US 20120225,047A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0225047 A1 Salonen (43) Pub. Date: Sep. 6, 2012

(54) NUTRIGENETIC BIOMARKERS FOR C40B 20/00 (2006.01) OBESITY AND TYPE 2 DABETES A638/17 (2006.01) A6IP3/10 (2006.01) (76) Inventor: Jukka T. Salonen, Helsinki (FI) A6IP3/04 (2006.01) C40B 40/06 (2006.01) (21) Appl. No.: 131509,903 CI2N 9/12 (2006.01) (22) PCT Filed: Nov. 16, 2010 (52) U.S. Cl...... 424/94.5:506/16:506/18: 530/350; 530/352:435/194; 514/6.9; 514/4.8: 506/2 (86). PCT No.: PCT/F10/50923 S371 (c)(1), (57) ABSTRACT (2), (4) Date: May 15, 2012 The present invention discloses , SNP markers and hap lotypes of Susceptibility or predisposition to obesity, type 2 (30) Foreign Application Priority Data diabetes (T2D) and subdiagnosis of obesity and T2D and related medical conditions. Particularly, the present invention Nov. 16, 2009 (FI) ...... 20096.188 provides T2D and obesity associated markers from SUCLA2. Methods for diagnosis, prediction of clinical Publication Classification course and efficacy of treatments for T2D, obesity and related (51) Int. Cl. phenotypes using polymorphisms in the risk genes and other A6 IK 38/45 (2006.01) related biomarkers are also disclosed. Kits are also provided C40B 40/10 (2006.01) for the diagnosis, selecting treatment and assessing prognosis C07K I4/47 (2006.01) of obesity and T2D. Patent Application Publication Sep. 6, 2012 Sheet 1 of 6 US 2012/0225,047 A1

Figure 1.

RS11792803 (AA+AG)

2OO 300 4OO 500 600 Carbohydrate intake (g)

RS11792803 (GG)

2OO 300 4OO 500 Carbohydrate intake (g) Patent Application Publication Sep. 6, 2012 Sheet 2 of 6 US 2012/0225,047 A1

Figure 2.

RS2847.666 (AA)

300 400 500 600 Glycemic load, cumulative per day

300 400 500 600 Glycemic load, cumulative per day Patent Application Publication Sep. 6, 2012 Sheet 3 of 6 US 2012/0225,047 A1

Figure 3.

RS10833641 (AA)

2OO 300 400 500 600 Carbohydrate intake (g)

RS10833641 (AC+CC)

2OO 300 400 Carbohydrate intake (g) Patent Application Publication Sep. 6, 2012 Sheet 4 of 6 US 2012/0225,047 A1

Figure 4.

RS17023900 (AA)

300 400 500 600 Glycemic load, Cumulative per day

400 500 600 Glycemic load, Cumulative per day Patent Application Publication Sep. 6, 2012 Sheet 5 of 6 US 2012/0225,047 A1

Figure 5.

RS3731572 (AA)

40 50 60 Glycemic index, cumulative per day

RS3731572 (AG)

40 50 60 70 Glycemic index, cumulative per day Patent Application Publication Sep. 6, 2012 Sheet 6 of 6 US 2012/0225,047 A1

i. 6 3. o O US 2012/0225,047 A1 Sep. 6, 2012

NUTRGENETIC BOMARKERS FOR brovascular stroke, and cancers of the breast, endometrium, OBESITY AND TYPE 2 DABETES prostate and colon (Burton & Foster 1985). 0006. The high prevalence of obesity, its significant con BACKGROUND OF THE INVENTION tribution to morbidity and mortality of several common chronic diseases and lack of obesity related biomarkers and 0001. Obesity risk assessment tests show unmet medical need both for obe sity related biomarkers as well as diagnostic methods and 0002 Obesity is an excessive accumulation of energy in kits. The present invention provides a number of new rela the form of body fat which impairs health. As the direct tionships between various polymorphic alleles and common measurement of body fat is difficult, Body Mass Index (BMI), obesity. Obesity associated biomarkers disclosed in this a simple ratio of weight to the square of height (kg/m), is invention provide the basis for improved risk assessment, typically used to classify overweight (BMD-25) and obese more detailed diagnosis and prognosis of obesity. (BMA-30) adults (Table 1). Consistent with this, the WHO has published international standards for classifying over 0007. Type 2 Diabetes weight and obesity in adults. The three major classes of obe 0008. The term diabetes mellitus (DM) (ICD/10 codes sity are monogenic, syndromic and polygenic obesity (or E10-E14) describes several syndromes of abnormal carbohy common obesity). Monogenic obesity is caused by a single drate metabolism that are characterized by hyperglycemia. It dysfunctional gene and is typically familial, rare and severe is associated with a relative or absolute impairment in insulin form of obesity. Syndromic obesity is also rare and severe secretion, along with varying degrees of peripheral resistance obesity form and there are about 30 Mendelian disorders, in to the action of insulin. The chronic hyperglycemia of diabe which patients are clinically obese and have mental retarda tes is associated with long-term damage, dysfunction, and tion, dysmorphic features and organ-specific developmental failure of various organs, especially the eyes, kidneys, nerves, abnormalities. Polygenic obesity is a complex, multi-facto heart, and blood vessels (ADA, 2003). According to the new rial chronic disease involving environmental (Social and cul etiologic classification of DM, four categories are differenti tural), genetic, physiologic, metabolic, behavioral and psy ated: type 1 diabetes (T1D), type 2 diabetes (T2D), other chological components and numerous genes seem to specific types, and gestational diabetes mellitus (ADA, 2003). contribute to the obesity phenotype (Mutch and Clement, 0009. In T1D, formerly known as insulin-dependent 2006). (IDDM), the pancreas fails to produce the insulin which is essential for survival. This form develops most frequently in TABLE 1. children and adolescents, but is being increasingly diagnosed WHO Classification of Obesity later in life. T2D, formerly named non-insulin-dependent (NIDDM), results from the body's inability to respond prop WHO Popular BMI erly to the action of insulin produced by the pancreas. T2D Classification Description (kg/m) Risk of co-morbidities occurs most frequently in adults, but is being noted increas Underweight Thin <18.5 Low (but risk of other ingly in adolescents as well (WHO, 2004). It is the common clinical problems est form of diabetes mellitus accounting for 90% of all cases increased) Normal range Normal 18.5-24.9 Average worldwide. Overweight >25.0 0010 Relationship Between Nutrition, Genes and Health Pre-obese Overweight 25-29.9 Increased Obese Class I Obese 30.0-34.9 Moderate 0011 Inter-individual genetic variation is a critical deter Obese Class II Obese 350-39.9 Sewere minant of differences in nutrient requirements. The common Obese Class III Morbidly Obese >40.0 Very severe est type of genetic variability is the single poly morphism (SNP), a single base substitution within the DNA sequence. These occur roughly once every 200 to 300 nucle 0003. Although obesity is not a recent phenomenon as the otides in the . Several genetic polymorphisms historical roots of obesity can be traced back to 25,000 years of importance to nutrition have been identified. As more such ago, the epidemic of obesity is a recent global health issue links between polymorphisms and disease conditions are across all age groups, especially in industrialized countries characterized, the scope for targeting dietary information and (American Obesity Association, 2006). According to WHO's recommendations to specific Subpopulations will increase. estimate there are more than 300 million obese people 0012 Nutrigenetics aims to understand how the genetic (BMD-30) world-wide. makeup of an individual determines or contributes to their 0004 Today, for example almost 65% of adult Americans response to diet, and thus considers underlying genetic poly (about 127 million) are categorized as being overweight or morphisms. It is the Science of identifying and characterizing obese. There is also evidence that obesity is increasing prob gene variants associated with differential responses to nutri lem among children, for example in the USA, the percentage ents, and relating this variation to disease states. Nutrigenet of overweight children (aged 5-14 years) has doubled in the ics will yield critically important information that will assist last 30 years, from 15% to 32%. clinicians and nutritionists in identifying the optimal diet for 0005. The degree of health impairment of obesity is deter a given individual, i.e. personalized nutrition. mined by three factors: 1) the amount offat 2) the distribution 0013 SNPs are important in explaining some of the varia offat and 3) the presence of other risk factors. Obesity is the tions in response to food components. Specific genetic poly second leading cause of preventable death in the U.S. It morphisms in humans change their metabolic responses to affects all major bodily systems—heart, lung, muscle and diet and other therapies and can have an important effect on bones—and is considered as a major risk factor for several disease risk. Inter-individual genetic variation is also a crucial chronic disease conditions, including coronary heart disease determinant of differences in nutrient requirements and tol (CHD), type 2 diabetes mellitus (T2D), hypertension, cere erances to nutrients. US 2012/0225,047 A1 Sep. 6, 2012

0014. It is already apparent that there are many polymor 0020 Still another object of the invention is to provide a phisms that influence risk of nutrition-related chronic dis method for prediction of clinical course, and efficacy and eases like obesity and type 2 diabetes. SNP analysis provides safety of therapeutic method(s) with current weight reduction a molecular tool for investigating the role of nutrition in and antidiabetic foods and other therapies for T2D using human health and disease, and their consideration in clinical, polymorphisms in the genes associated with Such response. metabolic and epidemiological studies and genetic screening can contribute enormously to the definition of optimal diets. 0021. Another object of the invention is providing novel 0015 The present invention is especially directed to pathways to elucidate the presently unknown modes of action genetic markers such as SNPs of gene SUCLA2. The prior art of known antiobesity and antidiabetic foods and diets. A such as Feitosa et al. (Diabetes. 2009:58(suppl 1):A304) dis major object of the invention are gene networks influencing closes that SUCLA2 is associated with waist/hip ratio and individual's response to a method of therapy with insulin that there is strong evidence that SUCLA2 is involved in the secretors or insulin sensitizers or insulin are presented. Such complex genetic architecture of coronary heart disease. How gene networks can be used for other methods of the invention ever, no disclosure of particular SNPs relating to T2D or comprising diagnostic methods for prediction of the response obesity is found in Feitosa et al. to a particular food, the efficacy and safety of aparticular food described herein and the treatment methods described herein. SUMMARY OF THE INVENTION 0022 Kits are also provided for the selection, prognosis 0016. This invention describes novel genes and markers and monitoring of the method of therapy for obesity and T2D. which are associated with individual's response to a method Better means for identifying those individuals who will ben of therapy Such as a known food, functional or non-functional efit more from the selected method of therapy for obesity or or diet or dietary pattern or Small molecule medicine or a T2D due to the better response and long-term glycemic con biological therapeutic product. It presents novel examples of trol and fewer adverse effects should lead to better preventive nutrigenetics for common traits such as obesity, type 2 dia and treatment regimens. Nutrigenetic information may be betes (T2D) and a T2D related condition. used to assist physician in choosing method of therapy for the 0017. This invention relates to genes and biomarkers asso particular patient ("personalized medicine'). ciated with a response to a method of therapy in weight 0023. In summary, the invention helps meet the unmet reduction and diabetes and their use in the treatment and medical needs and promotes public health in at least two prevention of obesity, T2D and a T2D related condition such major ways: 1) it provides novel means to predict individual’s as metabolic syndrome, insulin resistance, glucose intoler response and evaluate safety and efficiency of a selected ance, and T2D complications such as retinopathy, nephropa method of therapy with known weight reducing or antidia thy or neuropathy, coronary heart disease, cerebrovascular betic food or diet, as well as select the significant suitable disease, congestive heart failure, intermittent claudication or alternative method of antiobesity or antidiabetic therapy for other manifestations of arteriosclerosis. The present inven the individual (“personalized medicine') and 2) it provides tion provides novel genes and individual SNP markers asso functional food and other therapeutic targets that can be used ciated with a response to antiobesity and antidiabetic foods, further to screen and develop functional foods and other diets and other therapies. The invention further relates to therapeutic agents and therapies that can be used alone or in physiological and biochemical routes and pathways related to combination with the known antiobesity and antidiabetic these genes. therapies to treat, prevent or ameliorate the symptoms, sever 0018. The present invention relates to previously ity or progression of obesity and T2D or a T2D related con unknown associations between various genes, loci and biom dition in a given individual. arkers, and obesity and T2D. The detection of these biomar 0024. Accordingly in a first aspect, the present invention kers provides novel methods and systems for risk assessment provides methods and kits for diagnosing a susceptibility to and diagnosis of obesity, which will also improve risk assess high energy, carbohydrate or fat intake in an individual. The ment, diagnosis and prognosis of obesity related conditions methods comprise the steps of: (i) obtaining a biological comprising type 2 diabetes, diabetic complications, coronary sample from the individual, and (ii) detecting in the biological artery disease, myocardial infarction, stroke and hyperten sample the presence of one or more obesity and/or T2D Sion. associated biomarkers. These biomarkers may be SNP mark 0019. The major application of the current invention is its ers selected from Tables 6 through 17 of the invention or other use to predict an individual's response to a particular weight biomarkers of the genes that they are associated with Such as reducing or antidiabetic food/method of therapy. It is a well expressed RNA or or metabolites of the protein. The known phenomenon that in general, patients do not respond presence of obesity associated biomarkers in Subject's sample equally to the same food or method of therapy. Much of the is indicative of a susceptibility to high energy, carbohydrate differences in the response to a given food are thought to be or fat intake. The kits provided for diagnosing a Susceptibility based on genetic and protein differences among individuals in to high energy, carbohydrate or fat intake in an individual certain genes and their corresponding pathways. Our inven comprise wholly or in part protocol and reagents for detecting tion defines the genes associated with a response to known one or more biomarkers and interpretation Software for data method(s) of therapy in obesity, T2D and related conditions. analysis and risk assessment. In one embodiment of this Therefore, genes and gene variations which are the Subject of invention SNP markers being in linkage disequilibrium with current invention may be used as a nutrigenetic diagnostic to one or more SNP markers of this invention are used in meth predicta response to a method of therapy and guide choice of ods and kits for diagnosing a Susceptibility to obesity. In other method(s) of therapy for treating, preventing or ameliorating embodiment metabolites, expressed RNA molecules or the symptoms, severity or progression of obesity and T2D or expressed polypeptides, which are associated with one or a T2D related condition in a given individual ("personalized more SNP markers of this invention are used in disclosed nutrition”, “personalized prevention”). methods and kits. US 2012/0225,047 A1 Sep. 6, 2012

