 in a second sample from the subject after administration of a treatment regime to the subject;

(c) comparing the first and second level of ANGPTL3; and

(d) adjusting the treatment regime if the second level of ANGPTL3 is the same or lower than the first level.

[140] It is further encompassed by the present invention that the methods can further comprise step (a1 ) identifying a first level of BCAM in a first sample from the subject before administration of a treatment regime to the subject (especially in methods in which a first level of SIGLEC-14 and/or ANGPTL3 has been identified in a first sample from the subject before administration of a treatment regime to the subject in step (a)).

[141] It is further envisioned by the present invention that the methods can further comprise step (b1 ) identifying a second level of BCAM in a second sample from the subject after administration of a treatment regime to the subject (especially in methods in which a second level of SIGLEC-14 and/or ANGPTL3 has been identified in a second sample from the subject after administration of a treatment regime to the subject in step (b)). [142] It is further encompassed by the present invention that the methods can further comprise step (c1 ) comparing the first and second level of BCAM (especially in methods in which the first and second level of SIGLEC-14 and/or ANGPTL3 are compared in step (c)).

[143] It is further envisioned by the present invention that the methods can further comprise step (d1 ) adjusting the treatment regime if the second level of BCAM is the same or higher than the first level (especially in methods in which the treatment regime is adjusted if the second level of ANGPTL3 is the same or lower than the first level or in in methods in which the treatment regime is adjusted if the second level of SIGLEC-14 is the same or higher than the first level).

[144] It is further encompassed by the present invention that the methods can further comprise step (a2) identifying a first level of ANGPTL3 in a first sample from the subject before administration of a treatment regime to the subject (especially in methods in which a first level of SIGLEC-14 and/or BCAM has been identified in a first sample from the subject before administration of a treatment regime to the subject in step (a) and/or (a1 ) or vice versa).

[145] It is further envisioned by the present invention that the methods can further comprise step (b2) identifying a second level of ANGPTL3 in a second sample from the subject after administration of a treatment regime to the subject (especially in methods in which a second level of SIGLEC-14 and/or BCAM has been identified in a second sample from the subject after administration of a treatment regime to the subject in step (b) and/or (b1 ) or vice versa).

[146] It is further envisioned by the present invention that the methods can further comprise step (c2) comparing the first and second level of ANGPTL3 (especially in methods in which the first and second level of SIGLEC-14 and/or BCAM are compared in step (c) and/or (c1 ) or vice versa).

[147] It is further encompassed by the present invention that the methods can further comprise step (d2) adjusting the treatment regime if the second level of ANGPTL3 is the same or lower than the first level (especially in methods in which the treatment regime is adjusted if the second level of SIGLEC-14 and/or BCAM is the same or higher than the first level e.g. as in step (d) and/or (d1 ) or vice versa).

[148] It is further contemplated that the methods, uses (and kits) of the present invention includes obtaining a first, second, third, fourth, fifth, sixth or even more samples from a subject. These samples can, for example, be obtained concurrently or sequentially. Sequential obtaining can include obtaining 1 , 2, 3, 4, 5, 6 or more samples before administration of treatment regimen to the subject and/or 1 , 2, 3, 4, 5, 6 or more samples after administration of treatment regimen to the subject.

[149] As used herein the terms "treatment, treat, or treating" refer to a method of reducing or delaying the effects of neonatal chronic lung disease or symptoms of the disease. Thus, for example, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or symptoms of the disease. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Such a control may be a subject having chronic lung disease, which is not treated. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to control levels (e.g., in the absence of treatment). Treatment can also cause a delay in the onset of new symptoms or further progression of existing symptoms. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease or symptoms of the disease or condition.

[150] As used herein, the terms "prevent, preventing, and prevention" of neonatal chronic lung disease can e.g. refers to an action, for example, administration of a therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease, which inhibits or delays onset or exacerbation of one or more symptoms of the disease. As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.

[151] A "treatment regimen" as used herein means any treatment regimen, which can be beneficial for treating neonatal chronic lung disease. Thus, e.g. a treatment regimen can be a structured treatment plan designed to improve and maintain health. It can also mean a regulated system, as of medication, diet, or exercise, used to promote health or treat illness or injury.

[152] Exemplary treatment regimens can comprise treatment with vitamin A supplementation, diuretics, bronchodilators, corticosteroids or vasodilators.

[153] Exemplary diuretics are furosemide, chlorothiazide or thiazide diuretics plus aldosterone inhibitors (e.g. spironolactone).

[154] Exemplary bronchodilators are albuterol (a specific beta2-agonist), levalbuterol or methylxanthines.

[155] Corticosteroids may be administered systemically or via inhalation of corticosteroids. Exemplary corticosteroids include dexamethasone or glucocorticoid.

[156] Vasodilators can be administered systemically or via inhalation. Exemplary vasodilators include NO. For example, administration of NO can be via inhalation. Exemplary vasodilators can include Sildenafil.

[157] Further exemplary treatment regimens include variation of ventilator settings, extubation followed by non-invasive ventilation, corticosteroid administration, vitamin A or vitamin A analogue administration, caffeine administration, vasodilator administration, surfactant administration, application of adjusted surveillance, such as oxygen saturation levels, adjustment of oxygen administration levels.

[158] The present invention further relates to a kit for performing the methods and/or uses of the present invention, comprising binding molecules for SIGLEC-14, BCAM and/or ANGPTL3 and optionally means for detection.

[159] Such kits may additionally or alternatively comprise one or more extraction buffer/reagents and protocol; reverse transcription buffer/reagents and protocol; and qPCR buffer/reagents and protocol suitable for performing any of the methods of the present invention. In one embodiment, the kit of the present invention can be a kit-of-parts.

[160] The kit or kit-of-parts of the present invention can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 20, 30, 40, 50 or more primers. Also envisaged are kits or kit-of-parts comprising oligonucleotides (primers) specifically hybridizing to nucleic acid molecule(s) of any of SEQ ID NO: 2, 3, 5, 6, 8 or 9. The kit or kit-of-parts can further comprise nucleoside triphosphates.

[161] The buffer, which can be comprised in the kit or kit-of-parts can e.g. comprise a buffer, in which the reverse transcription can take place. In another embodiment, the buffer comprised in the kit or kit-of-part can e.g. comprise a buffer, suitable for the formation of primer/mRNA complexes. In further embodiments, the buffer comprised in the kit or kit-of- part comprises a buffer, suitable for the storage of the primers, control sequences and/or control samples.

[162] The kit or kit-of-parts of the present invention can further optionally comprise reagents for quantifications. The reagent for quantification can e.g. include dyes that bind to double stranded DNA. In addition, the kit or kit-of-parts of the present invention further optionally comprise one or more control values or control sequences. Also, the kit or kit-of-parts can further optionally comprise one or more templates, such as the test sample or control sample as described herein.

[163] The kit or kit-of-parts as described herein may additionally or alternatively comprise a binding molecule as described herein. The binding molecule may thus be an antibody.

[164] The present invention also relates to a use of a binding molecule for SIGLEC-14, BCAM and/or ANGPTL3 for identifying a subject being at risk for developing neonatal chronic lung disease.

[165] The present invention also relates to a use of a binding molecule for SIGLEC-14, BCAM and/or ANGPTL3 for preventing or treating neonatal chronic lung disease in a subject.

[166] The present invention also relates to a use of a binding molecule for SIGLEC-14, BCAM and/or ANGPTL3 determining the effectiveness of a treatment regime for neonatal chronic lung disease in a subject.

[167] Thus, a binding molecule for SIGLEC-14, BCAM and/or ANGPTL3 can be used in any of the methods and uses as described herein. [168] The present invention is further characterized by the following items:

[169] Item 1. A method for assessing the risk whether a subject develops neonatal chronic lung disease, comprising detecting SIGLEC-14 in a sample from said subject, wherein an increased level of SIGLEC-14 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[170] Item 2. The method of iteml , wherein said method further comprises detecting BCAM in a sample from said subject, wherein an increased level of BCAM as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[171] Item 3. The method of item 1 or 2, wherein said method further comprises detecting ANGPTL3 in a sample from said subject, wherein a reduced level of ANGPTL3 as compared to a control indicates the subject is at risk for developing neonatal chronic lung disease.

[172] Item 4. The method of any one of the preceding items, wherein neonatal chronic lung disease is bronchopulmonary dysplasia (BPD).

[173] Item 5. The method of any one of the preceding items, wherein the risk assessment takes place within the first two weeks of life of said subject.

[174] Item 6. The method of any one of the preceding items, wherein said subject is an infant.

[175] Item 7. The method of any one of the preceding items, wherein said subject is a preterm infant.

[176] Item 8. The method of item 6 or 7, wherein said infant is born at less than 32 weeks of gestational age.

[177] Item 9. The method of any one of the preceding items, wherein said sample is body fluid, a biopsy, cell material or tissue material.

[178] Item 10. The method of any one of the preceding items, wherein the sample is selected from the group consisting of blood, airway aspirate, tracheal aspirate, airway scrapping, bronchoalveolar lavage (BAL), lung tissue, and urine.

[179] Item 1 1. The method of any one of the preceding items, wherein SIGLEC-14, BCAM and/or ANGPTL3 is detected at the level of polypeptide.

[180] Item 12. The method of item 1 1 , wherein the level of polypeptide is determined using an assay selected from the group consisting of a Western blot, an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay ( IA), an immunohistochemistry (IHC) assay, a protein array, mass spectrometry (MS), MS/GC, antibody-enriched MS.

[181] Item 13. A method for preventing or treating neonatal chronic lung disease in a subject, the method comprising:

(a) determining whether the level of SIGLEC-14 is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control; and (b) subjecting the subject to a treatment regime.