0025. In one typical embodiment, the biomarker informa ecule which is related to a SNP marker set forth in Tables 6 tion obtained from the methods diagnosing a Susceptibility of through 17 and which is differentially present in samples an individual to high energy, carbohydrate or fat intake are taken from Subjects (patients) being obese compared to com combined with other information concerning the individual, parable samples taken from Subjects who are non-obese e.g. results from blood measurements, clinical examination, (BMI-30). An “organic biomolecule' refers to an organic questionnaires and/or interviews. molecule of biological origin comprising steroids, amino 0026. In one embodiment, the methods and kits of the acids, , Sugars, polypeptides, polynucleotides, invention are used in early diagnosis of obesity or T2D at or complex carbohydrates and lipids. A biomarker is differen before onset, thus reducing or minimizing the debilitating tially present between two samples if the amount, structure, effects of these conditions. In a preferred embodiment the function or biological activity of the biomarker in one sample methods and kits are applied in individuals who are free of differs in a statistically significant way from the amount, clinical symptoms and signs of obesity and/or T2D, but have structure, function or biological activity of the biomarker in family history of obesity and/or T2D or in those who have the other sample. multiple risk factors for obesity. 0036. A “haplotype.” as described herein, refers to a com 0027. In a second aspect, the present invention provides bination of genetic markers (“alleles'). A haplotype can com methods and kits for molecular diagnosis i.e. determining a prise two or more alleles and the length of a genome region molecular subtype of obesity in an individual. In one pre comprising a haplotype may vary from few hundred bases up ferred embodiment, molecular subtype of obesity in an indi to hundreds of kilobases. As it is recognized by those skilled vidual is determined to provide information of the molecular in the art the same haplotype can be described differently by etiology of obesity. When the molecular etiology is known, determining the haplotype defining alleles from different better diagnosis and prognosis of obesity can be made and nucleic acid strands. E.g. the haplotype GGC defined by the efficient and safe therapy for treating obesity in an individual SNP markers rs3936203, rs10933514 and rs4630763 of this can be selected on the basis of this subtype information. For invention is the same as haplotype rs393.6203, rs10933514, example, the food or other therapy that is likely to be effec and rs4630763 (CCG) in which the alleles are determined tive, can be selected without trial and error. In other embodi from the other strand, or haplotype rs393.6203, rs10933514, ment, biomarker information obtained from methods and kits and rs4630763 (CGC), in which the first allele is determined for determining molecular subtype of obesity in an individual from the other strand. The haplotypes described herein are is for monitoring the effectiveness of obesity treatment. In differentially present in individuals with obesity than in indi one embodiment, methods and kits for determining molecu viduals without obesity. Therefore, these haplotypes have lar subtype of obesity are used to select human subjects for diagnostic value for risk assessment, diagnosis and prognosis clinical trials testing efficacy of obesity therapies. The kits of obesity in an individual. Detection of haplotypes can be provided for diagnosing a molecular subtype of obesity in an accomplished by methods known in the art used for detecting individual comprise wholly or in part protocol and reagents nucleotides at polymorphic sites. Haplotypes found more for detecting one or more biomarkers and interpretation soft frequently in obese individuals (risk increasing haplotypes) ware for data analysis and obesity molecular Subtype assess as well as haplotypes found more frequently in non-obese ment. individuals (risk reducing haplotypes) have predictive value for predicting Susceptibility to obesity in an individual. BRIEF DESCRIPTION OF THE DRAWINGS 0037. A nucleotide position in genome at which more than 0028 FIG. 1. Linear regression between carbohydrate one sequence is possible in a population, is referred to herein intake and BMI in genotypes of RS11792803. as a “polymorphic site' or “polymorphism'. Where a poly 0029 FIG. 2. Linear regression between glycemic load morphic site is a single nucleotide in length, the site is referred and BMI for RS2847.666. to as a SNP. For example, if at a particular chromosomal 0030 FIG. 3. Linear regression between carbohydrate location, one member of a population has an adenine and intake and WHR in RS10833641 genotypes. another member of the population has a thymine at the same 0031 FIG. 4. Linear regression between glycemic load position, then this position is a polymorphic site, and, more and WHR in RS17023900 genotypes. specifically, the polymorphic site is a SNP. Polymorphic sites 0032 FIG. 5. Linear regression between glycemic index may be several nucleotides in length due to insertions, dele tions, conversions or translocations. Each version of the and WHR in RS373.1572 genotypes. sequence with respect to the polymorphic site is referred to 0033 FIG. 6. Linear regression between soluble carbohy herein as an “allele of the polymorphic site. Thus, in the drate intake (g/d) and BMI in RS16884.072 A/G and G/G previous example, the SNP allows for both an adenine allele genotypes. and a thymine allele. DETAILED DESCRIPTION OF THE INVENTION 0038. Typically, a reference nucleotide sequence is referred to for a particular gene e.g. in NCBI databases (www. 0034. The present invention relates to previously incbi.nlm.nih.gov). Alleles that differ from the reference are unknown associations between high energy, carbohydrate or referred to as “variant' alleles. The polypeptide encoded by fat intake, obesity and various biomarkers. These novel obe the reference nucleotide sequence is the “reference' polypep sity biomarkers provide basis for novel methods and kits for tide with a particular reference sequence, and risk assessment and diagnosis of obesity and obesity related polypeptides encoded by variant alleles are referred to as conditions. “variant' polypeptides with variant amino acid sequences. 0035. A “biomarker' in the context of the present inven Nucleotide sequence variants can result in changes affecting tion refers to a SNP marker disclosed in Tables 6 through 17 properties of a polypeptide. These sequence differences, or to a polymorphism which is in linkage disequilibrium with when compared to a reference nucleotide sequence, include one or more disclosed SNP markers, or to an organic biomol insertions, deletions, conversions and Substitutions: e.g. an US 2012/0225,047 A1 Sep. 6, 2012

insertion, a deletion or a conversion may resultina frame shift either equally useful as obesity biomarkers or even more generating an altered polypeptide; a Substitution of at least useful as causative variations explaining the observed obesity one nucleotide may result in a premature stop codon, amino association of SNP markers of this invention. acid change or abnormal mRNA splicing; the deletion of 0042. The term “gene.” as used herein, refers to an entirety several nucleotides, resulting in a deletion of one or more containing entire transcribed region and all regulatory regions amino acids encoded by the nucleotides; the insertion of of a gene. The transcribed region of a gene including all several nucleotides, such as by unequal recombination or and intron sequences of a gene including alternatively spliced gene conversion, resulting in an interruption of the coding and introns so the transcribed region of a gene contains sequence of a reading frame; duplication of all or a part of a in addition to polypeptide encoding region of a gene also sequence; transposition; or a rearrangement of a nucleotide regulatory and 5' and 3' untranslated regions present in tran sequence, as described in detail above. Such sequence scribed RNA. Each gene has been assigned a specific and changes alter the polypeptide encoded by an obesity Suscep unique nucleotide sequence by the Scientific community. By tibility gene. For example, a nucleotide change resulting in a using the name of a gene those skilled in the art will readily change in polypeptide sequence can alter the physiological find the nucleotide sequences of the corresponding gene and properties of a polypeptide dramatically by resulting in it's encoded mRNAS as well as amino acid sequences of it's altered activity, distribution and stability or otherwise affect encoded polypeptides although some genes may have been on properties of a polypeptide. Alternatively, nucleotide known with other name(s) in the art. sequence variants can result in changes affecting transcrip 0043. In certain methods described herein, an individual tion of a gene or translation of its mRNA. A polymorphic site who has increased risk for developing obesity is an individual located in a regulatory region of a gene may result in altered in whom one or more obesity associated polymorphisms transcription of a gene e.g. due to altered tissue specificity, selected from Tables 6 through 17 of this invention are iden altered transcription rate or altered response to transcription tified. In other embodiment also polymorphisms associated to factors. A polymorphic site located in a region corresponding one or more SNPs set forth in Tables 6 through 17 may be to the mRNA of a gene may result in altered translation of the used in risk assessment of obesity. The significance associ mRNA e.g. by inducing stable secondary structures to the ated with an alleleora haplotype is measured by an odds ratio. mRNA and affecting the stability of the mRNA. Such In a further embodiment, the significance is measured by a sequence changes may alter the expression of an obesity percentage. In one embodiment, a significant risk is measured Susceptibility gene. as odds ratio of 0.8 or less or at least about 1.2, including by 0039. The SNP markers to which we have disclosed novel not limited to: 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 1.2, 1.3, 1.4, obesity associations in Tables 6 through 17 of this invention 1.5, 1.6, 17, 1.8, 19, 2.0, 2.5, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, have been known in prior art with their official reference SNP 25.0, 30.0 and 40.0. In a further embodiment, a significant (rs) ID identification tags assigned to each unique SNP by the increase or reduction in risk is at least about 20%, including National Center for Biotechnological Information (NCBI). but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, Each rSID has been linked to specific variable alleles present 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. in a specific nucleotideposition in the human genome, and the In a further embodiment, a significant increase in risk is at nucleotide position has been specified with the nucleotide least about 50%. It is understood however, that identifying sequences flanking each SNP. For example the SNP having rs whether a risk is medically significant may also depend on a ID rS4737191 is SNP in 8, and variable alleles variety of factors such as subject's family history of obesity, are C and T. previously identified glucose intolerance, hypertriglyceri 0040 Although the numerical chromosomal position of a demia, hypercholesterolemia, elevated LDL cholesterol, low SNP may still change upon annotating the current human HDL cholesterol, elevated BP hypertension, cigarette Smok genome build the SNP identification information such as ing, lack of physical activity, and inflammatory components variable alleles and flanking nucleotide sequences assigned to as reflected by increased C-reactive protein levels or other a SNP will remain the same. Those skilled in the art will inflammatory markers. readily recognize that the analysis of the nucleotides present 0044) “Probes’ or “primers' are oligonucleotides that in one or more SNPs set forth in Tables 6 through 17 of this hybridize in a base-specific manner to a complementary invention in an individual's nucleic acid can be done by any strand of nucleic acid molecules. By “base specific manner method or technique capable of determining nucleotides is meant that the two sequences must have a degree of nucle present in a polymorphic site using the sequence information otide complementarity sufficient for the primer or probe to assigned in prior art to thers IDs of the SNPs listed in Tables hybridize to its specific target. Accordingly, the primer or 6 through 17 of this invention. As it is obvious in the art the probe sequence is not required to be perfectly complementary nucleotides present in polymorphisms can be determined to the sequence of the template. Non-complementary bases or from either nucleic acid strand or from both strands. modified bases can be interspersed into the primer or probe, 0041. It is understood that the obesity associated SNP provided that base substitutions do not inhibit hybridization. markers described in Tables 6 through 17 of this invention The nucleic acid template may also include “non-specific may be associated with other polymorphisms. This is because priming sequences' or “nonspecific sequences to which the the SNP markers listed in Tables 6 through 17 are so called primer or probe has varying degrees of complementarity. tagging SNPs (tagSNPs). TagSNPs are loci that can serve as Probes and primers may include modified bases as in proxies for many other SNPs. The use of tagSNPs greatly polypeptide nucleic acids (Nielsen PE et al., 1991). Probes or improves the power of association studies as only a Subset of primers typically comprise about 15, to 30 consecutive nucle loci needs to be genotyped while maintaining the same infor otides present e.g. in human genome and they may further mation and power as if one had genotyped a larger number of comprise a detectable label, e.g., radioisotope, fluorescent SNPs. These other polymorphic sites associated with the SNP compound, , or enzyme co-factor. Probes and primers markers listed in Tables 6 through 17 of this invention may be to aSNP marker disclosed in Tables 6 to 17 are available in the US 2012/0225,047 A1 Sep. 6, 2012

art or can easily be designed using the flanking nucleotide clinical risk factors to define an individual's risk relative to the sequences assigned to a SNPrs ID and standard probe and general population. Better means for identifying those indi primer design tools. Primers and probes for SNP markers viduals susceptible for obesity should lead to better preven disclosed in Tables 6 through 17 can be used in risk assess tive and treatment regimens, including more aggressive man ment as well as molecular diagnostic methods and kits of this agement of the risk factors related to obesity and related invention. diseases e.g. physicians may use the information on genetic 0045. The invention comprises polyclonal and mono risk factors to convince particular patients to adjust their life clonal antibodies that bind to a polypeptide related to one or style e.g. to stop Smoking, to reduce caloric intake and to more obesity associated SNP markers set forth in Tables 6 increase exercise. through 17 of the invention. The term “antibody” as used 0050. In one embodiment of the invention, diagnosing a herein refers to immunoglobulin molecules or their immuno Susceptibility to obesity in a subject, is made by detecting one logically active portions that specifically bind to an epitope or more SNP markers disclosed in Tables 6 through 17 of this (antigen, antigenic determinant) present in a polypeptide or a invention in the subject's nucleic acid. The presence of obe fragment thereof, but does not substantially bind other mol sity associated alleles of the assessed SNP markers (and hap ecules in a sample, e.g., a biological sample, which contains lotypes) in individual's genome indicates Subject's increased the polypeptide. Examples of immunologically active por risk for obesity. The invention also pertains to methods of tions of immunoglobulin molecules include F(ab) and F(ab') diagnosing a Susceptibility to obesity in an individual com fragments which can be generated by treating the antibody prising detection of a haplotype in an obesity risk gene that is with an enzyme Such as pepsin. The term "monoclonal anti more frequently present in an individual being obese (af body” as used herein refers to a population of antibody mol fected), compared to the frequency of its presence in a healthy ecules that are directed against a specific epitope and are non-obese individual (control), wherein the presence of the produced either by a single clone of B cells or a single hybri haplotype is indicative of a susceptibility to obesity. A hap doma cell line. Polyclonal and monoclonal antibodies can be lotype may be associated with a reduced rather than increased prepared by various methods known in the art. Additionally, risk of obesity, wherein the presence of the haplotype is recombinant antibodies, such as chimeric and humanized indicative of a reduced risk of obesity. In other embodiment of monoclonal antibodies, comprising both human and non the invention, diagnosis of susceptibility to obesity is done by human portions, can be produced by recombinant DNA tech detecting in the Subject's nucleic acid one or more polymor niques known in the art. Antibodies can be coupled to various phic sites being in linkage disequilibrium with one or more , prosthetic groups, fluorescent materials, lumines SNP markers and disclosed in Tables 6 through 17 of this cent materials, bioluminescent materials, or radioactive invention. Diagnostically the most useful polymorphic sites materials to enhance detection. are those altering the biological activity of a polypeptide 0046. An antibody specific for a polypeptide related to one related to one or more obesity associated SNP markers set or more obesity associated SNP markers set forth in Tables 6 forth in Tables 6 through 17. Examples of such functional through 17 of the invention can be used to detect the polypep polymorphisms include, but are not limited to frame shifts, tide in a biological sample in order to evaluate the abundance premature stop codons, amino acid changing polymorphisms and pattern of expression of the polypeptide. Antibodies can and polymorphisms inducing abnormal mRNA splicing. be used diagnostically to monitor protein levels in tissue Such Nucleotide changes resulting in a change in polypeptide as blood as part of a test predicting the Susceptibility to sequence in many cases alter the physiological properties of a obesity or as part of a clinical testing procedure, e.g., to, for polypeptide by resulting in altered activity, distribution and example, determine the efficacy of a given treatment regimen. stability or otherwise affect the properties of a polypeptide. 0047 “An obesity related condition' in the context of this Other diagnostically useful polymorphic sites are those invention comprises type 2 diabetes, coronary artery disease, affecting transcription of a gene or translation of it's mRNA myocardial infarction, stroke, hypertension, dyslipidaemias due to altered tissue specificity, due to altered transcription and metabolic syndrome. A T2D related condition' in the rate, due to altered response to physiological status, due to context of this invention comprises metabolic syndrome, altered translation efficiency of the mRNA and due to altered insulin resistance, glucose intolerance, and T2D complica stability of the mRNA. Thus presence of nucleotide sequence tions such as retinopathy, nephropathy or neuropathy, coro variants altering the polypeptide structure and/or expression nary heart disease, cerebrovascular disease, congestive heart rate of a gene related to one or more obesity associated SNP failure, intermittent claudication or another manifestation of markers set forth in Tables 6 through 17 of this invention in arteriosclerosis. As Obesity is the most important risk factor individual's nucleic acid is diagnostic for Susceptibility to and predursor of T2D, all examples and applications obesity. described in this invention concern, in addition to obesity, 0051. In diagnostic assays determination of the nucle also T2D and T2D related conditions. otides present in one or more obesity associated SNP markers 0048 Diagnostic Methods and Test Kits disclosed in this invention in an individual's nucleic acid can 0049. One major application of the current invention is be done by any method or technique which can accurately diagnosing a Susceptibility to obesity. The risk assessment determine nucleotides present in a polymorphic site. Numer methods and test kits of this invention can be applied to any ous suitable methods have been described in the art (see e.g. healthy person as a screening or predisposition test, although Kwok P-Y. 2001; Syvanen A-C, 2001), these methods the methods and test kits are preferably applied to high-risk include, but are not limited to, hybridization assays, ligation individuals (subjects who have e.g. family history of obesity, assays, primer extension assays, enzymatic cleavage assays, type 2 diabetes or hypertension, or previous glucose intoler chemical cleavage assays and any combinations of these ance or elevated level of any other obesity risk factor). Diag assays. The assays may or may not include PCR, Solid phase nostic tests that define genetic factors contributing to obesity step, a microarray, modified oligonucleotides, labeled probes might be used together with or independent of the known or labeled nucleotides and the assay may be multiplex or US 2012/0225,047 A1 Sep. 6, 2012

singleplex. As it is obvious in the art the nucleotides present in Tables 6 through 17. Status and/or function of a biological a polymorphic site can be determined from either nucleic acid network and/or a metabolic pathway can be assessed e.g. by strand or from both strands. measuring amount or composition of one or several polypep 0052. In another embodiment of the invention, a suscepti tides or metabolites belonging to the biological network and/ bility to obesity is assessed from transcription products or to the metabolic pathway from a biological sample taken related to one or more obesity associated SNP markers set from a subject. Risk to develop obesity is evaluated by com forth in Tables 6 through 17 of this invention. Qualitative or paring observed status and/or function of biological networks quantitative alterations in transcription products can be and or metabolic pathways of a subject to the status and/or assessed by a variety of methods described in the art, includ function of biological networks and or metabolic pathways of ing e.g. hybridization methods, enzymatic cleavage assays, healthy and obese subjects. RT-PCR assays and microarrays. A test sample from an indi 0055 Another major application of the current invention vidual is collected and the said transcription products are is diagnosis of a molecular Subtype of obesity in a Subject. assessed from RNA molecules present in the test sample and Molecular diagnosis methods and kits of this invention can be the result of the test sample is compared with results from applied to a person being obese. In one preferred embodi obese subjects (affected) and healthy non-obese subjects ment, molecular subtype of obesity in an individual is deter (control) to determine individual's susceptibility to obesity. mined to provide information of the molecular etiology of 0053. In another embodiment of the invention, diagnosis obesity. When the molecular etiology is known, better diag of a Susceptibility to obesity is made by examining expres nosis and prognosis of obesity can be made and efficient and Sion, abundance, biological activities, structures and/or func safe therapy for treating obesity in an individual can be tions of polypeptides related to one or more obesity associ selected on the basis of this subtype information. Physicians ated SNP markers disclosed in Tables 6 through 17 of this may use the information on genetic risk factors with or with invention. A test sample from an individual is assessed for the out known clinical risk factors to convince particular patients presence of alterations in the expression, biological activities, to adjust their life style and manage obesity risk factors and structures and/or functions of the polypeptides, or for the select intensified preventive and curative interventions for presence of a particular polypeptide variant (e.g., an isoform) them. In other embodiment, biomarker information obtained related to one or more obesity associated SNP markers set from methods and kits for determining molecular subtype of forth in Tables 6 through 17 of this invention. An alteration obesity in an individual is for monitoring the effectiveness of can be, for example, quantitative (an alteration in the quantity their treatment. In one embodiment, methods and kits for of the expressed polypeptide, i.e., the amount of polypeptide determining molecular subtype of obesity are used to select produced) or qualitative (an alteration in the structure and/or human subjects for clinical trials testing obesity foods. The function of a polypeptide i.e. expression of a mutant polypep kits provided for diagnosing a molecular Subtype of obesity in tide or of a different splicing variant or isoform). Alterations an individual comprise wholly or in part protocol and in expression, abundance, biological activity, structure and/or reagents for detecting one or more biomarkers and interpre function of a obesity susceptibility polypeptide can be deter tation Software for data analysis and obesity molecular Sub mined by various methods known in the art e.g. by assays type assessment. based on chromatography, spectroscopy, colorimetry, elec 0056. The diagnostic assays and kits of the invention may trophoresis, isoelectric focusing, specific cleavage, immuno further comprise a step of combining non-genetic informa logic techniques and measurement of biological activity as tion with the biomarker data to make risk assessment, diag well as combinations of different assays. An “alteration' in nosis or prognosis of obesity. Useful non-genetic information the polypeptide expression or composition, as used herein, comprises age, gender, Smoking status, physical activity, refers to an alteration in expression or composition in a test waist-to-hip circumference ratio (cm/cm), the Subject family sample, as compared with the expression or composition in a history of obesity, previously identified glucose intolerance, control sample and an alteration can be assessed either hypertriglyceridemia, low HDL cholesterol, HT and elevated directly from the polypeptide itself or its fragment or from BP. The detection method of the invention may also further Substrates and reaction products of said polypeptide. A con comprise a step determining blood, serum or plasma glucose, trol sample is a sample that corresponds to the test sample total cholesterol, HDL cholesterol, LDL cholesterol, triglyc (e.g., is from the same type of cells), and is from an individual eride, apolipoprotein B and AI, fibrinogen, ferritin, transfer who is not affected by obesity. An alteration in the expression, rin receptor, C-reactive protein and insulin concentration. abundance, biological activity, function or composition of a 0057 The score that predicts the probability of developing polypeptide related to one or more obesity associated SNP obesity may be calculated e.g. using a multivariate failure markers set forth in Tables 6 through 17 of this invention in time model or a logistic regression equation. The results from the test sample, as compared with the control sample, is the further steps of the method as described above render indicative of a susceptibility to obesity. In another embodi possible a step of calculating the probability of obesity using ment, assessment of the splicing variant or isoform(s) of a a logistic regression equation as follows. Probability of obe polypeptide encoded by a polymorphic or mutant gene sity=1/1+e (-(-a+X(biXi)), where e is Napier's constant, related to one or more obesity associated SNP markers set Xi are variables related to the obesity, bi are coefficients of forth in Tables 6 through 17 of this invention can be per these variables in the logistic function, and a is the constant formed directly (e.g., by examining the polypeptide itself), or term in the logistic function, and whereina and bi are prefer indirectly (e.g., by examining the mRNA encoding the ably determined in the population in which the method is to be polypeptide. Such as through mRNA profiling). used, and Xi are preferably selected among the variables that 0054 Yet in another embodiment, a susceptibility to obe have been measured in the population in which the method is sity can be diagnosed by assessing the status and/or function to be used. Preferable values for b, are between -20 and 20: of biological networks and/or metabolic pathways related to and for i between 0 (none) and 100,000. A negative coefficient one or more obesity associated SNP markers disclosed in b, implies that the marker is risk-reducing and a positive that US 2012/0225,047 A1 Sep. 6, 2012 the marker is risk-increasing. Xi are binary variables that can 0063 c) comparing the genetic marker data from the sub have values or are coded as 0 (zero) or 1 (one) such as SNP ject to genetic marker data from healthy and diseased people markers. The model may additionally include any interaction to make risk assessment, diagnosis or prognosis of obesity or (product) or terms of any variables Xi, e.g. biXi. An algorithm T2D. is developed for combining the information to yield a simple 0064. Accordingly, the invention is also directed to a test prediction of obesity as percentage of risk in one year, two kit for risk assessment, diagnosis or prognosis of obesity or years, five years, 10 years or 20 years. Alternative statistical T2D comprising: 0065 a) reagents, materials and protocols for assessing models are failure-time models such as the Cox's propor type and/or level of one or more T2D and/or obesity pheno tional hazards model, otheriterative models and neural net type associated genetic markers in a biological sample, working models. wherein the genetic markers are related to SUCLA2 gene, 0058 Diagnostic test kits (e.g. reagent kits) of this inven and; tion comprise reagents, materials and protocols for assessing 0.066 b) instructions and software for comparing the one or more biomarkers, and instructions and Software for genetic marker data from a subject to genetic marker data comparing the biomarker data from a subject to biomarker from healthy and diseased people to make risk assessment, data from obese and non-obese people to make risk assess diagnosis or prognosis of obesity or T2D. ment, diagnosis or prognosis of obesity. Useful reagents and materials for kits comprise PCR primers, hybridization EXPERIMENTAL SECTION probes and primers as described herein (e.g., labeled probes or primers), allele-specific oligonucleotides, reagents for Example 1. Genotyping and Statistical Analyses of genotyping SNP markers, reagents for detection of labeled the GWS Data molecules, restriction enzymes (e.g., for RFLP analysis), 0067 Genomic DNA Isolation and Quality Testing DNA polymerases, RNA polymerases, DNA ligases, marker 0068 High molecular weight genomic and mitochondrial enzymes, antibodies which bind to polypeptides related to DNA was purified from frozen blood samples using QIAamp one or more obesity associated SNP markers disclosed in DNA Blood Midi kits (Qiagen). Concentration of purified Tables 6 through 17, means for amplification and/or nucleic DNA in each sample was measured using NanoDrop acid sequence analysis of nucleic acid fragments containing ND-1000 spectrophotometer (NanoDrop Technologies, one or more obesity associated SNP markers set forth in Wilmington, Delaware USA) and aliquot was diluted to con Tables 6 through 17. In one embodiment, a kit for diagnosing centration 60 ng/ul. A sample was qualified if A260/A280 Susceptibility to obesity comprises primers and reagents for ratio was 21.7. detecting the nucleotides present in one or more SNP markers 0069 Genome-Wide Scanning. Using Illumina's Human selected from the Tables 6 through 17 of this invention in Hap550 individual's nucleic acid. 0070 The whole-genome genotyping of the DNA samples 0059 Yet another application of the current invention is was performed by using Illumina’s Sentrix Human Hap550 related to methods and test kits for monitoring the effective BeadChips and Infinium II genotyping assay. The Human ness of a treatment for obesity. The disclosed methods and Hap550 BeadChips contained over 550,000 SNP markers of kits comprise taking a tissue sample (e.g. peripheral blood which majority were tagSNP markers derived from the Inter sample or adipose tissue biopsy) from a Subject before start national HapMap Project. TagSNPs are loci that can serve as ing a treatment, taking one or more comparable samples from proxies for many other SNPs. The use of tagSNPs greatly the same tissue of the Subject during the therapy, assessing improves the power of association studies as only a Subset of expression (e.g., relative or absolute expression) of one or loci needs to be genotyped while maintaining the same infor more genes related to one or more obesity associated SNP mation and power as if one had genotyped a larger number of markers set forth in Tables 6 through 17 of this invention in SNPS. the collected samples of the subject and detecting differences 0071. The Infinium II genotyping with the Human Hap550 in expression related to the treatment. Differences in expres BeadChips were performed according to the “Single-Sample sion can be assessed from mRNAS and/or polypeptides BeadChip Manual process” described in detail in related to one or more obesity associated SNP markers set “InfiniumTM II Assay System Manual provided by Illumina forth in Tables 6 through 17 of this invention and an alteration (San Diego, Calif., USA). Briefly, 750 ng of genomic DNA in the expression towards the expression observed in the same from a sample was subjected to whole-genome amplification. tissue in healthy non-obese individuals indicates the treat The amplified DNA was fragmented, precipitated and resus ment is efficient. In a preferred embodiment the differences in pended to hybridization buffer. The resuspended sample was expression related to a treatment are detected by assessing heat denatured and then applied to one Sentrix Human biological activities of one or more polypeptides related to Hap550 BeadChip. After overnight hybridization mis- and one or more obesity associated SNP markers set forth in non-hybridized DNA was washed away from the BeadChip Tables 6 through 17 of this invention. and allele-specific single-base extension of the oligonucle 0060 Based on the results disclosed below, the present otides on the BeadChip was performed in a Tecan GenePaint invention is especially directed to a method for risk assess rack, using labeled deoxynucleotides and the captured DNA ment, diagnosis or prognosis of obesity or type 2 diabetes as a template. After staining of the extended DNA, the Bead (T2D) in a mammalian Subject comprising: Chips were washed and scanned with the BeadArray Reader 0061 a) providing a biological sample taken from the (Illumina) and genotypes from samples were called by using Subject; the BeadStudio software (Illumina). 0062 b) detecting one or more T2D and/or obesity asso 0072 Assessment of Diet ciated genetic markers in said sample, wherein the genetic 0073 Intake of nutrients was assessed by 177-item food markers are related to SUCLA2 gene, and; frequency questionnaire. US 2012/0225,047 A1 Sep. 6, 2012