[182] Item 14. The method of item 13, further comprising step (a1 ) determining whether the level of BCAM is increased in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control.

[183] Item 15. The method of item 13 or 14, further comprising step (a2) determining whether the level of ANGPTL3 is reduced in a sample from the subject suspected to be at a risk of neonatal chronic lung disease as compared to a control.

[184] Item 16. A method for determining the effectiveness of a treatment regime for neonatal chronic lung disease in a subject, the method comprising:

(a) identifying a first level of SIGLEC-14 in a first sample from the subject before administration of a treatment regime to the subject;

(b) identifying a second level of SIGLEC-14 in a second sample from the subject after administration of a treatment regime to the subject;

(c) comparing the first and second level of SIGLEC-14; and

(d) adjusting the treatment regime if the second level of SIGLEC-14 is the same or higher than the first level.

[185] Item 17. The method of item 16, further comprising step (a1 ) identifying a first level of BCAM in a first sample from the subject before administration of a treatment regime to the subject.

[186] Item 18. The method of item 16 or 17, further comprising step (b1 ) identifying a second level of BCAM in a second sample from the subject after administration of a treatment regime to the subject.

[187] Item 19. The method of any one of items 16 to 18, further comprising step (c1 ) comparing the first and second level of BCAM.

[188] Item 20. The method of any one of items 16 to 19, further comprising step (d1 ) adjusting the treatment regime if the second level of BCAM is the same or higher than the first level.

[189] Item 21. The method of any one of items 16 to 20, further comprising step (a2) identifying a first level of ANGPTL3 in a first sample from the subject before administration of a treatment regime to the subject.

[190] Item 22. The method of any one of items 16 to 21 , further comprising step (b2) identifying a second level of ANGPTL3 in a second sample from the subject after administration of a treatment regime to the subject.

[191] Item 23. The method of any one of items 16 to 22, further comprising step (c2) comparing the first and second level of ANGPTL3. [192] Item 24. The method of any one of items 16 to 23, further comprising step (d2) adjusting the treatment regime if the second level of ANGPTL3 is the same or lower than the first level.

[193] Item 25. The method of any one of items 13 to 24, wherein said treatment regime includes variation of ventilator settings, extubation followed by non-invasive ventilation, glucocorticoid administration, vitamin A or vitamin A analogue administration, caffeine administration, NO administration, surfactant administration, application of adjusted surveillance, such as oxygen saturation levels, adjustment of oxygen administration levels.

[194] Item 26. The method of any one of items 13 to 25, wherein neonatal chronic lung disease is bronchopulmonary dysplasia (BPD).

[195] Item 27. A kit for performing the method of any one of items 1 to 26, comprising binding molecules for SIGLEC-1 , BCAM and ANGPTL3 and optionally means for detection.

[196] Item 28. The kit of item 27, wherein said binding molecule is an antibody.

[197] Item 29. Use of a binding molecule for SIGLEC-14, BCAM and/or ANGPTL3 for identifying a subject being at risk for developing neonatal chronic lung disease.

[198] Item 30. The use of item 29, wherein neonatal chronic lung disease is bronchopulmonary dysplasia (BPD).

[199] List of sequences mentioned herein:

Uniprot MLPLLLLPLLWGGSLQEKPVYELQVQKSVTVQEGLCVLVPCSFSYPWRSWYSSPPLYVY

Number: FRDGEIPYYAEVVATNNPDRRVKPETQGRFRLLGDVQKKNCSLSIGDARMEDTGSYFFRV

ERGRDVKYSYQQNKLNLEVTALIEKPDIHFLEPLESGRPTRLSCSLPGSCEAGPPLTFSW

Q08ET2

TGNALSPLDPETTRSSELTLTPRPEDHGTNLTCQVKRQGAQVTTERTVQLNVSYAPQNLA

HUMAN ISIFFRNGTGTALRILSNGMSVPIQEGQSLFLACTVDSNPPASLSWFREGKALNPSQTS

SIGLEC14 SGTLELPNIGAREGGEFTCRVQHPLGSQHLSFILSVQRSSSSCICVTEKQQGSWPLVLTL

IRGALMGAGFLLTYGLTWIYYTRCGGPQQSRAERPG

gi1148762979 ACTCACCCTCCGGCTTCCTGTCGGGGCTTTCTCAGCCCCACCCCACGTTTGGACATTTGG ref | NM_0010 AGCATTTCCTTCCCTGACAGCCGGACCTGGGACTGGGCTGGGGCCCTGGCGGATGGAGAC

ATGCTGCCCCTGCTGCTGCTGCCCCTGCTGTGGGGGGGGTCCCTGCAGGAGAAGCCAGTG

98612.11 TACGAGCTGCAAGTGCAGAAGTCGGTGACGGTGCAGGAGGGCCTGTGCGTCCTTGTGCCC Homo sapiens TGCTCCTTCTCTTACCCCTGGAGATCCTGGTATTCCTCTCCCCCACTCTACGTCTACTGG sialic acid TTCCGGGACGGGGAGATCCCATACTACGCTGAGGTTGTGGCCACAAACAACCCAGACAGA binding Ig like AGAGTGAAGCCAGAGACCCAGGGCCGATTCCGCCTCCTTGGGGATGTCCAGAAGAAGAAC lectin 14 TGCTCCCTGAGCATCGGAGATGCCAGAATGGAGGACACGGGAAGCTATTTCTTCCGCGTG

GAGAGAGGAAGGGATGTAAAATATAGCTACCAACAGAATAAGCTGAACTTGGAGGTGACA

(SIGLEC14),

GCCCTGATAGAGAAACCCGACATCCACTTTCTGGAGCCTCTGGAGTCCGGCCGCCCCACA

mRNA AGGCTGAGCTGCAGCCTTCCAGGATCCTGTGAAGCGGGACCACCTCTCACATTCTCCTGG

ACGGGGAATGCCCTCAGCCCCCTGGACCCCGAGACCACCCGCTCCTCGGAGCTCACCCTC ACCCCCAGGCCCGAGGACCATGGCACCAACCTCACCTGTCAGGTGAAACGCCAAGGAGCT CAGGTGACCACGGAGAGAACTGTCCAGCTCAATGTCTCCTATGCTCCACAGAACCTCGCC ATCAGCATCTTCTTCAGAAATGGCACAGGCACAGCCCTGCGGATCCTGAGCAATGGCATG TCGGTGCCCATCCAGGAGGGCCAGTCCCTGTTCCTCGCCTGCACAGTTGACAGCAACCCC CCTGCCTCACTGAGCTGGTTCCGGGAGGGAAAAGCCCTCAATCCTTCCCAGACCTCAATG TCTGGGACCCTGGAGCTGCCTAACATAGGAGCTAGAGAGGGAGGGGAATTCACCTGCCGG GTTCAGCATCCGCTGGGCTCCCAGCACCTGTCCTTCATCCTTTCTGTGCAGAGAAGCTCC TCTTCCTGCATATGTGTAACTGAGAAACAGCAGGGCTCCTGGCCCCTCGTCCTCACCCTG ATCAGGGGGGCTCTCATGGGGGCTGGCTTCCTCCTCACCTATGGCCTCACCTGGATCTAC TATACCAGGTGTGGAGGCCCCCAGCAGAGCAGGGCTGAGAGGCCTGGCTGAGCCCCTCCC GCTCAAGACAGAACTGAGGTGTGGACACTTAGCCCTGTGGGACACATGCAGGACATCACT GTCAGCTTCTTTCTGGAAGCTCACATCCCACTGACTACCCCTCTTTTCCTTCCTGCCCCA TACCCCTTCTACTTATTCCCCTCTGCTTGTGAGTCTTGCCCCACCACACCTGCATCCCCA TCTGCACCCCATCCCCTCTCCACCTGCCCTTCTCTTCCCTCTCCATCCACCATCTCCAGC CCTGTGAAGGGAATGTACTTTCGGTCTTATACCCCCATTACCCATTACCCAAAAGTTACC TTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTCACTCTGTTGCACAGGCTGGAGTTCA GTGGCACAATCTCCGTTCACTGCAACCTCCACCTCTGGGGTTCAAGCAATTCTCCTGCCT CAGCCTCCCTAGTAGCTGGGATTACAGGTGCCTGCCACCACATCCAGTTAATTTTTTTTT TTTGTATGTTAGTAGAGATGGGGTTTTACCATGTTGGCCAGGTCTCGAACTCCTGACCTC AAGCAATCCACTGCATTGGCCTCCCAAAGTGCTGGCATTACAGGTATGAGCCACCGTGCC TGGCTGCCAAAAGTTACCTTCTTAACACTTGAATTTCTGGTCTCCTCAGCTTCCCTATCC ATATAGGCACAGAGAGGCAGCATTTGTTTTCCAGTTAAAACTCTACCTCATTGTGATTAT TATCCAATACAATTGTTACAAAATAAGTAAAACTTTTATGAAACAATACAACATAACTGA TTTTACTCTTTAA

S gi 1616134111 GCCGGACCTGGGACTGGGCTGGGGCCCTGGCGGATGGAGACATGCTGCCCCTGCTGCTGC E gb | AY854038. TGCCCCTGCTGTGGGGGGGGTCCCTGCAGGAGAAGCCAGTGTACGAGCTGCAAGTGCAGA