0074. Initial SNP Selection for Statistical Analysis ing WHR residualD3 or WHR residual-3 were excluded 0075 Prior to the statistical analysis, SNP quality was from the WHR analysis. Our data set included 1203 subjects assessed on the basis of three values: the call rate (CR), minor with adjusted WHR values. allele frequency (MAF), and Hardy-Weinberg equilibrium I0084. The data were analyzed using PLINK-program (H-W). The CR is the proportion of samples genotyped suc where the sample means of the two groups with different cessfully. It does not take into account whether the genotypes alleles were compared with the t-test. are corrector not. The call rate was calculated as: CR number 0085. It was invented that one can use the ratio of BMI and of samples with Successful genotype call/total number of WHR to dietary energy intake as a measure of “energy intol samples. The MAF is the frequency of the allele that is less erance' or “energy efficiency'. The same can be technically frequent in the study sample. MAF was calculated as: done by examining the interaction of BMI and WHR with MAF-min(p,q), where p is frequency of the SNP allele A energy intake. and q is frequency of the SNP allele B; p=(number of samples with 'AA'-genotype--0.5*number of samples with “AB'-genotype)/total number of samples with successful Example 2. SUCLA2 Gene Polymorphisms Modify genotype call: q=1-p. SNPs that are homozygous (MAF-0) the Association Between Energy Intake and Obesity cannot be used in genetic analysis and were thus discarded. I0086. In our 550k GWS data set, SNPs in the gene sucla2 H-W equilibrium is tested for controls. The test is based on were associated with the ratio of BMI and WHR to dietary the standard Chi-square test of goodness of fit. The observed energy intake and also modified the energy intakexBMI and genotype distribution is compared with the expected geno energy intakexWHR interactions. The gene works in the type distribution under H-W equilibrium. For two alleles this Krebs cycle. The function and possibly activity of the gene distribution is p2, 2pd, and q2 for genotypes AA, AB and can be assessed by measuring its metabolites in urine (see, BB, respectively. If the SNP is not in H-W equilibrium it can e.g., prior art technologies disclosed in US 5,508,204 and be due to genotyping error or some unknown population Williams et al., 2005, J. Pharm. Biomed. Anal. 38(3):465 dynamics (e.g. random drift, selection). 471). The invention concerns the diagnostic use of markers in 0076 Markers with CR>90%, MAD1%, and H-W equi the Sucla2 gene, the Sucla2 gene as target for obesity drugs librium Chi-square test statistics-27.5 (the control group) and the use of Sucla2 metabolites in monitoring energy effi were used in the statistical analysis. A total of 315.917 ciency and tolerance, energy consumption and physical activ Illumina300K SNPs fulfilled the above criteria and 534,022 ity. These markers can be either genetic, RNA, protein mark Illumina550K SNPS. ers or metabolites of Sucla2. 0077. Single SNP Analysis BMI and WHR (Binary Traits) I0087. Over 550,000 gene-tagging SNP markers were 0078. In our study the obese cases (based on BMI) had typed in 1062 subjects from East Finland. BMID=30 and at least 1 obese relative and the obese controls had BMI<=27 and no obese relatives. Based on these selec I0088. If we compare two persons with a same BMI: tion criteria there were 128 obese cases and 522 controls. 0089 high value in BMI/E means that a person with the 0079 Obesity was also defined based on WHR (waist to same BMI gets (eats) less energy in her/his diet than the hip circumference ratio). In this case the obese cases (based person with a low BMI/E on WHR) had WHRD=0.92 (men) or WHR>=0.83 (women) 0090 low value means that a person with the same BMI and at least one obese relative. Obese controls (based on takes in more energy i.e. can eat more and still does not WHR) had WHR<=0.91 (men) and WHR<=0.82 (women) get any more obese and no obese relatives. Based on these selection criteria there 0091) If we compare two persons with a same Energy were 311 cases (105 men and 206 women) and 303 controls intake: (92 men and 211 women). The analyses were done for both 0092 high value in BMI/E means that the person has genders combined and separately for men and women. higher BMI than the person with a low BMI/E, both at 0080. Differences in allele distributions between cases the same energy intake and controls were screened for all SNPs. The screening was (0093. The person with a high BMI/E value tends to store carried out using the standard Chi-square independence test the energy easier or at lower energy intake levels thana person with 1 df (allele distribution, 2x2 table). SNPs that gave a with a low BMI/E. I.e. the lower the ratio, the larger is the P-value less than 0.001 (Chi-square with 1 df of 10.23 or proportion of energy used out of taken energy. A high BMI/E more) were considered statistically significant and reported in ratio denotes energy intolerance, i.e. BMI tends to rise easier Tables 6 through 17. Odds ratio was calculated as ad/bc. or at a lower energy intake levels. where a is the number of minor alleles in cases, b is the (0094. Mean values of BMI/E (Ratio of BMI to Energy number of major alleles in cases, c is the number of minor Intake) in Subjects with Different RS12873870 Genotypes: allele in controls, and d is the number of major alleles in AA and AG Versus GG Genetypes (GG Vs Other) controls. Minor allele was defined as the allele for a given SNP that had smaller frequency than the other allele in the control group. TABLE 2 I0081. Single SNP Analysis BMI and WHR (Continuous The distribution of BMI/E in all 1062 subjects Traits) according to RS12873870 genotype. 0082) 10-based logarithm transformation was used for Std. Std. Error BMI values and samples with log(BMI)>1.6 were discarded GENOTYPE N Mean Deviation Mean from the analysis as outliers. Our data set included 1191 GG 989 O.O14777 0.005373 O.OOO170837 log(BMI) samples. AA or AG 73 O.O18O16 O.OO606 O.OOO709282 0083 WHR values were first adjusted for gender, smok ing, physical activity, alcohol g/week, and age. Samples hav US 2012/0225,047 A1 Sep. 6, 2012

0095 P-value for difference: 9.71 E-07. Allele A carriers 0101. A high CRP is associated with obesity and elevated are energy intolerant, as compared with the GG homozy leptin and elevated leptinto adiponectin ratio. This is consis gotes. tent with a relationship between obesity, glucose homeosta 0096. Difference in BMI/E Between Genders sis, and inflammation. Our invention also suggests that the RS 12873870 genotype may be a very early predictor of obesity, TABLE 3 insipient insulin resistance, glucose intolerance and T2D. The distribution of BMIE in all 1062 subjects in men and women. Individuals with defective SUCLA2 function could be prone for fat accumulation due to reduced functioning of the Krebs Std. Std. Error cycle. GENDER N Mean Deviation Mean MEN 378 O.O14171 O.OOS273 0.000271204 TABLE 4 WOMEN 684 O.O15457 O.OOSS44. O.OOO21, 1975 Statistical significance of associations or differences in all 1062 subjects. 0097. P-value: 2.43E-04. 0098 Women are more energy intolerant (less energy tol- ANOVA Table F Sig. st R men and the least tolerant are those women bmi per energy * GENOTYPE 24.26882 9.71E-07 with either AA or AG genotype of RS 12873870. whir per energy * GENOTYPE 13.8493 O.OOO2O8 0099 Comparison of Different Collected and Measured Starch (g) * GENOTYPE 13.55887 O.OOO243 Measurements Between RS12873870 A-and GG Genotypes Total energy intake (kJ) * GENOTYPE 12.S9436 O.OOO404 0100 Thus, the RS12873870 genotype was also associ- (g) : ENOype s o ated with many obesity-related traits such as hsCRP (C-reac- Ocilt (g) tive protein), height and dietary intakes of energy, starch, total GENOTYPESerum High sensitivity CRP (mg/l)* 9.98.9428 O.OO1614 Sugars, fat, protein, insoluable fiber, and cholesterol. The Insoluable fiber (g) * GENOTYPE 9.628912 O.OO1966 individuals with the rare (mutant) allele, A, had lower intakes Total Sugars (in food and added) (g) * S.O46629 O.O24878 of all energy nutrients, but were more obese and had much GENOTYPE higher serum CRP. It can be speculated that there was Height cm * GENOTYPE 4.385863 O.O36446 enhanced inflammatory response in them or metabolic Dietary cholesterol (mg) * GENOTYPE 4.061926 0.044111 changes in the liver and pancreatic carbohydrate metabolism, which manifested as elevated CRP.

TABLE 5 The distribution of different traits in all 1062 subjects according to RS12873870 genotype. Serum High Body sensitivity BMI WHR Weight Height Mass CRP GENOTYPE energy energy kg Cl Index (mg/l)

GG Mean O.O14777 O.OOO48S 74.6667 166.3077 26.93844 192975 N 989 989 112O 1120 112O 112O Std. 0.005373 OOOO16 14.S1929 8.617389 4.544O74 3.22724 Deviation AA+ AG Mean O.O18O16 O.OOOSS8 74.34382 164.309 27.589.28 3.06348 N 73 72 89 89 89 89 Std. O.OO606 O.OOO17 12.26919 9.259364 4.39697 3.61529 Deviation Total Mean O.O15 O.OOO489 74.64293 16.6.16OS 26.9863S 2.01320 N 1062 1061 1209 1209 1209 1209 Std. O.OOS481 OOOO162. 14.36145 8.677937 4.5348.23 3.26919 Deviation Total Total Sugars Water energy Protein (natural and unsoluable GENOTYPE (kJ) Fat (g) (g) Starch (g) added) (g) fiber (g)

GG 84.46.393 63.37012 92.81.167 1409142 114.2726 21.74235 994 994 994 994 994 994 328S906 28.83443 36.03632 62.36105 59.337 10.62OOS AA+ AG 7O66.014 S2.35541 79.22973 113.74OS 98.47568 17.84459 74 74 74 74 74 74 2299.894 20.67073 26.02831 43.26.289 42.86433 7.256233 Total 83SO.749 62.60693 91.87O6 139.0314 113.1781 2147228 1068 1068 1068 1068 1068 1068 3245.495 28.47SO1 35.59227 61.6O282 58.468 1046644 US 2012/0225,047 A1 Sep. 6, 2012 10

0102 Information on RS12873870 Association with I0123 Synonyms: EC 6.2.1.5; ATP-specific succinyl-CoA SUCLA2 and BMI Per Energy Intake synthetase subunit beta; Succinyl-CoA synthetase, betaA (0103 SUCLA2, GeneID:8803, mRNA NM 003850.2, chain; SCS-betaA; Renal carcinoma antigen NY-REN-39 genomic reference NC 000013. 10, position 0104 Chr 13 (13q12.2-q13.3), 0.124 , Gene: The protein encoded by this gene is an 0105 Start: 47,414,792 by from pter ATP-specific SCS beta subunit that dimerizes with the SCS 0106 End: 47,473,463 by from pter alpha subunit to form SCS-A, an essential component of the 01.07 Size: 58,672 bases tricarboxylic acid cycle. SCS-A hydrolyzes ATP to convert 0108 Orientation: minus strand Succinate to Succinyl-CoA. Defects in this gene are a cause of 0109 Analysis results BMI per energy intake myopathic mitochondrial DNA depletion syndrome. A 0110. Significant SNP: of this gene has been found on chromosome 6.

Marker l P MAF CR Chr pos gene RS12873870 1062 111E-06 0.037634 1 13 47443974 SUCLA2 intron Minor allele A if major allele G Total of4 intragenic SNPs are in Illumina 550kassay, RS12873870 is potentially obesity-associated, p for BMI 0.003105

0111 NCBI dbSNP for RS12873870 0.125 Map: This gene SUCLA2 maps on , (O112 MAF CEU: 0.067 (NOTE: within populations at 13q12.2-q13.3 according to Entrez genotyped in HapMap project, this SNP is polymorphic 0.126 Gene. In AceView, it covers 228.41 kb, from only in Caucasian population) 47510103 to 47281697 (NCBI 36, March 2006), on the 0113 Alleles: C/T forward: A/G reverse reverse Strand. 0114 LD in HapMap CEU population: RS12873870 is in I0127. AceView (shortened): RefSeq annotates one repre D'=1 with a number of markers, but is in relatively low R' sentative transcript (NM included in with all markers. The highest R=0.38 with 9 SNPs that are I0128 AceView variant.c), but Homo sapiens cDNA intronic/flanking 5"/3' to SUCLA2, and intronic/flanking 5' to sequences in GenBank, filtered against clone rearrangements, MED4 (gene ID: 29079). Thus, the observed association of coaligned on the genome and clustered in a minimal non RS12873870 indicates association of SUCLA2 gene. redundant way by the manually supervised AceView pro 0115 LD Block Structure: SUCLA2 shares an LD block gram, support at least 17 spliced variants. Alternative mRNA with the neighboring 5' genes NUDT15 (Nucleoside diphos expression and splicing: The gene contains 36 different gt-ag phate-linked moietyxmotif 15) and MED4. RS12873870 is introns. Transcription produces 20 different mRNAs, 17 an outlier in the LD block having very little linkage to other alternatively spliced variants and 3 unspliced forms. 659 by of markers in the block. this gene are antisense to spliced gene blaspey, 399 to 0116 LD in the Eastern Finnish Population: Similar to NUDT15, raising the possibility of regulated alternate HapMap CEU population; significant R2-0.405 with expression. Protein coding potential: 13 spliced and the rs9285.165 (intergenic, p=0.06498 in BMI per energy) unspliced mRNAS putatively encode good , alto 0117 Gender Distribution of RS12873870 Minor Allele gether 14 different isoforms (10 complete, 2 COOH com (AA/AG) Frequency in the Eastern Finnish Population: plete, 2 partial). 0118. There is gender specificity in SUCLA2 RS I0129. Several transcripts of various sizes are coded for 12873870 minor allele (A) inheritance. The minor allele A is SUCLA2 gene thus suggesting existence of multiple protein more frequent in females than in males. variants. 0119 SUCLA2 Markers in Affymetrix 100K Mapping I0130 SwissProt: Pathway: Carbohydrate metabolism; tri Assay: carboxylic acid cycle. 0120 100 K assay has two SNPs for SUCLA2 gene, I0131 Protein length is 463 amino acids. It is a precursor intronic RS2182374 and a locus-region SNP RS7335797. protein; it contains a 52 amino acid long mitochondrial sort Neither of these SNPs is in Illumina 500 kassay. ing sequence, and a 411 amino acids long Succinyl-CoA 0121 Information on 2 Gene: ligase ADP-forming subunit beta, mitochondrial sequence. 0122) The SUCLA2 gene encodes the beta-subunit of the Molecular weight: 50317 Da. ADP-forming succinyl-CoA synthetase (SCS-A: EC 6.2.1. (0132) Tissue Specificity: Widely expressed. SUCLA2 is 5). SCS is a enzyme that catalyzes the predominant in catabolic tissues, such as , heart, and reversible synthesis of Succinyl-CoA from Succinate and . Expression as well as the amount of the CoA. The reverse reaction occurs in the Krebs cycle, while protein and enzymatic activity of SCS-A varies considerably the forward reaction may produce Succinyl-CoA for activa between tissues in one species but also between species tion of and synthesis. GTP-specific (Lambeth DO, Tews KN, Adkins S. Frohlich D, Milavetz B (SCS-G: EC 6.2.1.4) and ATP-specific (SCS-A) isoforms of I. Expression of two succinyl-CoA synthetases with different SCS catalyze GTP-dependent and ATP-dependent reactions, nucleotide specificities in mammalian tissues. J Biol Chem. respectively. SCS is composed of an invariant alpha subunit 2004 Aug. 27:279(35):36621-4). and a beta subunit that determines the enzyme’s nucleotide 0.133 Posttranslational Modification: phosphoprotein specificity. (Rattus norvegicus): the alpha-Subunit of Succinyl-CoA syn US 2012/0225,047 A1 Sep. 6, 2012 thetase undergoes autophosphorylation at a histidine residue. 0146 What is encephalomyopathy'? Coprovision of exogenous succinate and CoA results in pro 0147 Mitochondrial encephalomyopathy-aminoacidopa nounced dephosphorylation of the phosphorylated alpha thy: A very rare syndrome characterized mainly by muscle subunit of succinyl-CoA synthetase (source BRENDA data and brain disease and an amino acid disorder. Medical Symp base) toms include: Developmental delay, Neurological problems, 0134) Pathways for SUCLA2 Deafness, Exercise intolerance, Lactic acidosis, Increased 0135 KEGG pathway: CS-Branched dibasic acid level of amino acids in plasma, Muscle wasting, Reduced metabolism (0.0660) reflexes, Ataxia, and Poorly muscled build. 0.136 KEGG pathway: Citrate cycle (TCA cycle) (00020) 0148 Muscle Tissue in Subjects with SUCLA2 Defi 0137 KEGG pathway: Propanoate metabolism (0.0640) ciency: Histology of muscle tissue showed a very consistent 0138 KEGG pathway: Reductive carboxylate cycle (CO2 and characteristic pattern in all seven patients from whom a fixation) (00720) muscle biopsy was available. The findings included (i) 0139 Reactome Event: Pyruvate metabolism and TCA increased variability of fibre diameter with scattered hyper cycle (71406) trophic, spherical fibres with an increased mitochondrial con 0140 SUCLA2 Associated Phenotypes: tent, (ii) a marked type I fibre predominance (>95%) and (iii) 0141 OMIM: Deficiency of SUCLA2 is associated with extensive intracellular fat accumulation in type I fibres (Os encephalomyopathy and mitochondrial DNA depletion. tergaard E. Hansen F J, Sorensen N. Duno M. Vissing J, 0142 Literature References for the Phenotype: Larsen P L. Faeroe O, Thorgrimsson S. Wibrand F. Chris 0143 1. The mitochondrial DNA (mtDNA) depletion syn tensen E. Schwartz M. Mitochondrial encephalomyopathy drome is a quantitative defect of mtDNA resulting from dys with elevated methylmalonic acid is caused by SUCLA2 function of one of several nuclear-encoded factors respon mutations. Brain. 2007 March;130(Pt3):853-61). We pro sible for maintenance of mitochondrial deoxyribonucleoside pose as part of this invention that intracellular fat accumula triphosphate (dNTP) pools or replication of mtDNA. Mark tion may be due to muscular atrophy that is caused by edly decreased succinyl-CoA synthetase activity due to a decreased mtDNA in SUCLA2 deficiency. deleterious mutation in SUCLA2, the gene encoding the beta 0149 Role of SCS-A in TCA Cycle: Subunit of the ADP-forming Succinyl-CoA synthetase ligase, 0150. SCS-A plays a significant role in . was found in muscle mitochondria of patients with encepha Entrez, Gene and other databases present the function of lomyopathy and mtDNA depletion. Succinyl-CoA synthetase SUCLA2 in hydrolyzing ATP to convert succinate to succi is invariably in a complex with mitochondrial nucleotide nyl-CoA. However, it appears that SCS-A complex in fact diphosphate kinase; hence, the authors propose that a defect catalyzes the reverse reaction in the citric acid cycle. Succi in the last step of mitochondrial dNTPsalvage is a novel cause nyl-CoA-ADP->succinate+CoA-ATP (D. O. Lambeth, K. of the mtDNA depletion syndrome (Elpelego, et al.: Defi N. Tews, S. Adkins, D. Frohlich, and B.I. Milavetz: Expres ciency of the ADP-forming succinyl-CoA synthase activity is sion of Two Succinyl-CoA Synthetases with Different Nucle associated with encephalomyopathy and mitochondrial DNA otide Specificities in Mammalian Tissues. J. Biol. Chem. depletion. Am J Hum Genet. 2005 June.76(6):1081-6. Epub Aug. 27, 2004; 279(35): 36621-36624.) SCS-A is not a rate 2005 Apr. 22.). limiting enzyme in Krebs cycle. Its activity is regulated by the 0144. 2. “The hallmark of the condition, elevated methyl amount of succinyl-CoA. In KEGG TCA-pathway, enzyme malonic acid, can be explained by an accumulation of the Succinyl-CoA hydrolase (EC 3.1.2.3) is presented as func Substrate of the enzyme. Succinyl-CoA, which in turn leads to tionally similar enzyme for conversion of Succinyl-CoA to elevated methylmalonic acid, because the conversion of Succinate. This enzyme, however, has been described only in methylmalonyl-CoA to succinyl-CoA is inhibited. (Oster organisms in lower taxonomy, and thus cannot be considered gaard E. Hansen FJ, Sorensen N. Duno M, Vissing J. Larsen as relevant substitute for SCS-A/SCS-G enzymes. PL, Faeroe O, Thorgrimsson S. Wibrand F. Christensen E. 0151. Invention Concerning the Role of SUCLA2 in Schwartz M. Mitochondrial encephalomyopathy with Energy Intolerance: elevated methylmalonic acid is caused by SUCLA2 muta 0152 Both SCS-A and SCS-G are localized in beta cell tions. Brain. 2007 March: 130(Pt3):853-61.) mitochondria, and it has been proposed that GTP generated 0145 3. Succinate-CoA ligase catalyses the reversible by the activation of SuccinylCoA synthetase could promote conversion of succinyl-CoA and ADP or GDP to succinate key functional roles in the mitochondrial metabolism leading and ATP or GTP. It is a mitochondrial matrix enzyme and at to insulin secretion (Kowluru A. Diabetologia. 2001 January; least the ADP-forming enzyme is part of the Krebs cycle. The 44(1):89-94. Adenine and guanine nucleotide-specific Succi substrate specificity is determined by the beta subunit of nyl-CoA synthetases in the clonal beta-cell mitochondria: succinate-CoA ligase, which is encoded by either SUCLA2 implications in the beta-cell high-energy phosphate metabo or SUCLG2. In patients with severe hypotonia, deafness and lism in relation to physiological insulin secretion. PMID: Leigh-like syndrome, mutations have been found in 11206416). SUCLA2. Mutations have also been reported in SUCLG1, 0153. Knockdown of SCS-A (by si-RNA) in rat INS which encodes the alpha subunit found in both enzymes, in 1832/13 insulinoma cells and in cultured rat islets increases patients with severe infantile acidosis and lactic aciduria. glucose-stimulated insulin secretion (GSIS) by two-fold, Elevated methylmalonate and methylcitrate and severe whereas suppression of GTP-specific SCS (SCS-G) reduces mtDNA depletion were found in both disorders. The mtDNA GSIS by 50%. Increasing the rate of GTP synthesis by reduc depletion may be explained by the interaction of Succinate ing the expression of SCS-ATP results in increased oxygen CoA ligase with nucleoside diphosphate kinase, which is consumption and cytosolic calcium with a concomitant involved in mitochondrial nucleotide metabolism (Oster increase in insulin secretion, which is unassociated with an gaard E. Disorders caused by deficiency of Succinate-CoA increase in the ATP/ADP ratio or NAD(P)H. Conversely, if ligase. J Inherit Metab Dis. 2008 Apr. 4.). GTP synthesis is decreased by silencing SCS-GTP, then oxy US 2012/0225,047 A1 Sep. 6, 2012 gen consumption, ATP synthesis, and NAD(P)H levels acetylcarnitine carrier complex. Cytosolic elevated levels of increase while cytosolic calcium does not, leading to acetyl-CoA can result in increased conversion acetyl-CoA to impaired GSIS. Taken together, these data suggest that TCA Malonyl-CoA by the action of acetyl-CoA carboxylase cycle-generated mtGTP regulates insulin secretion by (ACC). Malonyl-CoA is a potent inhibitor of CPTI (carnitine increasing cytosolic calcium (Kibbey R. G. Pongratz R L. palmitolyltransferase I), and this inhibition could result in Romanelli AJ, Wollheim C B, Cline G. W. Shulman G. I. decreased mitochondrial fatty acid oxidation. Decreased fatty Mitochondrial GTP regulates glucose-stimulated insulin acid oxidation, in turn, results in abnormal fatty acid metabo secretion. Cell Metab. 2007 April:5(4):253-64.). lism and storage. In this model, decreased overall activity of 0154 Therefore, it is plausible that alterations in SUCLA2 the citric acid cycle would yield elevated levels of fat, and function would influence glucose-induced insulin secretion. subjects with defective SUCLA2 function would be prone for In particular, decreased function of SCS-A could provide fat accumulation due to reduced functioning of the Krebs more availability of succinyl-CoA for SCS-G to promote cycle. Furthermore, it has been proposed that malonyl-CoA GTP production and Subsequently glucose stimulated insulin serves as an intermediary in a signaling circuit that regulates secretion. Hypothetically, subjects RS12873870 minor allele feeding behavior (Dowell P. Hu Z., Lane MD. Annu. Rev. A genotype could have such alterations in their insulin secre Biochem. 74, 515-534, 2005). Interestingly, in our BMI/En tion that would promote energy intolerance. ergy Intake—dataset, Subjects with minor allele hetero/ho 0155. In Krebs cycle, SCS-A catalyzes the synthesis of mozygotia in RS12873870 were not significantly different in succinate+CoA+ATP from succinyl-CoA and ADP. Thus, BMI from control group. They however eat less and yet increased expression or activity of SCS-A could lead to accu maintain normal BMI. This could potentially be explained by mulation of succinate in Krebs cycle, which is substrate for more efficient intake of energy from the food that might fumarate production. Obesity-associated gene FTO that accompany with lower threshold for feeling satiety. encodes for a 2-Oxoglutarate-dependent nucleic acid dem (0160 Potential Sites for Modulation of SCS-A ethylase is an enzyme that is inhibited by Krebs cycle inter 0.161 Interacting Partners of Succinyl-CoA Synthase mediates, in particular by fumarate, but also by Succinate (SCS): (Gerken Tet al., The obesity-associated FTO gene encodes a 0162 Nucleoside diphosphate kinase (NDPK; alias 2-oxoglutarate-dependent nucleic acid demethylase. Sci mNDPK; NDPK-D, encoded by gene NME4). This ence. 2007 Nov 30:318(5855): 1469-72.). Modulation of FTO association has been proposed to enable intramitochon activity has been Suggested in disease states with elevated drial generation of GTP which (unlike ATP) cannot be fumarate/succinate levels. -->Thus, it is possible that a single transported into mitochondria via classical nucleotide point mutation affecting expression or activity of SUCLA2 translocase. (Kowluru A. Tannous M. Chen HQ. Local gene may have a function that could indirectly relate with ization and characterization of the mitochondrial iso function of FTO that could result in energy intolerance. form of the nucleoside diphosphate kinase in the pan Increased production of succinate and fumarate, in particular, creatic beta cell: evidence for its complexation with could inhibit FTO function. In animal models, FTO has been mitochondrial succinyl-CoA synthetase. Arch Biochem shown to be regulated by feeding and starvation. With this Biophys. 2002 Feb. 15:398(2):160-9. PMID: aspect, it is of interest that subjects with SUCLA2 1183 1846). This complex formation has also been pro RS12873870 minor allele Agenotype seem to eat less and yet posed to underlie the mitochondrial DNA defect in maintain same BMI as those with the major allele genotype. SUCLA2 gene phenotype (Ostergaard E. Disorders 0156 Thus, minor allele A might relate to lower threshold caused by deficiency of Succinate-CoA ligase. J Inherit for Satiety, or increased tendency to gain weight which would Metab Dis. 2008 Apr. 4.) NDPK is responsible for intra be counteracted by intentional lower calorie consumption. cellular di-and triphosphonucleoside homeostasis, plays 015.7 Interestingly, increased fumarate has been shown to multifaceted role in cellular energetic, signaling, prolif induce adipogenesis in vitro. S-(2-Succinyl)cysteine (2SC), eration, differentiation, and tumor invasion. NDPK-D the product of chemical modification of proteins by the Krebs localizes in inner mitochondrial membrane and is Sug cycle intermediate, fumarate, is significantly increased dur gested to function for mitochondrial membrane lipid ing maturation of 3T3-L1 fibroblasts to adipocytes (Nagai R. transfer in liposomes that mimic mitochondrial mem Brock J. W. Blatnik M, Baatz, J. E. Bethard J. Walla M D, brane contents (Epand RF, Schlattner U. Wallimann T. Thorpe SR, Baynes J. W. Frizzell N. Succination of protein Lacombe ML, Epand RM. Novel lipid transfer property thiols during adipocyte maturation: a biomarker of mitochon of two mitochondrial proteins that bridge the inner and drial stress. J Biol Chem. 2007 Nov 23:282(47):34219-28.). outer membranes. Biophys J. 2007 Jan. 1:92(1): 126 0158. In contrast, decreased expression or activity of 37.). NDPK-D has also been recently shown to bind with SCS-A could lead to accumulation of succinyl-CoA in Krebs high affinity to cardiolipin, and to couple with mitochon cycle. As succinyl-CoA functions as feedback inhibitor of drial oxidative respiration (Tokarska-Schlattner M, Krebs cycle by inhibiting citrate synthase (Laloue, Bryla and Boissan M. Munier A, Borot C. Mailleau C. Speer O. Williamson: Feedback inhibitions in the control of citric acid Schlattner U, Lacombe M L. The nucleoside diphos cycle activity in rat heart mitochondria. JBC, 1972), it is phate kinase D (NM23-H4) binds the inner mitochon possible that reduced SCS-A function leads to reduced over drial membrane with high affinity to cardiolipin and all energy production in Krebs cycle. Concomitantly, Krebs couples nucleotide transfer with respiration. J Biol cycle intermediates forward from succinate would be below Chem. 2008 Jul 17. Epub ahead of print). normal levels as a result of decreased SCS-A activity. In (0163 TRIM28 interacts with the SUCLA2 gene. PMID combination, these two pathways when converging could 17542650. An interaction between TRIM28 and the result in elevated levels of mitochondrial acetyl-CoA. SUCLA2 gene was demonstrated by ChIP-on-chip 0159. Increased mitochondrial levels of acetyl-CoA could assay.TRIM28: Tripartite motif-containing 28. ID: result in transportation of acetyl-CoA to cytosol via carnitine 101.55. GO Terms Molecular Function.transcription fac US 2012/0225,047 A1 Sep. 6, 2012 13