AGTCGGTGACGGTGCAGGAGGGCCTGTGCGTCCTTGTGCCCTGCTCCTTCTCTTACCCCT

Q i | GGAGATCCTGGTATTCCTCTCCCCCACTCTACGTCTACTGGTTCCGGGACGGGGAGATCC

Homo sapiens CATACTACGCTGAGG TGTGGCCACAAACAACCCAGACAGAAGAGTGAAGCCAGAGACCC SIGLEC14 AGGGCCGATTCCGCCTCCTTGGGGATGTCCAGAAGAAGAACTGCTCCCTGAGCATCGGAG (SIGLEC14) ATGCCAGAATGGAGGACACGGGAAGCTATTTCTTCCGCGTGGAGAGAGGAAGGGATGTAA mRNA, AATATAGCTACCAACAGAATAAGCTGAACTTGGAGGTGACAGCCCTGATAGAGAAACCCG

ACATCCACTTTCTGGAGCCTCTGGAGTCCGGCCGCCCCACAAGGCTGAGCTGCAGCCTTC

N complete cds

CAGGATCCTGTGAAGCGGGACCACCTCTCACATTCTCCTGGACGGGGAATGCCCTCAGCC

0 CCCTGGACCCCGAGACCACCCGCTCCTCGGAGCTCACCCTCACCCCCAGGCCCGAGGACC

ATGGCACCAACCTCACCTGTCAGGTGAAACGCCAAGGAGCTCAGGTGACCACGGAGAGAA CTGTCCAGCTCAATGTCTCCTATGCTCCACAGAACCTCGCCATCAGCATCTTCTTCAGAA ATGGCACAGGCACAGCCCTGCGGATCCTGAGCAATGGCATGTCGGTGCCCATCCAGGAGG GCCAGTCCCTGTTCCTCGCCTGCACAGTTGACAGCAACCCCCCTGCCTCACTGAGCTGGT TCCGGGAGGGAAAAGCCCTCAATCCTTCCCAGACCTCAATGTCTGGGACCCTGGAGCTGC CTAACATAGGAGCTAGAGAGGGAGGGGAATTCACCTGCCGGGTTCAGCATCCGCTGGGCT CCCAGCACCTGTCCTTCATCCTTTCTGTGCAGAGAAGCTCCTCTTCCTGCATATGTGTAA CTGAGAAACAGCAGGGCTCCTGGCCCCTCGTCCTCACCCTGATCAGGGGGGCTCTCATGG GGGCTGGCTTCCTCCTCACCTATGGCCTCACCTGGATCTACTATACCAGGTGTGGAGGCC CCCAGCAGAGCAGGGCTGAGAGGCCTGGCTGAGCCCCTCCCGCTCAAGACAGAACTGAGG TGTGGACACTTAGCCCTGTGGGACACATGCAGGACATCACTGTCAGCTTCTTTCTGGAAG CTCACATCCCACTGACTACCCCTCTTTTCCTTCCTGCCCCATACCCCTTCTACTTATTCC CCTCTGCTTGTGAGTCTTGCCCCACCACACCTGCATCCCCATCTGCACCCCATCCCCTCT CCACCTGCCCTTCTCTTCCCTCTCCATCCACCATCTCCAGCCCTGTGAAGGGAATGTACT TTCGGTCTTATACCCCCATTACCCATTACCCAAAAGTTACCTTTTTTTTTTTTTTTTTTT TTTTTTGAGACAGAGTCTCACTCTGTTGCACAGGCTGGAGTTCAGTGGCACAATCTCCGT TCACTGCAACCTCCACCTCTGGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCTAGTAGC TGGGATTACAGGTGCCTGCCACCACACCCAGTTAATTTTTTTTTTTTGTATGTTAGTAGA GATGGGGTTTTACCATGTTGGCCAGGTCTCGAACTCCTGACCTCAAGCAATCCACTGCAT TGGCCTCCCAAAGTGCTGGCATTACAGGTATGAGCCACCGTGCCTGGCTGCCAAAAGTTA CCTTCTTAACACTTGAATTTCTGGTCTCCTCAGCTTCCCTATCCATATAGGCACAGAGAG GCAGCATTTGTTTTCCAGTTAAAACTCTACCTCATTGTGATTATTATCCAATACAATTGT TACAAAATAAGT AAACTTT TGAAACAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

s BCAM_ MEPPDAPAQARGAPRLLLLAVLLAAHPDAQAEVRLSVPPLVEVMRGKSVILDCTPTGTHD

E HUMAN HYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDE RDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPA

Q l (Uniprot

PKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAH

number: YSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPE

P50895 YTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAY

LDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGP LSLSSITFDSNG TYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPD PKLSWSQLGGSPAEPIPGRQGWVSSSL LKV SALSRDGI SCEASNPHGNKRHVFHFGTV N SPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGS 0 EQPEQTGLLMGGASGGARGGSGGFGDEC _

S gi 1386645004 CTCTGGCTCCCAGCCCCGCAGCGGCCGAGCTGCAGCCCGGGCTCAGTCTCCGCCGCCGCC

E NM_0010132 GTGAACATGGAGCCCCCGGACGCACCGGCCCAGGCGCGCGGGGCCCCGCGGCTGCTGTTG

CTCGCAGTCCTGCTGGCGGCGCACCCAGATGCCCAGGCGGAGGTGCGCTTGTCTGTACCC

Ql 57.2

CCGCTGGTGGAGGTGATGCGAGGAAAGTCTGTCATTCTGGACTGCACCCCTACGGGAACC

Homo sapiens CACGACCATTATATGCTGGAATGGTTCCTTACCGACCGCTCGGGAGCTCGCCCCCGCCTA blood group) GCCTCGGCTGAGATGCAGGGCTCTGAGCTCCAGGTCACAATGCACGACACCCGGGGCCGC (BCAM), AGTCCCCCATACCAGCTGGACTCCCAGGGGCGCCTGGTGCTGGCTGAGGCCCAGGTGGGC transcript GACGAGCGAGACTACGTGTGCGTGGTGAGGGCAGGGGCGGCAGGCACTGCTGAGGCCACT

GCGCGGCTCAACGTGTTTGCAAAGCCAGAGGCCACTGAGGTCTCCCCCAACAAAGGGACA

N variant 2,

CTGTCTGTGATGGAGGACTCTGCCCAGGAGATCGCCACCTGCAACAGCCGGAACGGGAAC

0 m NA CCGGCCCCCAAGATCACGTGGTATCGCAACGGGCAGCGCCTGGAGGTGCCCGTAGAGATG

AACCCAGAGGGCTACATGACCAGCCGCACGGTCCGGGAGGCCTCGGGCCTGCTCTCCCTC ACCAGCACCCTCTACCTGCGGCTCCGCAAGGATGACCGAGACGCCAGCTTCCACTGCGCC GCCCACTACAGCCTGCCCGAGGGCCGCCACGGCCGCCTGGACAGCCCCACCTTCCACCTC ACCCTGCACTATCCCACGGAGCACGTGCAGTTCTGGGTGGGCAGCCCGTCCACCCCAGCA GGCTGGGTACGCGAGGGTGACACTGTCCAGCTGCTCTGCCGGGGGGACGGCAGCCCCAGC CCGGAGTATACGCTTTTCCGCCTTCAGGATGAGCAGGAGGAAGTGCTGAATGTGAATCTC GAGGGGAACTTGACCCTGGAGGGAGTGACCCGGGGCCAGAGCGGGACCTATGGCTGCAGA GTGGAGGATTACGACGCGGCAGATGACGTGCAGCTCTCCAAGACGCTGGAGCTGCGCGTG GCCTATCTGGACCCCCTGGAGCTCAGCGAGGGGAAGGTGCTTTCCTTACCTCTAAACAGC AGTGCAGTCGTGAACTGCTCCGTGCACGGCCTGCCCACCCCTGCCCTACGCTGGACCAAG GACTCCACTCCCCTGGGCGATGGCCCCATGCTGTCGCTCAGTTCTATCACCTTCGATTCC AATGGCACCTACGTATGTGAGGCCTCCCTGCCCACAGTCCCGGTCCTCAGCCGCACCCAG AACTTCACGCTGCTGGTCCAAGGCTCGCCAGAGCTAAAGACAGCGGAAATAGAGCCCAAG GCAGATGGCAGCTGGAGGGAAGGAGACGAAGTCACACTCATCTGCTCTGCCCGCGGCCAT CCAGACCCCAAACTCAGCTGGAGCCAATTGGGGGGCAGCCCCGCAGAGCCAATCCCCGGA CGGCAGGGTTGGGTGAGCAGCTCTCTGACCCTGAAAGTGACCAGCGCCCTGAGCCGCGAT GGCATCTCCTGTGAAGCCTCCAACCCCCACGGGAACAAGCGCCATGTCTTCCACTTCGGC ACCGTGAGCCCCCAGACCTCCCAGGCTGGAGTGGCCGTCATGGCCGTGGCCGTCAGCGTG GGCCTCCTGCTCCTCGTCGTTGCTGTCTTCTACTGCGTGAGACGCAAAGGGGGCCCCTGC TGCCGCCAGCGGCGGGAGAAGGGGGCTCCGTGAGTGGCCTGCTATCTGCAATGACCACCA TAGCTTAACCCCATCCCCACCCTCAACCCCAAGCTCAACCCATAACCTCAACCACATCTT ATCCTCCACCCCACATCCCACCACATCCACCTCCATCCCCAACCCATCCTCATCCCCAAC TACAGCCCCAAACCCAGCCCCAGACTAATCCACAGCCATCCCCAACTCATCCTCATCCCC AACTGCAGCCCCAAACCCAACCCAGGGCCATCCCCAAACCCATCCCCAAGCCAAACTCAA CACCATCCCCTTCCCTAATCACCTCTCCTGCCCCTAGCTCAGCCTCATCCCCAACTCCAT GCCTGTCTCCAATCCCAACCCTGCCCCCAATCTCCCCTCAACTCCAATCCATAACCCCCT TCAAACCATCCTCAACTAAGCTCCTCTCCAGCCCTGGCCACATCCCCATCCTCCCCCAAC CTCCAGCCCCAACACCCATCATCCCCCTGAGCTCACCCTTAACTCCAATTTATCCTCCAA GCCTATCTCTCACCATCCAGCCCTCACCTAGCCATTTCCCAACCCTAGTCCTCCACCGTC CCGAAGCCAACTTCACCCATGTCCCCATCCCCAATCTTGTCCCCATTCCTGACTCCAAAT CCAACCCCTAAGCCTCCCCAAGCTCCTCCTCATCTAACCTTGTCTCCAGCCCCAACCACA TGGCCGTCCTCACCTCCAATCCGTAACCATCCCAGACCGATCCCAAAGCCAACTCCAGCT ACATTCCCACCTCCAACCCCAATAGCATCTGCATCCCCATGCCCAACCCTAACCCCAGTG CCATTACCAGCCCCTACCCCATCCCTATGCCATCCCCACCCAGCACACCCCCATCCTCAC CTCCATCCCCAGCCGCATCCTAGTCCCACCCACCCCCATCCTCAGTTCCTCCCCCTGCCG TGTGCACCTCCAACACGACGCCTCCGCCCGCTGCCTCCTCCCCCCAGGCCGCCAGGGGAG CCAGGGCTGAGCCACTCGGGGTCGGAGCAACCAGAGCAGACCGGCCTTCTCATGGGAGGT GCCTCCGGAGGAGCCAGGGGTGGCAGCGGGGGCTTCGGAGACGAGTGCTGAGCCAAGAAC CTCCTAGAGGCTGTCCCTGGACCTGGAGCTGCAGGCATCAGAGAACCAGCCCTGCTCACG CCATGCCCGCCCCCGCCTTCCCTCTTCCCTCTTCCCTCTCCCTGCCCAGCCCTCCCTTCC TTCCTCTGCCGGCAAGGCAGGGACCCACAGTGGCTGCCTGCCTCCGGGAGGGAAGGAGAG GGAGGGTGGGTGGGTGGGAGGGGGCCTTCCTCCAGGGAATGTGACTCTCCCAGGCCCCAG AATAGCTCCTGGACCCAAGCCCAAGGCCCAGCCTGGGACAAGGCTCCGAGGGTCGGCTGG CCGGAGCTATTTTTACCTCCCGCCTCCCCTGCTGGTCCCCCCACCTGACGTCTTGCTGCA GAGTCTGACACTGGATTCCCCCCCCTCACCCCGCCCCTGGTCCCACTCCTGCCCCCGCCC TACCTCCGCCCCACCCCATCATCTGTGGACACTGGAGTCTGGAATAAATGCTGTTTGTCA CA CAACACCAAAAAAAAAAAAAAAAAA