tor activity GO:3700. transcription corepressor activity and 1.30) (Hansson O. Donsmark M. Ling C, Nevsten P. GO:3714-protein binding GO:5515.Zinc ion binding Danfelter M. Andersen J. L. Galbo H. Holm C. Transcriptome GO:8270.sequence-specific DNA binding GO:43565. and proteome analysis of soleus muscle of hormone-sensitive metal ion binding GO:46872.electron transporter activ lipase-null mice. J Lipid Res. 2005 December:46(12):2614 ity GO:5489 Cellular Component. intracellular 23.). HSL is encoded in humans by the LIPE (HSL, GeneID: GO:5622. nucleus GO:5634 Biological Process.epithe 3991, mRNA NM 005357; genomic reference lial to mesenchymal transition GO:1837...transcription NC 000019.9) gene. HSL is thus an activator of SCS-A, and GO:6350.regulation of transcription from RNA poly recombinant HSL or analogs of HSL can be used as SCS-A merase II promoter GO:6357-positive regulation of agonists and to boost the Krebs cycle. In the Krebs cycle, gene-specific transcription GO:43.193.electron trans SCS-A catalyzes the synthesis of succinate+CoA+ATP from port GO:6118 succinyl-CoA and ADP. Thus, increased expression or activ (0164 Pol II interacts with the SUCLA2 promoter. An ity of SCS-Acould lead to accumulation of succinate in Krebs interaction between Pol II (RNA polymerase II) and cycle, which is substrate for fumarate production. HSL may SUCLA2 promoter was demonstrated by chromatin be activated by two mechanisms: immunoprecipitation and genomic microarray hybrid 0171 In the first, it is phosphorylated by perilipin A, ization (chip-CHIP). PMID 12808131. causing it to move to the Surface of the lipid droplet, (0165 E2F1 interacts with the SUCLA2 promoter. where it may begin hydrolyzing the lipid droplet. Peril PMID 12808131. E2F transcription factor 1; retinoblas ipin A is encoded in humans by the PLIN1 gene (Ge toma-associated protein 1; pRB-binding protein 3. E2F1 neID: 5346, mRNANM 002666.4; genomic reference (RBP3) is a member of the E2F transcription factor NC 000015.9). family. E2F1 displays preferential binding to retinoblas 0172 Alternately, it may be activated by a cAMP-de toma protein pRB in a cell-cycle dependent manner, and pendent protein kinase, encoded in humans by the is involved in cell proliferation and p53-dependent/in PRKACA gene (GeneID: 5566, mRNANM 002730.3: dependent apoptosis. NCBI Entrez 1869. genomic reference NC 000019.9). This pathway is sig (0166 TAFII250 interacts with the SUCLA2 promoter. nificantly less effective than the first, which is necessary PMID 12808131. TAF1 RNA polymerase II, TATA box to lipid mobilization in response to cyclic AMP, which binding protein (TBP)-associated factor. Note that the itself is provided by beta adrenergic stimulation of the listed GeneID refers to multiple variants encoded by the glucagon receptor. same gene. The precise molecular variant involved in 0173 Thus, also recombinant forms or analogs of peril this interaction is not specified. NCBI Entrez Gene Id ipin A or cAMP-dependent protein kinase may be used as 6872. agonists of SCS-A and to boost the Krebs cycle. Any biom (0167 HNF4-alpha interacts with the SUCLA2 pro arker or metabolite of the interacting proteins or activators moter region. PMID 14988562. Hepatocyte nuclear fac can be used as biomarkers of Sucla2. tor 4-alpha; transcription factor 14, hepatic nuclear fac tor. Mutations in this gene have been associated with CONCLUSIONS monogenic autosomal dominant non-insulin-dependent (0174) 1. Marker RS12873870 supports association of diabetes mellitus type I. Three transcript variants encode SUCLA2 gene in BMI/energy data set. three isoforms. This protein represents variant 2 and (0175 2. Association of RS12873870 could relate to dif isoform b. NCBI ID: 3172. DNA binding GO:3677. ferential function or expression of SUCLA2 protein. Several transcription factor activity GO:3700. RNA polymerase transcripts have been described that in theory could have II transcription factor activity GO:3702.steroid hor tissue specific roles. In addition, tissue specificity of mone receptor activity GO:3707.receptor activity SUCLA2 mRNA and protein has been described in humans. GO:4872.ligand-dependent nuclear receptor activity (0176 3. SUCLA2 encodes for the beta subunit of ATP GO:4879.steroid binding GO:5496NOTE: this is a well specific succinyl-CoA ligase (SCS) that provides a part of the known molecule in diabetes mellitus. required ATP for citric acid cycle. (0168 ALAS2 interacts with SUCLA2 as identified by 0177 4. SCS has been shown to affect glucose stimulated two hybrid. This is an elemental interaction record from insulin secretion in vitro. MIPS. PMID 10727444. The first and the rate-limiting (0178) 5. Subjects with RS12873870 minor allele appear as enzyme of heme biosynthesis is delta-aminolevulinate energy intolerant. Minor allele Subjects are not significantly synthase (ALAS), which is localized in mitochondria. different from the major allele genotype subjects by BMI, but 5-aminolevulinic acid synthase, erythroid-specific, they consume less energy for maintaining BMI. Distribution mitochondrial precursor. NCBI ID: 28588. ALAS2 of muscle/fat ratio in the Subjects under study is not known; it interacts with SUCLA2 as identified by coimmunopre is possible that although BMI is not different, muscle/fat— cipitation. This is an elemental interaction record from ratio could be affected. CRP levels are higher in subjects with MIPS RS 12873870 minor allele genotype. 0169. In other species, e.g. yeast, bacteria and fruit fly, 0179 6. Potential sites for manipulation: transportation of also other interacting molecules have been described. citric acid cycle intermediates; modulation of SCS-A or However, the only small molecules are CoA, Mg2+, and SCS-G activity; modulation of methylmalonyl-CoA levels ADP. Other molecule types belong to proteins, genes and DNA. Example 3: Interactions Between SNPs, Intake of 0170 Hormone-sensitive lipase (HSL), a key enzyme in Energy Nutrients and Obesity (BMI or WHR) i.e. fatty acid mobilization in adipocytes knock-out mice showed How SNPs Modify the Effect of Intakes of Energy, increased expression in transcriptome analysis of soleus Fat and Carbohydrates on BMI and WHR muscle of HSL-null mice of succinyl-CoA synthetase, (1.25 0180 Linear Regression Between a Trait and a SNP US 2012/0225,047 A1 Sep. 6, 2012 14

0181 Subjects were from the Jukka T. Salonen's popula 0200. The results of the two subsets were compared with tion study collected from the East-Finland founder popula the following statistic, that has a standard normal distribution: tion. Effect of the SNP variation were tested based on a simple linear regression where a dummy variable is a dose of the B1 - B2 minor allele e.g. if the minor allele is A and the major allele is 3. G then GG=0, AG=1, and AA=2. All calculations were based VSE + SE; on PLINK-Statistical package (http://pngu.mgh.harvard.edu/ purcell/plink?) implemented in the BCISNPmax environment (Biocomputing platforms Ltd). 0201 where the subscripts correspond to different subsets 0182 Following results, quality measurements and anno within a particular SNP. Explanations for the other abbrevia tions in the tables are following: tation information are presented: 0202 P-value P-value corresponding to Z-value 0183 BETA: Regression coefficient (0203 HW Hardy-Weinberg equilibrium 0184 R2: Regression r-squared 0204 MAF minor allele frequency 0185. P: Wald test asymptotic p-value 0205 CR call rate 0186 HWE: Hardy-Weinberg equilibrium calculated for 0206 CHR chromosome hypertension controls 0207 Position chromosomal position in by 0208 Gene gene if the SNP is intragenic 0187 MAF: minor allele frequency 0209 GeneID corresponding gene ID 0188 CR: call rate 0210 Class indicating if the intragenic SNP is intronic etc. (0189 CHR: chromosome 0211 BMI per Energy IntakexSNP Interaction (“Energy (0190. POSITION: chromosomal position Intolerance') (0191 GENE: gene if the SNP is intragenic 0212 Regression model (ln(BMI)-mu+energy--e, where energy intake in food) within different genotype groups. The (0192 GENE ID: gene ID model was separately used for samples with minor allele (0193 CLASS: classification of the intragenic SNP present and samples that are homozygous for the major allele. 0194 Interaction between food intake and SNP 0213 SUMMARY: The closest gene of the significant (0195 Each SNP was analyzed separately. The data were SNP on chromosome is (KLF4) Kruppel-like factor 4 (gut), split into two subsets based on the SNP genotype: the first set Gene ID:9314; mRNA NM 004235.4, genomic reference included samples with minor allele of the SNP present and the NC 000009.11.

TABLE 6 Continuous variable: InCBMI) adjusted for age, HT-status, average weekly exercise. SNP P HW MAF CR CHR Position Gene GeneD class

RS117928O3 1.67E-08 O.224966 0.079385 0.995037 9 109563610

SNP B1 SE1 P1 n1 B2 SE2 P2 n2 Z. P

RS117928O3 -O.OOO12 2.56E-OS 4.06E-06 153 3.19E-OS 9.58E-06 OOOO91 896 -5.6439 1.67E-08 other subset included samples that were homozygous for wild 0214. This SNP and associated biomarkers can be used for (major) allele. The following information was obtained for nutrigenetic diagnostics for the selection of individuals for each SNP and Subset: low-energy food products. 0.196 B–regression coefficient 0215 BMI Per Fat IntakexSNP Interaction (“Fat Intoler 0.197 SE=standard error of the coefficient ance 0198 t—t-test statistic for B=0 vs Bz.0 0216 SUMMARY: The closest gene of the significant (0199 P=P-value of the test statistic SNP on chromosome is (KLF4) Kruppel-like factor 4 (gut).

TABLE 7

Continuous variable: ln(BMI), adjusted for age, HT-status, average weekly exercise.

SNP P HW MAF CR CHR Position Gene GeneD class

RS117928O3 3.56E-08 O.224966 O.O7938S 0.995037 109563610 RS2O4638O 4.6SE-07 O.108.188 O.O34326 1 3 178299225 TBL1XR1 79718 intron RS2142100 5.38E-O7 O.O95324 O.O11993 1 21 35806214 RS2834947 5.38E-O7 O.O82899 O.O1158 1 21 357988.18 RS11626428 6.26E-07 O.S691.68 O.147181 0.997519 14 9496.0193 C14orfA9 161176 intron RS6936924 7.76E-O7 O.O82899 O.O11589 O.999.173 6 34673 137 C6orf106 64771 intron

US 2012/0225,047 A1 Sep. 6, 2012 17

0228. The SNPs and associated markers can be used for NC 000011.9). The significant intronic SNP is located in nutrigenetic diagnostics for the selection of individuals for DNAH11 (dynein, axonemal, heavy chain 11), however the low-carbohydrate food products. MAF of this SNP is very low thus the results are unreliable. 0229 FIG. 2 shows linear regression between glycemic load and BMI for RS2847.666. 0230. The SNP rs2847.666 which is located in the MS4A2 gene, modifies the effect of dietary glycemic index on BMI. Almost half of the people are major allele (A) homozygotes (upper panel), and in them a high glycemic load appears to increase BMI, while in the minor allele (G) carriers, the higher the glycemic load, the lower the BMI (lower panel). 0231 BMI Per Glycemic IndexxSNP-Interaction

TABLE 10 Results from the glycemic indexx SNP -interaction for BMI. SNP P-value HW MAF CR Alleles CHR Position Gene GeneID class

RS10762971. 4.92E-08 O.O82899 O.O124O7 1 ACG 54751285 RS173O1783 3.04E-06 0.964O84 O.O21919 1. CT 60847990 RS9452215 4.15E-06 3.5637 O.O96774 1 ACG 98.809954 RS9452223 4.15E-06 3.5637 O.O96774 1 ACG 96813514 RS83O994 6.14E-06 0.267951 O.314309 1 ACG 2 1.7E--08LRP2 4036 coding-synonymous RS6571713 6.31E-06 1.572395 O.06906S 1. CT B489946O

SNP B1 SE1 P1 n1 B2 SE2 P2 n2 Z. P-value

RS10762971 -0.128O1 O.O2O656 2.1OE-06 24 -O.O1218 OOO4927 O.O136O1 1032 -5.45442 4.92E-08 RS173O1783 -0.10467 O.O19676 3.15E-06 45 -0.00994 OOO4969 O.O45822 1011 -4.6682 3.04E-06 RS9452215 O.028983 0.010241 O.OO5164. 187 -O.O244 O.OO5436 8.13E-O6 869 4604056 4.15E-06 RS9452223 O.028983 0.010241 O.OO5164. 187 -O.O244 O.OO5436 8.13E-O6 869 4604056 4.15E-06 RS83O994 O.OO4844 OOO651 O.457179 S75 -0.03881 O.OO713 8.32E-08 481 4.5218O3 6.14E-O6 RS6571713 O.O3598 0.011786 O.OO2732 1.35 -0.02231 O.OOS261 2.45E-0S 921 4S16036 6.31E-06

0232. The SNPs and associated markers can be used for nutrigenetic diagnostics for the selection of individuals for low-carbohydrate food products.

TABLE 11 Continuous variable:WHRad usted for: gender, age, Smoker, alcohol use, average weekly exercise. SNP P HW MAF CR CHR Position Gene GeneID class

RS10833641 3.59E-09 OO28118 O49247S O.989247 11 21796469 RS461758S 2.31E-07 O.861317 O.44347S O.995037 11 21839.434 RS7807695 4.84E-O7 O.108.639 O.O1571S 1 7 21836OSO DNAH11 8701 intron RS1524783 6.68E-O7 O.768.707 O.461538 1 3 83883488 RS17269759 9.27E-O7 O.108.639 O.O1861 1 2 114269008

SNP B1 SE1 P1 n1 B2 SE2 P2 n2 Z. P

RS10833641 -3.19E-OS 1.06E-OS O.OO2739 785 O.OOO 103 2.03E-OS 6.83E-07 26S -5.90209 3.59E-09 RS4617585 -2.91E-OS 1.08E-OS O.OO72S6 743 8.58E-OS 1.94E-OS 1.36E-OS 313 -S.17224 2.31E-O7 RS7807695 O.OOO178 3.69E-OS 4.07E-OS 29 - 1.38E-OS 9.68E-06 0.155456 1032 S.O32SS6 4.84E-07 RS1524783 -2.88E-OS 1.07E-OS O.OO7002 7SO 8.29E-OS 1.98E-OS 3.66E-OS 311 -4.97O34 6.68E-O7 RS17269759 O.OOO286 S.94E-OS 2.4SE-OS 37 -8.82E-06 9.57E-06 0.35731 1024 4.906586 9.27E-O7

0233 WHR Per Energy IntakexSNP Interaction (“Energy 0235 Possibly for nutrigenetic diagnostics for the selec Intolerance') tion of individuals for low-energy food products. 0234 SUMMARY: The closest genes on chromosome 11 0236 WHR Per Fat IntakexSNP Interaction (“Fat Intoler for the significant SNP are ANO5 (anoctamin 5, GeneID: ance 203859, mRNA NM 213599.2, genomic reference 0237 SUMMARY: The closest genes on chromosome 11 NC 000011.9) and NELL1 (NEL-like 1 (chicken), GeneID: for the significant SNP are ANO5 (anoctamin 5) and NELL1 4745, mRNA NM 006157.3, genomic reference (NEL-like 1 (chicken)). The significant intronic SNP is

US 2012/0225,047 A1 Sep. 6, 2012 21

TABLE 15-continued Results from the glycemic indexx SNP - interaction for WHR. RSS29674 -O.O3O83 O.OO7551 S.32E-OS 433 O.O148O3 O.OO68S1 O.O3109997 629 -4.47SO1 7.65E-06 RS1071905 O.OO8403 O.OOS901. O.154877 749 -0.04271 O.OO9853 1.97E-OS 313 4.450S72 8.57E-06 RS11796366 0.168609 O.O39349 O.OOO651 1S -O.OO778 O.OOS133 0.13009609 1045 4.444892 8.8E-06 RS1733.8297 0.033229 O.OO9931 O.OOO927 295 -0.01806 O.OOS91 O.OO232618 767 4.437696 9.1E-06 RS2969.018 -0.03794 O.OO8864 2.41E-OS 352 O.O10O3 O.OO619 O.10562459 710 -4.437O6 9.13E-06

0248 FIG. 5 shows linear regression between glycemic (0256 Finding: SNPs in the DNAH11 gene modify the index and WHR in RS373.1572 genotypes. association between energy intake and glucose load and 0249. In the major allele (A) homozygotes (upper panel), WHR there was only a weak association between the dietary glyce- (0257 SNP Energy intake interaction for WHR mic index and WHR, whereas in the minor allele (G) carriers the glycemic index had a strong association with WHR (lower 0258 Energy intake from Food survey panel). (0259. Dependent Variable: WHR residual (0250 BMI/Carbohydrate Intake 0260 Regression model WHR=Energy intake

TABLE 16 Results from the carbohydrate intolerance (BMI/carbohydrate intake). MARKER N BETA P-value HW MAF CR CHR POSITION GENE GENE ID CLASS

RS735984 1065 O.3124 1.79E-06 8.30 O.12 1.OOOO 11 4322O897 RS1021797 1065 O.4191 2.74E-O6 OO1 OO6 1.OOOO 12 26457699 ITPR2 3709 intron RS2305299 1065 O.288 2.92E-O6 OSO 0.14 10000 10 45456999 ANUBL1 93550 intron RS9858834 1065 O.32S5 3.45E-06 O.O1 O.10 1.OOOO 3 176771673 NAALADL2 254827 intron RS436488 1065 O.2638 3.6OE-06 0.12 0.17 1.OOOO 19 61056102 NLRP4 147945 intron RS12O3O971 1065 O.2O27 3.72E-06 O.O3 O.4O 1.OOOO 1 692O3851 RS16825963 1059 0.323 S.12E-06 O.OO O.10 O.995O 3 176764272 NAALADL2 254827 intron RS7780636 1065 O.4089 7.81E-06 6.2O O.OS 1.OOOO 7 70664399 WBSCR17 64.409 intron RS11633.874 10SS -0.191S 8.57E-06 0.04 O.48 0.9901 15 66719213 CORO2B 10391 intron RS1005316 1065 O.2257 9.4OE-06 0.16 0.22 1.OOOO 17 66SO1964

0251 WHR/Carbohydrate Intake

TABLE 17 Results from the carbohydrate intolerance (WHR/carbohydrate intake MARKER N BETA P-value HW MAF CR CHR POSITION GENE GENE ID CLASS RS9858834 1065 O.3377 1.45E-06 O.O1 O.10 1.OOOO 3 176771673 NAALADL2 254827 intron RS16825963 1059 O.3325 2.7OE-06 O.OO O.10 O.995O 3 176764272 NAALADL2 254827 intron RS1426499 1065 O.2O31 2.8OE-06 1.01 O.41 1.OOOO 7 131885330 PLXNA4B 91584 intron RS2305299 1065 O.2862 3.36E-06 OSO 0.14 1.OOOO 10 45456999 ANUBL1 93550 intron RS4442796 1065 O.2853 3.54E-06 0.25 0.15 1.OOOO 16 68345609 NOB1 28987 intron RS4816047 1065 O.1974 6.18E-O6 O24 O.41 1.OOOO 20 807322O PLCB1 23236 intron RS291.7682 1065 O.2794 6.8OE-06 O15 O14 1.OOOO 16 68323.792

0252. Further Genes of the Invention 0261 Model was separately used for samples with minor 0253 DNAH11 as Energy and Carbohydrate Intolerance allele present and samples that are homozygous for wild Gene (major) allele 0254. Data for DNAH 11 Association: 0262. Adjusted variables were Gender, Smoker, Age, 0255 Analysis of WHR by Energy intake by SNP inter- Alcohol use, absolute ethanol grams/day, actions 0263 Average weekly exercise (hours). US 2012/0225,047 A1 Sep. 6, 2012 22

TABLE 1.8 The most significant marker. SNP P HW MAF CR CHR Position Gene GeneID class

RS7807695 4.84E-07 O.108.639 O.O1571S 1 7 21836OSO DNAH11 8701 intron

0264 There are 160 SNPs for DNAH11 gene on Illumina 5OOK.

TABLE 19 All significant markers intragenic for DNAH11 in WHR Energy intake SNP interaction analysis: SNP P HW MAF CR Alleles CHR Position Gene GeneID class RS4722O54 O.OOO639 14.09977 0.027709 1 AG 7 21773017 DNAH11 8701 intron RS1026833O O.OOO639 14.09977 0.027709 1 AG 7 21774561 DNAH11 8701 intron RS78.07695 4.84E-07 0.108639 O.O1571S 1 CT 7 21836OSO DNAH11 8701 intron

0265 R of less significant SNPs wrt RS7807695:

SNP R’ in 550K EF data R’ in HapMap CEU data RS4722O54 O.O11 O.186 RS10268330 O.O11 O.OO8

0266 No high R2 SNPs in 550k assay for RS7807695 (R2 Max EF=0.026, RS6954331) 0267. The lowest P-values for DNAH11 in BMI/WHR analyses:

Subjects SNP BMI Analysis

BMI analysis Eastern Finnish women RS109SO880 O.OO1972 Min P BMI RS7807695 MEN RS7807695 0.4 WHR Analysis WHR analyses P VE MEN WHR RS6978629 O.OO2983 WHR RS7807695 P VE WOMEN WHR RS7807695 O.O1529

TABLE 20

Glucose load interaction.

SNP n1 n2 P MAF Alleles Gene GeneD class

RS7807695 29 1033 2.12E-08 O.O1571S CT DNAH11 8701 intron

0268. The regression coefficient is positive in the smaller (0271 MAF in HapMap CEU population: 0.092 for minor group and negative in the larger group. In the carriers of the allele C. MAF in EF population: 0.015715. minor allele a high glucose load is strongly associated with 0272) Our data set included 29 individuals with heterozy gote minor allele genotype. No minor allele homozygotes WHR, while in the others there is a weak inverse association. were observed. 0269. RS7807695 (0273 LD Block Structure of DNAH11 Region: Hapmap 0270 DNAH11 is a large gene in at posi CEU population chr7:21353669-22103668 by (750 kb) tion 21836050 bp. The marker is located in the intron 65 of (0274 RS7807695 is in weak linkage in HapMap CEU DNAH11 gene. population with other SNPs within 750kb window (including US 2012/0225,047 A1 Sep. 6, 2012