s gi 1386645003 CTCTGGCTCCCAGCCCCGCAGCGGCCGAGCTGCAGCCCGGGCTCAGTCTCCGCCGCCGCC

E ref | NM_0055 GTGAACATGGAGCCCCCGGACGCACCGGCCCAGGCGCGCGGGGCCCCGCGGCTGCTGTTG

CTCGCAGTCCTGCTGGCGGCGCACCCAGATGCCCAGGCGGAGGTGCGCTTGTCTGTACCC

Q 81.4

CCGCTGGTGGAGGTGATGCGAGGAAAGTCTGTCATTCTGGACTGCACCCCTACGGGAACC

Homo sapiens CACGACCATTATATGCTGGAATGGTTCCTTACCGACCGCTCGGGAGCTCGCCCCCGCCTA

1 basal cell GCCTCGGCTGAGATGCAGGGCTCTGAGCTCCAGGTCACAATGCACGACACCCGGGGCCGC

D adhesion AGTCCCCCATACCAGCTGGACTCCCAGGGGCGCCTGGTGCTGGCTGAGGCCCAGGTGGGC molecule GACGAGCGAGACTACGTGTGCGTGGTGAGGGCAGGGGCGGCAGGCACTGCTGAGGCCACT

GCGCGGCTCAACGTGTTTGCAAAGCCAGAGGCCACTGAGGTCTCCCCCAACAAAGGGACA

N (Lutheran

CTGTCTGTGATGGAGGACTCTGCCCAGGAGATCGCCACCTGCAACAGCCGGAACGGGAAC

0 blood group) CCGGCCCCCAAGATCACGTGGTATCGCAACGGGCAGCGCCTGGAGGTGCCCGTAGAGATG

(BCAM), AACCCAGAGGGCTACATGACCAGCCGCACGGTCCGGGAGGCCTCGGGCCTGCTCTCCCTC

6 transcript ACCAGCACCCTCTACCTGCGGCTCCGCAAGGATGACCGAGACGCCAGCTTCCACTGCGCC variant 1, GCCCACTACAGCCTGCCCGAGGGCCGCCACGGCCGCCTGGACAGCCCCACCTTCCACCTC m NA ACCCTGCACTATCCCACGGAGCACGTGCAGTTCTGGGTGGGCAGCCCGTCCACCCCAGCA

GGCTGGGTACGCGAGGGTGACACTGTCCAGCTGCTCTGCCGGGGGGACGGCAGCCCCAGC CCGGAGTATACGCTTTTCCGCCTTCAGGATGAGCAGGAGGAAGTGCTGAATGTGAATCTC GAGGGGAACTTGACCCTGGAGGGAGTGACCCGGGGCCAGAGCGGGACCTATGGCTGCAGA GTGGAGGATTACGACGCGGCAGATGACGTGCAGCTCTCCAAGACGCTGGAGCTGCGCGTG GCCTATCTGGACCCCCTGGAGCTCAGCGAGGGGAAGGTGCTTTCCTTACCTCTAAACAGC AGTGCAGTCGTGAACTGCTCCGTGCACGGCCTGCCCACCCCTGCCCTACGCTGGACCAAG GACTCCACTCCCCTGGGCGATGGCCCCATGCTGTCGCTCAGTTCTATCACCTTCGATTCC AATGGCACCTACGTATGTGAGGCCTCCCTGCCCACAGTCCCGGTCCTCAGCCGCACCCAG AACTTCACGCTGCTGGTCCAAGGCTCGCCAGAGCTAAAGACAGCGGAAATAGAGCCCAAG GCAGATGGCAGCTGGAGGGAAGGAGACGAAGTCACACTCATCTGCTCTGCCCGCGGCCAT CCAGACCCCAAACTCAGCTGGAGCCAATTGGGGGGCAGCCCCGCAGAGCCAATCCCCGGA CGGCAGGGTTGGGTGAGCAGCTCTCTGACCCTGAAAGTGACCAGCGCCCTGAGCCGCGAT GGCATCTCCTGTGAAGCCTCCAACCCCCACGGGAACAAGCGCCATGTCTTCCACTTCGGC ACCGTGAGCCCCCAGACCTCCCAGGCTGGAGTGGCCGTCATGGCCGTGGCCGTCAGCGTG GGCCTCCTGCTCCTCGTCGTTGCTGTCTTCTACTGCGTGAGACGCAAAGGGGGCCCCTGC TGCCGCCAGCGGCGGGAGAAGGGGGCTCCGCCGCCAGGGGAGCCAGGGCTGAGCCACTCG GGGTCGGAGCAACCAGAGCAGACCGGCCTTCTCATGGGAGGTGCCTCCGGAGGAGCCAGG GGTGGCAGCGGGGGCTTCGGAGACGAGTGCTGAGCCAAGAACCTCCTAGAGGCTGTCCCT GGACCTGGAGCTGCAGGCATCAGAGAACCAGCCCTGCTCACGCCATGCCCGCCCCCGCCT TCCCTCTTCCCTCTTCCCTCTCCCTGCCCAGCCCTCCCTTCCTTCCTCTGCCGGCAAGGC AGGGACCCACAGTGGCTGCCTGCCTCCGGGAGGGAAGGAGAGGGAGGGTGGGTGGGTGGG AGGGGGCCTTCCTCCAGGGAATGTGACTCTCCCAGGCCCCAGAATAGCTCCTGGACCCAA GCCCAAGGCCCAGCCTGGGACAAGGCTCCGAGGGTCGGCTGGCCGGAGCTATTTTTACCT CCCGCCTCCCCTGCTGGTCCCCCCACCTGACGTCTTGCTGCAGAGTCTGACACTGGATTC CCCCCCCTCACCCCGCCCCTGGTCCCACTCCTGCCCCCGCCCTACCTCCGCCCCACCCCA TCATCTGTGGACACTGGAGTCTGGAATAAATGCTGTTTGTCACATCAACACCAAAAAAAA AAAAAAAAAA

S Uniprot MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDF E Number: VHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLEL

NSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQ Q Q9Y5C1

TVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHD

Homo sapiens GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWE

1 ANGPTL3 NYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTL

D HLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKP RAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE

N

0

7

S gi 1452408443 ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAAAACAGTTCCACGTTGCTTGA E ref | NM_0144 AATTGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAG

TTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAA

Q 95.3

AATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGG

Homo sapiens GACATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAA

1 angiopoietin AACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATCAAAG like 3 AAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAA

(ANGPTL3), AGAA AT GT C AC T T GAAC T C AAC T C AAAAC T T GAAAGCC T C C T AGAAGAAAAAAT T C T AC

T TCAACAAAAAGT GAAAT AT T T AGAAGAGCAAC T AAC T AAC T TAAT T C AAAATCAACC TG

N mRNA

AAACTCCAGAACACCCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAAACAAGATAATA

0 GCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAGCATA

G T C AAAT AAAAGAAAT AGAAAAT CAGCT CAGAAGGACT AGT AT TCAAGAAC C C AC AGAAA TTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAA TAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTG AACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACT GTGATGTTATATCAGG AG CCA GGACA AATTCAACATCGAATAGATGGATCACAAA ACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTT GGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTG AGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATC ACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCC CGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACT GTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAA ATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATCTT GGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAA CAGATTCAGAAAGCT T T GAAT GAACT GAGGCAAAT T AAAAGGC AAT AAT T T AAAC AT A ACCTCATTCCAAGTTAATGTGGTCTAATAATCTGGTATTAAATCCTTAAGAGAAAGCTTG AGAAATAGAT T T T T T T TAT CT T AAAGT CACT GT C TAT T T AAGAT T AAAC A ACAATCACA TAACCTTAAAGAATACCGTTTACATTTCTCAATCAAAATTCTTATAATACTATTTGTTTT AAATTTTGTGATGTGGGAATCAATTTTAGATGGTCACAATCTAGATTATAATCAATAGGT GAACTTATTAAATAACTTTTCTAAATAAAAAATTTAGAGACTTTTATTTTAAAAGGCATC ATATGAGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTA AACTTGACTAAATACAGAGGACTGGTAATTGTACAGTTCTTAAATGTTGTAGTATTAATT TC AAAAC TAAAAAT CGT CAGCACAGAGT AT GT GT AAAAAT C T GT AAT AC AAAT T T T T AAA CTGATGCTTCATTTTGCTACAAAATAATTTGGAGTAAATGTTTGATATGATTTATTTATG AAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGTTCGCTGTCTTTAAACAAAT GGAGATGACTACTAAGTCACATTGACTTTAACATGAGGTATCACTATACCTTATTTGTTA AAATATATACT GT AT ACAT T T T AT AT AT T T T AAC AC T TAAT AC TAT GAAAACAAATAAT T GTAAAGGAATCTTGTCAGATTACAGTAAGAATGAACATATTTGTGGCATCGAGTTAAAGT TTATATTTCCCCTAAATATGCTGTGATTCTAATACATTCGTGTAGGTTTTCAAGTAGAAA TAAACCTCGTAACAAGTTACTGAACGTTTAAACAGCCTGACAAGCATGTATATATGTTTA AAATTCAATAAACAAAGACCCAGTCCCTAAATTATAGAAATTTAAATTATTCTTGCATGT TTATCGACATCACAACAGATCCCTAAATCCCTAAATCCCTAAAGATTAGATACAAATTTT TTACCACAGTATCACTTGTCAGAATTTATTTTTAAATATGATTTTTTAAAACTGCCAGTA AGAAATTTTAAATTAAACCCATTTGTTAAAGGATATAGTGCCCAAGTTATATGGTGACCT ACCTTTGTCAATACTTAGCATTATGTATTTCAAATTATCCAATATACATGTCATATATAT T T T TATATGT CACAT AT AT AAAAGAT AT GT AT GAT C T AT GT GAAT C C T AAGT AAAT AT T T TGTTCCAGAAAAGTACAAAATAATAAAGGTAAAAATAATCTATAATTTTCAGGACCACAG ACTAAGCTGTCGAAATTAACGCTGATTTTTTTAGGGCCAGAATACCAAAATGGCTCCTCT C T T C C C C CAAAAT T GGAC AAT T T C AAAT GCAAAAT AAT TCATTATTTAATATATGAGTTG CTTCCTCTATT

s gi 149297891 GGCACGAGGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCT

E gb | AF152562. CTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAG

CCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAG

Q i | TTGGGACATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTT

Homo sapiens CAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATC angiopoietin- AAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAG related G T AAAGAAT AT GT C AC T T GAAC T C AAC T C AAAAC T T GAAAGCC T C C T AGAAGAAAAAAT T protein 3 C TAC TTCAACAAAAAGT GAAAT AT T T AGAAGAGCAACT AAC T AAC T TAAT T CAAAAT CAA

CCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAAACAAGAT

N (ANGPTL3)

AATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAG

0 mRNA, CATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAAGGACTAGTATTCAAGAACCCACA complete cds GAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAAT

GAAATAAGAAAT GT AAAACAT GAT GGCAT T C C T GC T GAAT GT AC C AC CAT T TATAACAGA GGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTC TACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCA CAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAA TTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGA ATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGA AATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCA ATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTC AACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAAC CTAAATGGTAAATA AACAAACCAAGAGCAAAATCT AGCCAGAGAGGAGAAGAGGATTA TCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCAT CCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAATTTAAAC ATTAACCTCATTCCAAGTTAATGTGGTCTAATAATCTGGTATTAAATCCTTAAGAG

Uniprot MLPLLLLPLLWGGSLQEKPVYELQVQKSVTVQEGLCVLVPCSFSYPWRSWYSSPPLYVY Number: FRDGEIPYYAEVVATNNPDRRVKPE QGRFRLLGDVQKKNCSLS IGDARMEDTGSYFFRV

ERGRDVKYSYQQNKLNLEVTALIEKPDIHFLEPLESGRPTRLSCSLPGSCEAGPPLTFS

Homo sapiens

TGNALSPLDPETTRSSELTLTPRPEDHGTNLTCQMKRQGAQVTTERTVQLNVSYAPQTIT SIGLEC-5 IFRNGIALEILQNTSYLPVLEGQALRLLCDAPSNPPAHLSWFQGSPALNATPISNTGILE Uniprot No. LRRVRSAEEGGFTCRAQHPLGFLQIFLNLSVYSLPQLLGPSCSWEAEGLHCRCSFRARPA 015389 PSLCWRLEEKPLEGNSSQGSFKVNSSSAGPWANSSLILHGGLSSDLKVSCKAWNIYGSQS

GSVLLLQGRSNLGTGVVPAALGGAGVMALLCICLCLIFFLIVKARRKQAAGRPEKMDDED PIMGTITSGSRKKPWPDSPGDQASPPGDAPPLEEQKELHYASLSFSEMKSREPKDQEAPS TTEYSEIKTSK

Homo sapiens GTGCGCGTCCACAGCTCTCACTCACCCTCCGGCTTCCTGTCGGGGCTTTCTCAGCCCCAC sialic acid CCCACGTTTGGACATTTGGAGCATTTCCTTCCCTGACAGCCGGACCTGGGACTGGGCTGG

GGCCCTGGCGGATGGAGACATGCTGCCCCTGCTGCTGCTGCCCCTGCTGTGGGGGGGGTC

binding Ig like

CCTGCAGGAGAAGCCAGTGTACGAGCTGCAAGTGCAGAAGTCGGTGACGGTGCAGGAGGG

lectin 5 CCTGTGCGTCCTTGTGCCCTGCTCCTTCTCTTACCCCTGGAGATCCTGGTATTCCTCTCC (SIGLEC5), CCCACTCTACGTCTACTGGTTCCGGGACGGGGAGATCCCATACTACGCTGAGGTTGTGGC mRNA CACAAACAACCCAGACAGAAGAGTGAAGCCAGAGACCCAGGGCCGATTCCGCCTCCTTGG NCBI GGATGTCCAGAAGAAGAACTGCTCCCTGAGCATCGGAGATGCCAGAATGGAGGACACGGG AAGCTATTTCTTCCGCGTGGAGAGAGGAAGGGATGTAAAATATAGCTACCAACAGAATAA

Reference

GCTGAACTTGGAGGTGACAGCCCTGATAGAGAAACCCGACATCCACTTTCTGGAGCCTCT

Sequence: GGAGTCCGGCCGCCCCACAAGGCTGAGCTGCAGCCTTCCAGGATCCTGTGAAGCGGGACC NM_003830.3 ACCTCTCACATTCTCCTGGACGGGGAATGCCCTCAGCCCCCTGGACCCCGAGACCACCCG

CTCCTCGGAGCTCACCCTCACCCCCAGGCCCGAGGACCATGGCACCAACCTCACCTGTCA GATGAAACGCCAAGGAGCTCAGGTGACCACGGAGAGAACTGTCCAGCTCAATGTCTCCTA TGCTCCACAGACCATCACCATCTTCAGGAACGGCATAGCCCTAGAGATCCTGCAAAACAC CTCATACCTTCCGGTCCTGGAGGGCCAGGCTCTGCGGCTGCTCTGTGATGCTCCCAGCAA CCCCCCTGCACACCTGAGCTGGTTCCAGGGCTCCCCTGCCCTGAACGCCACCCCCATCTC CAATACCGGGATCTTGGAGCTTCGTCGAGTAAGGTCTGCAGAAGAAGGAGGCTTCACCTG CCGCGCTCAGCACCCGCTGGGCTTCCTGCAAATTTTTCTGAATCTCTCAGTTTACTCCCT CCCACAGTTGCTGGGCCCCTCCTGCTCCTGGGAGGCTGAGGGTCTGCACTGCAGATGCTC CTTTCGAGCCCGGCCGGCCCCCTCCCTGTGCTGGCGGCTTGAGGAGAAGCCGCTGGAGGG GAACAGCAGCCAGGGCTCATTCAAGGTCAACTCCAGCTCAGCTGGGCCCTGGGCCAACAG CTCCCTGATCCTCCACGGGGGGCTCAGCTCCGACCTCAAAGTCAGCTGCAAGGCCTGGAA CATCTATGGGTCCCAGAGCGGCTCTGTCCTGCTGCTGCAAGGGAGATCGAACCTCGGGAC AGGAGTGGTTCCTGCAGCCCTTGGTGGTGCTGGTGTCATGGCCCTGCTCTGTATCTGTCT GTGCCTCATCTTCTTTTTAATAGTGAAAGCCCGCAGGAAGCAAGCAGCTGGGAGACCAGA GAAAATGGATGATGAAGACCCCATTATGGGTACCATCACCTCGGGTTCCAGGAAGAAGCC CTGGCCAGACAGCCCCGGAGATCAAGCATCTCCTCCTGGGGATGCCCCTCCCTTGGAAGA ACAAAAGGAGCTCCATTATGCCTCCCTTAGTTTTTCTGAGATGAAGTCGAGGGAGCCTAA GGACCAGGAGGCCCCAAGCACCACGGAGTACTCGGAGATCAAGACAAGCAAGTGAGGATT TGCCCAGAGTTCAGTCCTGGCTGGAGGAGCCACAGCCTGTCTGGGGGAAAGGACAAGTCA GGGACCACTTGCTGAAGCACGAAGAGCCCTTGTGGCAATGTTAACATTAACTGATGTTTA AGTGCTCCAAGCAGATGGAATTAGAGAGGTGGGCTCAAATCTAGGCCCTGGCACTGTCAT CAAGCAATTCACTGCATCCCTCTGTGCCTCAGTTTCCCATTCTGTAAATCAGAGATCATG CATGCTACCTCAAAGGTTGTTGTGAACATTAAAGAAATCAACACATGGAAATCAACCAAC ATGGGTCCTGGAACAGGGCGTTGTGCTCAGTGCTTTCTGGTCTCTCTTCCTTGAATAGAA AGGTCCTGCTGGCAAGTTCTCTCAAGGCTGGGGATGACCAGGCACAAAAAACAGGGCAGC AATATGTTGGTGTCACTCCCCTTCCCAAAACTCTTCGAAGACTCCCTAGGAAAGACCAGC CCCTCAGCCTGGCACTTGGTTCATGATGTGGGATCTTATATCCTTGCCAGAGTCATATCT TTGCCCACTTTTACCTGCAATCCTTGCATCATATTCCTTTGGCTCCAGTCCTTCATTTAT GAGACCCATAGGAATCCTTCCAACAGCCAAAGAGTTGAGTCTAACTCTTTCCTGCCCAAA CCCATTCACGGCCCCCTGGCCTTAGACAATATATCACAAGCATCTCCCCTGACACATAAA GTC [200] Figure 1 : Identification of plasma biomarkers by unbiased proteomics screening.

A) Assignment of the 12 significantly regulated proteins to the GO categories identified by enrichment analysis performed on the protein screening results in the exploration cohort. B) Separation of disease groups in the exploration cohort by protein expression of BCAM, Siglec-1 and ANGPTL3. C, D) Sensitivity and specificity for the identification of BPD, i.e BPD grade 0 (no), 1 (mild), 2 (moderate) and 3 (severe) by Siglec-14 plasma concentration in the first week of life (C) and up to 28 days of life (D). E, F) Validation of the results in the confirmation cohort by ELISA for SIGLEC-14, BCAM, ANGPTL-3 within the study group (area under the ROC 0.83 (E)) and by the use of the model identified in the exploration cohort (area under the ROC curve 0.76 (F)).

[201] Figure 2: Immunostaining for the identified BPD biomarker proteins in human preterm lungs.

[202] Pulmonary expression of SILGEC-14 (A), BCAM (B) and ANGPTL3 (C) in preterm infants with mild (1 ), moderate (2) or severe (3) BPD as compared to an infant without BPD. Red arrows indicate positive stain. Magnification 400x.

[203] Figure 3: Increased T2 relaxation times characterize infants with BPD.

B) Principal component analysis (PCA) applied on the statistically selected variables in the exploration cohort. Each triangle (mild (1 ), moderate (2) or severe (3) BPD) or circle (no (0) BPD) represents the individual data point for one preterm infant at the corrected age of 36 weeks postmenstrual age (PMA.). B) Imaging and clinical variables identified by the statistical model to best describe the outcome variable mild, moderate and severe BPD in preterm infants: T2 relaxation times (upper left lung), lung volume (right lung, coronar image analysis) and the clinical variable postmenstrual gestational age (GA) C) Representative T2- weighted MR images (i, ii) with echo times (TE) between 26 and 92 ms and calculated T2 maps of two subjects with BPD 0 (iii) and BPD 3 (iv). T2 relaxation time is increased in the infant with severe BPD. D) Validation of the MRI findings in the confirmation cohort with a significant increase in T2 relaxation time values in the lungs of infants with BPD with modest regional variation.

[204] Figure 4: Decreased T1 relaxation times characterize infants with BPD.

C) Principal component analysis (PCA) applied on the statistically selected variables in the exploration cohort. Each triangle (moderate (2) and severe (3) BPD) or circle (no (0) and mild (1 ) BPD) represents the individual data point for one preterm infant at the age of 36 weeks GA. B) Selected imaging and clinical variables identified by the statistical model best describing the outcome variable moderate and severe BPD in the preterm infant: T2 relaxation times (lower right lung) and T1 relaxation times (lower right and lower left lung). C) T1 -weighted MR images (i, ii) without inversion pulse (Tl = ∞) and inversion times (Tl) between 25 and 2600 ms. Calculated T1 maps of two subjects with BPD 0 (iii) and BPD 3 (iv). T1 relaxation time is decreased in the infant with severe BPD.

[205] Figure 5: Functional differences in BPD lungs support structural findings by MRI.

D) Principal component analysis (PCA) applied on the statistically selected variables in the exploration cohort. Each triangle (mild (1 ), moderate (2) and severe (3) BPD) or circle (no (0) and mild (1 ) BPD) represents the individual data point for one preterm infant at the age of 36 weeks PMA. B) Lung function and clinical variables identified by the statistical model to best describe the outcome variable mild, moderate and severe BPD in the preterm infant: FRC_p_pre = plethsymographic Functional Residual Capacity before inhalation of nebulized salbutamol, FRC_p_post = plethysmographic Functional Residual Capacity after inhalation, Crs.SO_pre = Compliance (single occlusion technique) before inhalation.

[206] Figure 6: Affinity enrichment to identify target proteins

[207] Proteome-wide affinity enrichment coupled with quantitative LC-MSMS confirmed SIGLEC1 as top target protein for the respective aptamers. Sequence Coverages are depicted in Figure 6.

[208] Figure 7: Affinity enrichment to identify target proteins

[209] Proteome-wide affinity enrichment coupled with quantitative LC-MSMS confirmed BCAM as top target protein for the respective aptamers. Sequence Coverages are depicted in Figure 7.

[210] Examples

[211] Patients and Methods

1. Study population

[212] Preterm infants with or without later development of BPD and a GA < 32 weeks born at the Perinatal Center of the Ludwig-Maximilians-University, Campus Grosshadern were prospectively included in the study (exploration cohort, n=43). A second, independent study cohort was recruited at the Perinatal Center of the University Hospital Giessen and Marburg in Giessen (confirmation cohort, n=21 ). Patient characteristics of both cohorts are given in Table 1 . Approval by the local Ethics Committee prior to the start of the study and written informed parental consent for all study infants was obtained (exploration cohort #195-07; confirmation cohort #135/12, last supplemented July 30th, 2014). The study registration was performed with the German Registry for Clinical Studies (DRKS00004600).

[213] The following definitions were applied: BPD was defined according to Jobe and Bancalari (Jobe and Bancalari, Am J Respir Crit Care Med. 2001 ; 163(7): 1723-9) and graded as follows: mild (oxygen supplementation at 28 days postnatally), moderate (oxygen supplementation below 30% or ventilator support at 36 weeks postmenstrual age), or severe (oxygen supplementation above 30% or ventilator support at 36 weeks postmenstrual age). Days with ventilator support were recorded as endotracheal (invasive) mechanical ventilation, nasal intermittent mandatory ventilation or nasal intermittent positive pressure ventilation and/or nasal continuous positive airway pressure in days.

A course of antenatal corticosteroids was recorded as "complete" if two doses of betamethasone were given >24 hours before birth with the last dose administered no later than 7 days before birth. Chorioamnionitis was defined as the presence of inflammatory alterations of the chorionic plate at histologic examination or signs of infection in both mother and infant (Franz et al., Acta Paediatr. 2001 ; 90(9): 1025-32). Intrauterine growth restriction was defined as birth weight below the 10 ^th percentile. Postnatally, diagnosis and severity of RDS (respiratory distress syndrome) was scored on anterior-posterior (a. -p.) chest radiographs according to Couchard et al (Couchard et al., Ann Radiol (Paris). 1974; 17(7): 669-83). Systemic infections were diagnosed according to Sherman et al. (Sherman et al., Pediatrics. 1980; 65(2): 258-63) with one or more clinical and laboratory signs of infection (C- reactive protein > 2mg/dl). [215] Table 1. Patient characteristics

Exploration cohort Confirmation cohort

43 21

Gestational age (weeks PMA) 27.3 (24.1-30.6) 25.6 (24.4-29.6) Birth weight (g) 915 (415-1770) 810 (340-1470) Gender (female/male) 25/18 14/7 pH, umbilical artery 7.34 (6.95-7.47) 7.34 (7.01-7.48) Antenatal corticosteroid 39 (90%) 20 (95.2%) Chorioamnionitis 22 (51.2%) 15 (71.4%) Early onset infection 10 (23.3%) 4 (19%) RDS > grade 3 13 (30.2%) 3 (14.3%)

Days of mechanical ventilation 38 (0-78) 24 (2-74)

- Endotracheal mechanical ventilation (n/days) 3 (0-41 ) 2 (0-32)

- Pharyngeal ventilation/CPAP (n/days) 34 (0-55) 18 (2-56) PDA 23 (53.5%) 15 (71 ,4%)

Postnatal Steroids 20 (46.5%) 1 (4.8%) ROP > grade 3 2 (4.7%) 6 (28.6%) IVH > grade 3 1 (2.3%) 3 (14.3%) ICU stay (days) 63 (30-127) 36 (5-93) BPD

- None 21 (48.8%) 4 (19%)

- Mild 13 (30.2%) 9 (42.9%)

- Moderate 4 (9.3%) 2 (9.5%)

- Severe 5 (1 1.6%) 6 (28.6%)

Data are given as median and range or number and percent of total in group. GA, gestational age; NS-pH, umbilical cord pH; ANCS, antenatal corticosteroids; RDS, respiratory distress syndrome; ROP, retinopathy of prematurity; IVH, intraventricular haemorrhage; ICU, intensive care unit; BPD, bronchopulmonary dysplasia, PMA, postmenstrual age

[216] 2. Biomarker analysis in preterm plasma samples

[217] Serial whole blood samples (200μΙ minimum each) obtained during routine laboratory blood drawings were collected in the first week of life, i.e. day 0-7 using

EthylenDiaminTetraAcetate (EDTA) neonatal collection tubes. An additional sample at day

28 of life was obtained in the exploration cohort. Samples were pseudonymized and processed for proteomic screening by centrifugation (1000g, 5 minutes) before supernatants were aliquoted and stored at -80 °C. Samples were analyzed by the SOMAscan assay

(SomaLogic, Boulder, USA), providing sensitive quantitative and reproducible proteomic screening by the use of 1 129 individual high affinity molecules (SOMAmer ^® - slow off-rate modified DNA aptamer - reagents). In brief, after a centrifugation step at 14000g for 7 minutes, biological samples were incubated with the mixture of the 1 129 SOMAmer ^® reagents on a 96 well plate for two sequential bead-based immobilization and washing steps before quantification of protein target-bound SOMAmer ^® reagents on a custom Agilent hybridization array (Gold et al., PLoS One. 2010; 5(12): e15004; Rohloff et al., Mol Ther Nucleic Acids. 2014; 3: e201 .

[218] Confirmation of protein expression was performed by the use of ELISA measurements in plasma samples obtained in the first week of life and processed as described above. ELISA measurements were performed according to the manufacturer's instructions: Siglec 5/Siglec 14 (DY1072, R&D systems), BCAM (EHBCAM, Thermofischer

Scientific), ANGPTL3 (ELH-ANGPTL3, Raybiotech).

[219] 3. Immunohistochemistry in human preterm lung tissue

[220] In order to verify the expression of the identified proteins in the human lung, immunohistochemistry (IHC) was performed using tissue sections from paraformaldehyde fixed and paraffin embedded autopsy lungs obtained from preterm infants with BPD (n= 5) and one infant that died from a non-pulmonary cause born at the Department of Pediatric

Surgery, Erasmus Medical Center - Sophia Children's Hospital, Rotterdam. After deparaffinization and blocking (5% Goat/Horse serum, 1 % BSA , 0.05% Tween in PBS)., adjacent sections were stained with anti-human SIGLEC-1 (1 :50, #MAB10721 ; R&D systems), anti-human BCAM (1 :200, #sc-99188; Santa Cruz Biotech) and anti-human

ANGPTL3 (1 :50,#600-401-Y15; Rockland antibodies and assays) overnight followed by secondary antibody incubation (1 :300) and visualization of the stain using Vector ABC reagent and DAB solution.

[221] 4. Pulmonary magnetic resonance imaging in preterm infants at 36 weeks GA

[222] 4.1 Imaging protocols. Pulmonary MRI measurements were performed in non- sedated, spontaneously breathing infants positioned in supine position in a vacuum mattress using a newly developed protocol for a 3-Tesla whole-body MRI scanner (Magnetom Skyra, Siemens Healthcare, Eriangen, Germany). MRI was performed with a size-adapted number of coil elements from the 32-channel spine array coil, an 18-channel flexible body array coil and the 20-channel head-and-neck array coil. The protocol included pulse sequences for the qualitative and quantitative assessment of morphology, volume, and structural changes of the lung. In detail, the following pulse sequences were used: i) T2-weighted single-shot fast- spin-echo (ssFSE) sequences in coronal, axial, and sagittal orientation; spatial resolution 1 .9x 1 .3x4.0 mm ³ , 20 slices with a field of view (FOV) of 340x255 mm ² . The echo time (TE) was 57 ms; the acquisitions were ECG-triggered with a minimum repetition time (TR) of 2 RR intervals; 2 signal averages were acquired, ii) Single-slice T2-mapping and T1 -mapping using ssFSE sequences (T2-mapping: TR = 2000 ms, TE = 26, 41 , 61 , 92 ms; T1 -mapping: TR/TE = 3000 ms/26 ms, Tl = 25, 150, 400, 800, 1600, 2600 ms and without inversion pulse) in coronal orientation (8 averages); spatial resolution: 2.3x2.3x20.0 mm ³ . FOV: 300x300 mm ² ; the slice was positioned in a representative central lung area. The total acquisition time of the three T2-weighted ssFSE sequences was about 5 minutes (depending on the cardiac rate of the infant); the total acquisition time of the T2 and T1 mapping sequences was also about 2 and 3 minutes, respectively. MRI measurements in the independent confirmation cohort followed the same protocol and were performed in lightly sedated, spontaneously breathing infants.

[223] 4.2 Image analysis. In order to ensure equal pulmonary imaging conditions, a consensus panel of experienced radiologists and neonatologists ruled out significant atelectasis, air leaks or pleural effusion. For T2- and T1 -mapping analysis, the lung was manually, virtually segmented into four lung quadrants (upper left, upper right, lower left, and lower right quadrant) using the software "Osirix MD" (version 6.5) (Rosset et al., J Digit Imaging. 2004; 17(3): 205-16). T2 and T1 relaxation time values were calculated by nonlinear exponential fitting and evaluated for these four lung quadrants and for the total lung. Free-breathing average total lung volume was measured by manual lung segmentation in axial and coronal acquisitions with the "editor" tool in the open-source software "3D Slicer" (version 4.3.1 r22599) (Fedorov et al., Magn Reson Imaging. 2012; 30(9): 1323-41 ); the left and right main bronchi were excluded; further exclusion of airways was limited by the small size of the segmented lungs. To reduce the influence of measurement errors, the arithmetic mean of the two volumes derived from axial and coronal images was used (sagittal slices were not analysed due to the lack of discernibility between tissues next to the mediastinum).

[224] 5. Infant Lung Function Testing (ILFT) in preterm infants at 36 weeks GA

[225] 5.1 Lung function protocol. ILFT was performed in parallel to MRI measurements at

36 weeks GA or immediately prior to hospital discharge. All studies were standardized according to the recommendations of the American Thoracic Society (ATS) and European

Respiratory Society (ERS) (Bates et al.,. Eur Respir J. 2000; 16(6): 1 180-92; Stocks et al.,

Eur Respir J. 2001 ; 17(2): 302-12). All infants were spontaneously breathing room air under light sedation (chloral hydrate, 30-40 mg/kg; orally) and had been free of respiratory infection for more than 3 weeks, presenting with normal blood gas values; inhalation therapy was terminated three days prior to the study. Body weight and crown-heel length was documented using a calibrated infant stadiometer. Oxygen saturation levels (Sp0 ₂ ; Masimo

Radical-7 pulse oximeter, Irvine, CA) and vital signs were monitored. Lung function measurements were recorded during relaxed quiet sleep in supine position using the Jaeger

MasterScreen BabyBody device (v4.6; CareFusion, San Diego, CA; Rendell-Baker Soucek face mask (size 0 or 1 ; Rusch UK Ltd., High Wycombe, UK), sealed with a rim of therapeutic putty. Measurements of tidal breathing, passive respiratory mechanics and functional residual capacity (FRC _P ) were performed before and 15 min after inhalation of salbutamol (1 .25 mg salbutamol/2.5 ml_ administered via PARI JuniorBOY N (PARI, Starnberg, Germany) to verify lung function on two different levels of airway condition. Total respiratory compliance (C _rs ), was assessed in single occlusion technique (SOT) using five to eight regular tidal breaths to establish a stable end-expiratory level (EEL) before activating the balloon shutter. FRC _P was measured as described previously (Stocks et al., Eur Respir J. 2001 ; 17(2): 302-12; Hulskamp et al., Pediatr Pulmonol. 2006; 41 (1 ): 1-22).

[226] 5.2 ILFT analysis. The following quality criteria were applied (Nguyen et al., Pediatr Pulmonol. 2012): For C _re and FRC _P a minimum of 5 technically satisfactory measurements were obtained and the mean of 3-5 valid measurements was calculated. The "within subject within-occasion coefficient of variability (CV)" was reported as [SD/mean] x 100.

[227] 6. Statistical Analysis

[228] For statistical analysis, protein measurements obtained from samples that did not pass the SOMAscan™ quality control were initially removed from analysis. Sample processing was found to be not biased by EDTA or potential hemolysis as shown previously (SOMA Scan™ Technical white paper 2014 SomaLogic IS-, Rev. 2 DCN 14-160). Principal component analysis (PCA) was done using the prcomp function within the R framework on the log-transformed data and showed significantly diverging overall expression patterns for the excluded samples. In a next step, confounder effects were subtracted from the protein expression patterns by applying lasso regression analysis to each measured protein expression profile including all clinical variables compiled in the study except for the disease outcomes. The corrected expression profile of a protein was then generated from the residuals of the regression model. A generalized additive model was fitted to the corrected expression profile of each protein including the disease grades as predictor variable as well as the patients modelled as random effects and the measurement time points. The resulting p-values for the association between disease and protein expression were finally corrected for multiple testing by the Benjamini-Hochberg procedure. Functional enrichment analysis was performed by applying Gene Set Enrichment Analysis (GSEA) (Subramanian et al., Proc Natl Acad Sci U S A. 2005; 102(43): 15545-50) on the corrected protein expression profiles using functional annotations from Gene Ontology (GO) (Ashburner et al., Nat Genet. 2000; 25(1 ): 25-9). A category was considered to be associated with altered protein expression caused by BPD were if the p-value of GSEA was less than 0.1. The Akaike information criterion (AIC) was used to estimate the quality of each model, i.e. model size, specificity and sensitivity, relative to each of the other models to provide a means for model selection and was used to identify the protein set best describing BPD. For visualization of the association between SIGLEC14 expression and disease grades, we subtracted the confounder effects of gestational age, gender, AIS, early onset infection, RDS and steroids from SIGLEC1 expression by including them into a linear model and calculated the residuals.

[229] MRI and lung function data were log2-transformed and missing values were initially imputed by sampling from a normal distribution with the sample mean and standard deviation of the observed values for each variable. A penalized regression analysis was used to identify MRI and lung function variables best describing the disease outcome. To model binary disease outcomes all MRI and lung function patterns described above were included in separate logistic regression models, respectively. For modelling count variables, i.e. days of oxygen supplementation and days of mechanical ventilation (MV), Poisson regression was used. By applying penalization in combination with leave-one-out cross-validation, MRI and lung function patterns could be identified describing the disease outcome best. High correlation among some predictor variables led to the usage of an elastic net approach (Zou et al., J R Statist Soc. 2005; 67(Part 2): 301-20) including both, L1 and L2 penalty, which were tuned according to the minimum prediction error. To account for confounder effects, clinical variables indicated in Table 1 were included in the statistical models, reflecting the panel of known risk factors for disease development (Bose et al., Arch Dis Child Fetal Neonatal Ed. 2008; 93: F455-F61 ; Bhandari et al., Semin Fetal Neonatal Med. 2010; 15: 223-9; Gortner et al., Neonatology. 201 1 ; 99: 1 12-7; Bhandari et al., Semin Perinatol. 2006; 30: 219-26; Gortner et al., Klin Padiatr. 2013; 225: 64-9).

[230] For validation of the main findings in the independent cohort we i) confirmed the imaging variables of interest by applying student's t-test and ii) fitted the protein expression data of the confirmation cohort in an individual logistic regression model as well as in the model established for the exploratory cohort and calculated the area under the receiver operating characteristic curve (ROC) in order to assess the predictive power of the respective model.

[231] Example 1 : BPD can be detected in infants within the first week of postnatal life by a specific biomarker profile

[232] In order to empirically identify plasma proteins that can serve as early markers of BPD in the first week of postnatal life, a non-hypothesis driven approach by the use of sophisticated proteomic screening in serial samples obtained from preterm infants with and without BPD was followed. 46 high quality samples from 18 patients were available for analysis in the exploration cohort (no/mild BPD (n=12), moderate/severe BPD (n= 6)), and 20 samples from 17 patients in the confirmation cohort (no/mild BPD (n=12), moderate/severe BPD (n=5)), respectively.

[233] Functional enrichment analysis showed overrepresentation of the following GO categories in infants with BPD: 'immune functionV'defense response' (GO:0006955), 'extracellular matrix' (ECM) (GO 030198) including 'peptidase activity (GO:0008233), 'cellular proliferation' (GO:0008284), 'cellular migration' (GO:0030334) and 'adhesion' (GO:0007155) as well as 'organ development' including the central nervous system (GO:0007399) and 'angiogenesis' (GO:0001525).

[234] Twelve proteins were found to be significantly regulated in infants later developing BPD when compared to preterm infants without BPD, reflecting the categories described by functional enrichment analysis above (Fig 1 A, p<0.1 ): FGF19 (FDR=0.062); SIGLEC-14 (FDR=0.062); TNFRSF19 (FDR=0.053); BCAM (FDR=0.062); COLEC12 (FDR=0.062); PES1 (FDR=0.062); EFNA 5 (FDR=0.062); EFNA 4 (FDR=0.096); HDGFRP2 (FDR=0.096); ANGPTL3 (FDR=0.096); EPAH2 (FDR=0.062) and SIGLEC-1 (FDR=0.062).

[235] AIC analysis revealed the combination of increased proteins levels for SIGLEC-14 and BCAM in combination with decreased levels for ANGPTL3 to describe the outcome variable 'BPD' with high specificity and sensitivity (Fig. 1 B). Out of these, increased levels of SIGLEC-14 were identified to best predict the outcome variable 'BPD' as a single marker by the use of both, statistical modelling and linear regression analysis with a characteristic expression in infants with mild, moderate and severe BPD in the first week of life (Fig. 1 C). The finding was confirmed using the count variable 'oxygen supplementation' and 'ventilator support' for statistical analysis. The expression of SIGLEC-14 was furthermore found to correlate with elevated T2 relaxation times (p<0.05), while an association with WBCs could not be observed.

[236] In follow up plasma samples, protein expression of all three markers, i.e. SIGLEC-14 (Fig. 1 D) and BCAM remained up-regulated at 28 days up to 32-36 weeks GA in preterm infants with BPD while the expression of ANGPTL3 stayed down-regulated in these patients.

[237] ELISA measurements for SIGLEC-14, BCAM and ANGPTL3 confirmed the findings outlined above in samples obtained from the independent validation cohort as a result of an individual logistic regression analysis (Figure 1 E) as well as the use of the model established in the exploratory cohort (Figure 1 F). [238] In order to confirm the expression of all three proteins in the lungs of preterm infants with BPD, valuable human tissue sections were employed and showed expression of all proteins in the lung's periphery of these infants (Figure 2). In more detail, all sections stained from preterm infants with BPD showed increased expression of SIGLEC-14 (Figure 2A) and BCAM (Figure 2B,) whereas ANGPTL3 (Figure 2C) expression was decreased as compared to the protein expression observed in the lung of a non-BPD infant (Figure 2A-C).

[239] Overall, characteristic protein expression levels for SIGLEC-14, BCAM and ANGPTL3 in plasma specimen were shown to indicate the development of BPD with high specificity and sensitivity. Out of these, increased levels of SIGLEC-14 were identified to best predict the outcome variable 'BPD' as a single marker in the first week of life. Expression levels remained stable until term, allowing for reliable identification of infants at risk for the disease. Biomarker expression in plasma samples was confirmed by IHC in the lungs of BPD infants

[240] Example 2: Advanced MRI analysis enables detection of characteristic structural changes in BPD lungs

[241] In order to identify structural characteristics associated with BPD in the preterm lung at the time of diagnosis, infants with and without BPD were investigated by advanced imaging protocols using MRI at 36 weeks GA. High-quality image and lung function data were available from 40 preterm infants in the exploration cohort, and from 21 infants in the confirmation cohort, respectively.

[242] Thereby characteristic structural pulmonary changes in premature infants with BPD at 36 weeks were identified by using a statistical model to identify the imaging variables best describing the outcome 'BPD' under consideration of a broad panel of clinical variables (Fig. 3A). The analysis revealed increased transverse relaxation times (T2) in characteristic regions of the right and left lung to be significantly associated with the diagnosis of BPD (Fig. 3B, C). In addition, an increase in lung volume was observed in infants with BPD (Fig. 3B). Together with the clinical variable GA, identified by the model to describe the outcome variable BPD, T2 relaxation times could unequivocally separate diseased from non-diseased infants (Fig. 3B).

[243] The analysis in an independent study cohort confirmed the results, showing a significant increase in T2 relaxation times in the group of infants with BPD when compared to results obtained from infants with no disease (Fig. 3D, p<0.01 ).

[244] When comparing moderate and severe BPD cases with results obtained from infants with no or mild BPD (Fig. 4A), increased T2 relaxation times were again confirmed to best describe the outcome variable BPD in characteristic regions of the right and left lung (Fig. 4B). In addition, the diagnosis of moderate or severe BPD was found to be significantly associated with decreased T1 relaxation times in characteristic regions of the lung (Fig. 4B, C).

[245] Statistical modelling with the count outcome variables 'days of oxygen' or 'days of MV (mechanical ventilation)' confirmed the results obtained by the nominal variable 'BPD'.

[246] Overall, increased T2 relaxation times were shown to identify diseased lungs in infants with BPD, reflecting enhancement of the signal derived from the interstitial tissue. In severe cases, increased T2 relaxation times are paralleled by shortening in T1 relaxation time, indicating emphysematous changes.

[247] Example 3: Lung function analysis reflects the structural changes in the lungs of infants with BPD

[248] In order to support the structural changes observed in BPD lungs by analysis of pulmonary function, standardized bodyplethysmographic measurements at 36 weeks GA were performed. Results from ILFT were available in all infants studied by MRI.

[249] Here, statistical analysis showed a significant correlation between MRI parameters and lung function variables (p<0.01 ). By the use of the statistical model described above, elevated FRC _P levels together with decreased levels for C _rs before and after inhalation were identified to best describe the outcome variable BPD in infants with moderate or severe disease as compared to the group of infants with no or mild BPD (Fig. 5A, 5B), supporting the functional relevance of the structural findings. Statistical modelling with the count variables 'days of oxygen' or 'days of MV confirmed the results.

[250] Overall, characteristic changes in lung function with elevated FRCp and decreased Crs were shown to be significantly associated with BPD in infants with moderate or severe disease as compared to the group of infants with no or mild BPD.

[251] Example 4: Proteome-wide affinity of aptamers to target proteins

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