the neighboring genes SP4 and CDCA7L). RS7807695 has 0284. Shah et al. have recently shown that loss of Bardet D'=1 with 208 SNPs, however, the highest R2 values are: Biedl syndrome proteins (that relate to obesity) alters the morphology and function of motile cilia in airway epithelia (Shah A S, et al. Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway marker1 marker2 D' r 2 LOD Location epithelia. Proc Natl AcadSci USA. 2008 Mar 4:105(9):3380 rsf807695 rS2893060 0.662 0.328 5.01 DNAH11 intron 74 5. PMID: 18299575). Therefore, it is possible that also rsf807695 rS17145.742 0.633 0.322 4.63 DNAH11 intron 70 DNAH11 may play a similar role in ciliary disorders as what rsf807695 rS4392794 0.682 0.285 4.97 DNAH11 intron 76 has been shown for BBS proteins. Moreover, although cilia rsf807695 rS2O74329 0.588 0.264 4.36 DNAH11 intron 71 are broadly classified as 9+2 type motile cilia and 9+0 type rsf807695 rS1139224 0.769 0.251 4.63 DNAH11 intron 79 sensory immotile cilia, there are examples of 9+2 sensory rsf807695 rS17145715 0.584 0.241 4.09 DNAH11 intron 68 cilia and 9+0 motile cilia (reviewed in Bisgrove B W, Yost H rsf807695 rS10269223 0.545 0.204 3.31 DNAH11 intron 75 J. The roles of cilia in developmental disorders and disease. Development. 2006 Nov;133(21):4131-43. PMID: 0275. The highest R of RS7807695 in HapMap CEU 17021045; and in Christensen ST et al., Sensory Cilia and population to neighboring genes is R=0.082 to rs10238945 Integration of Signal Transduction in Human Health and Dis ease. Traffic. 2007 Feb:8(2): 97-109.). Several signaling class (CDCA7L intron). receptors have been located in motile cilia, including receptor 0276 Conclusions About the Data: tyrosine kinases, Hedgehog, Wnt and steroid signaling and (0277. The associated SNP RS7807695 pinpoints to the ion channel/calcium signaling (reviewed in Christensen STet gene DNAH11, in addition there are two other markers al., 2007). As research with the subject is currently in heavy (RS4722054 and RS 10268330) in this large gene that are hits progress more signaling pathways in cilia are likely to be in the analysis. reported, and may well relate to obesity-associated mecha 0278 DNAH11 GeneID: 8701 nisms. 0279 DNAH11 and Obesity: 0285. As a mechanism of obesity, appetite or absorption of nutrients are possibilities with relation to ciliary mechanism 0280 DNAH11 encodes for a dynein heavy chain family of obesity. Choroid plexus, the area on the ventricles of the protein that is a microtubule-dependent motor ATPase and brain where cerebrospinal fluid (CSF) is produced by modi participates in motility of flagella and cilia. DNAH11 is not fied ependymal cells, is a central site with motile cilia in the presently known to play any role in intracellular dynein func human brain. There are four choroid plexus in the brain, one tion. DNAH11 is expressed in tissues that have flagella or in each of the ventricles. Choroid plexus is immunoreactive cilia. DNAH11 has been shown in human to associate to for leptin protein (Couce M. E. et al., Localization of leptin disorders involving perturbed or absent beating of primary receptor in the human brain. Neuroendocrinology. 1997 Sep motile cilia, such as in PCD and KS. The disorders are char tember;66(3): 145-50.), and circulating leptin is transported acterized by respiratory infections, reduced fertility, and situs into the brain by binding to megalin at the choroid plexus inversus, due to dysfunction of monocilia at the embryonic epithelium (Dietrich MO, et al. Megalin mediates the trans node and randomization of left-right body asymmetry. port of leptin across the blood-CSF barrier. Neurobiol Aging. 0281. Until recently, ciliary structures were thought to be 2008 June:29(6):902-12. PMID: 17324488). Furthermore, in present mainly in structures with dense ciliary content, for amouse model interference of normal energy homeostasis by example in epithelial lining of lungs and ear, olfactory cells, disrupting cilia on throughout the central nervous in spermatozoa, and ovaries. Recent studies have greatly system and on pro-opiomelanocortin-expressing cells in the increased understanding of ciliary function in several cell hypothalamus (lining next to Ventricular choroid plexus, types and tissues. For example, in brain cilia play roles in resulted in obesity (Davenport J R et al., Curr Biol. 2007 Sep. Hedgehog-signaling, and in neural stem cell generation 18:17(18): 1586-94). (Hedgehog signaling and primary cilia are required for the 0286. In conclusion, DNAH11 may play a role in obesity formation of adult neural stem cells. Nat Neurosci. 2008 and energy and carbohydrate intolerance by modulating the Mar;11(3):277-84. PMID: 18297065). function of motile cilia. This could be due to alterations for 0282. Cilia seem to play a role in obesity, mainly based on example in ciliary beating, protein transport or localization in the evidence that genes mutated in patients with BBS encode cilia. These alterations may affect chemosensory mecha for proteins that have ciliary function. In animal models, nisms and/or intracellular or neuroendocrine signaling. ciliary disruption has been shown to result in obesity, poten Potential sites of action are both peripheral and central. tially through central nervous system action. It has been pro 0287 Markers in the DNAH11 gene also significantly posed that pro-opiomelanocortin expressing cells in hypo modified the association between dietary glucose load and thalamus could relay the pathways for regulating Satiety WHR (2.12x10). This enzyme-coding gene is also associ responses. Other locations for ciliary dysfunction in obesity ated with obesity, T2D and CHD in several of our studies. A are also likely. large proportion of individuals are susceptible to obesity 0283 DNAH11 appears to mainly relate to motile cilia because of high carbohydrate intake, they are carbohydrate which seem to have functions somewhat different from intolerant. This intolerance can theoretically be cured/attenu immotile, sensory primary cilia. Motile cilia are found in ated by functional foods against this target or its binding or great numbers on the surface of the epithelial cells lining the functional partners. airways and reproductive tracts and on epithelial cells of the 0288 CDKAL1 as Energy and Carbohydrate Intolerance ependyma and choroid plexus in the brain. DNAH11 has been Gene especially shown to affect the motility of airway epithelial (0289 BMI: Carbohydrate IntakexSNP Interaction motile cilia, whereas it has been shown not to inherently 0290 Continuous Variable: ln(BMI) affect the motility of sperm. The function of DNAH11 outside 0291 Adjusted for: Age, HT-status, average weekly exer of motile cilia has not been explored. cise US 2012/0225,047 A1 Sep. 6, 2012 24

TABLE 21 Regression model (ln(BMI) = mu + carboh + e, where carboh is carbohydrate intake in food) within different genotype groups. Unstandardized Standardized Coefficients Coefficients B Std. Error Beta t Sig. (Constant) 3.036151 0.035271 86.08079 () Age O.OO1751 O.OOOS26 O.O91377 3.327731 O.OOO902 HT status O.104684 O.OO869 O.329856 12.04586 1.24E-31 Average weekly -OOO211 O.OOO705 -0.08.207 -298647 (O.OO2879 exercise (hours) Dependent Variable: BMI In The model was separately used for samples with minor allele present and samples that are homozygous for the major allele, B = regression coefficient SE = se of the coefficient P = P-value of the test statistic

TABLE 22 Single-SNP associations of SNPs related to the CDKAL1 gene with the carbohydate intake - BMI interaction.

SNP P HW MAF CR CHR Position Gene GeneD class

RS16884O72 S.O772E-06 0.127074 0.208023 1 6 2O763482 CDKAL1 S4901 intron RS7364.25 S.O772E-06 0.127074 0.208023 1 6 2O772291 CDKAL1 S4901 intron RS10484632 6.589E-06 O.OO7833 O.216239 O.998.346 6 2O7SS639 CDKAL1 S4901 intron RS13 1944O7 6.6385E-06 OOO2442. O.196443 1 6 2O7389.32 CDKAL1 S4901 intron

SNP B1 SE1 P1 n1 B2 SE2 P2 n2 Z. P

RS16884O72 -0.001785.14 O.OOOS28 O.OOO796 393 O.OO 1033 O.OOO32O59 O.OO133622 663 -4561.78 RS736425 -0.001785.14 O.OOOS28 O.OOO796 393 O.OO 1033 O.OOO32O59 O.OO133622 663 -4561.78 RS10484632 -0.00174339 O.OOOS22 O.OOO91S 403 0.001023 O.OOO323O4 O.OO16164 651 -4.50675 RS13 1944O7 -O.OO184241 O.OOOS43 O.OOO767 375 O.OOO993 O.OOO31776 O.OO1862S 681 -4.50516

0292 FIG. 6 shows linear regression between soluble car bohydrate intake (g/d) and BMI in RS 16884072 A/G and G/G genotypes. Carbohydrate intake vs BMI in subjects with the 0293 RS16884072 A/A genotype (upper figure) and in the combined group of subjects with A/G or G/G genotype (BMI is used as y value i.e. ordinant instead of ln(BMI) in these figures.)

TABLE 23

The Smallest P-values for SNPs in BMI and WHR analyses:

SNP MIN P BMI ANALYSIS 2 MIN P WHR ANALYSIS WHR

RS16884O72 O.2288 WOMEN 0.151045 P BIN VE MEN WHR RS7364.25 O.140558 BINARY BOTH 0.151045 P BIN VE MEN WHR GENDERS RS10484632 O.174052 BINARY BOTH 0.085541 P BIN VE ALL WHR GENDERS RS13 1944O7 O.1262 MEN 0.030742 P BIN VE MEN WHR US 2012/0225,047 A1 Sep. 6, 2012

0294 D' and R2 Values for Most Significant Markers in centration (Pascoe L. et al. 2007). Diabetes-associated vari BMI: Carbohydrate IntakexSNP Interaction Analysis ants in CDKAL1 impair insulin secretion and conversion of

L1 L2 D' LOD r 2 CIlow CIhi Dist

RS16884O72 RS73642S 1 418.OS 1 O.99 1 88.09 RS16884O72 RS10484632 1 384.31 O.954 O.99 1 7843 RS16884O72 RS13 1944O7 O.968 318.8 O.871 O.94 O.99 245SO

0295 The most significant markers are in linkage in the proinsulin to insulin (Kirchhoff K et al. 2008). Therefore, Eastern Finnish population some CDKAL1 alleles are likely to increase the risk of type 2 0296 CDKAL1, GenelD: 54901, CDK5 Regulatory Sub diabetes by impairing insulin secretion. unit Associated Protein 1-like 1 (mRNA: NM 017774.2: (0300 BMI Per Carbohydrate IntakexSNP Interaction Genomic Reference NC 000006.11) Analysis vs T2D Association 0297 CDKAL1 gene encodes a 579-residue, 65-kD pro 0301 The important role of CDKAL1 in glucose induced tein, which function is unknown. However it shares consid insulin secretion may explain the result obtained in this analy erable domain and amino acid homology with CDK5RAP1, sis (carbohydrate intake). The most significant markers from an inhibitor of CDK5 (cyclin-dependent kinase 5, GeneID: BMI: Carbohydrate intakexSNP interaction analysis are 1020) activation (OMIM). CDK5 has been implicated in the located near to the region that is shown to be associated with regulation of pancreatic beta cell function through formation T2D. Haploview image below presents the location and of p35/CDK5 complexes that down-regulate insulin expres P-values of T2D associated markers that were included into a sion (Ubeda et al., 2006). CDK5RAP1 is expressed in neu replication study. Two SNPs that were significantly associ ronal tissues, where it inhibits cyclin-dependent kinase 5 ated with T2D in the T2D replication are associated in BMI: (CDK5) activity by binding to the CDK5 regulatory subunit Carbohydrate intakexSNP interaction analysis (table below). p35. In pancreatic beta cells, CDK5 has been shown to have a Therefore, it cannot be said whether these two associations role in the loss of beta cell function under glucotoxic condi are related with each other.

TABLE 24 Association of the strongest T2D related SNPs with the CH-BMI interaction. P for association P for with T2D BMI*Charbohydrate MARKER POSITION GENE X2 (replication) intake interaction

RS1569699 2O787289 CDKAL1 34.81 3.62771E-09 O.OS1111S2 RS7756992 2O787688 CDKAL1 34.62 3.99569E-09 O.O4738437 tions. Furthermore, inhibition of the CDK5/p35 complex pre 0302 References vents a decrease of insulin that results from 0303 Barret J C et al. Genome-wide association defines glucotoxicity. Steinthorsdottir et al. (2007) speculated that more than 30 distinct suspectibility loci for Crohn's disease CDKAL1 may have a role in the inhibition of the CDK5/p35 Nat Genet 2008: 40:955-962 complex in pancreatic beta cells similar to that of 0304 Diabetes Genetics Initiative of Broad Institute of CDK5RAP1 in neuronal tissue. Reduced expression of Harvard and MIT, Lund University, and Novartis Institutes CDKAL1 or reduced inhibitory function thus could lead to an for BioMedical Research: Genome-wide association analysis impaired response to glucotoxicity. identifies loci for type 2 diabetes and triglyceride levels. 0298. In genomewide association studies, the Wellcome Science 316: 1331-1336, 2007. Trust Case Control Consortium (2007), Diabetes Genetics (0305 Kirchhoff Ket al. Polymorphisms in the TCF7L2, Initiative of Broad Institute of Harvard and MIT, Lund Uni CDKAL1 and SLC30A8 genes are associated with impaired versity, and Novartis I, Zeggini et al. (2007), and Scott et al. proinsulin conversion. Diabetologia. 2008 April:51(4):597 (2007) identified association of single-nucleotide polymor 6O1 phisms (SNPs) within intron 5 of the CDKAL1 gene with 0306 Pascoe Let al. Common variants of the novel type 2 susceptibility to type 2 diabetes (OMIM). Barret et al. (2008) diabetes genes CDKAL1 and HHEX/IDE are associated with have identified the same genomic region associated with decreased pancreatic beta-cell function). Diabetes. 2007 Crohn's disease. However, the associated alleles for these two December:56(12):3101-4 diseases were not correlated. We have also replicated 0307 Scott, L. J.; Mohlke. K. L.; Bonnycastle, L. L.; CDKAL1 T2D associated region in our replication study. Willer, C.J.; Li.Y.; Duren, W. L.; Erdos, M. R.: Stringham, H. 0299 Further studies have shown that CDKAL1 diabetes M.; Chines, P. S.; Jackson, A. U.: Prokunina-Olsson, L.; associated alleles are associated with decreased pancreatic Ding, C.-J., and 29 others: A genome-wide association study beta-cell function, including decreased beta-cell glucose sen of type 2 diabetes in Finns detects multiple susceptibility sitivity that relates insulin secretion to plasma glucose con variants. Science 316: 1341-1345, 2007 US 2012/0225,047 A1 Sep. 6, 2012 26

0308 Steinthorsdottir et al. A variant in CDKAL1 influ- R. B.; Rayner, N. W.; Freathy, R. M.; Barrett, J. C.; Shields, ences insulin response and risk of type 2 diabetes. Nat Genet. B.; and 15 others: Replication of genome-wide association 2007 June;39(6):770-5. signals in UK samples reveals risk loci for type 2 diabetes. - Science 316: 1336-1341, 2007. 0309 M. Ubeda, J. M. Rukstalis, J. F. Habener, Inhibition 0311 VWF von Willebrand Factor of cyclin-dependent kinase 5 activity protects pancreatic beta 0312 BMI Per Carbohydrate IntakexSNP Interaction cells from glucotoxicity. J. Biol. Chem. 281, 28858 (2006) 0313 Continuous Variable: ln(BMI) 0310 Zeggini, E.; Weedon, M. N.; Lindgren, C.M.; Fray- 0314 Adjusted for: Age, HT-status, average weekly exer ling, T. M.: Elliott, K. S.; Lango, H.; Timpson, N.J.; Perry, J. cise

TABLE 25

Regression model (ln(BMI) = mu+ carboh + e, where carboh is carbohydrate intake in food) within different genotype groups.

Unstandardized Standardized Coefficients Coefficients B Std. Error Beta t Sig.

(Constant) 3.036151 0.035271 86.08079 O Age O.OO1751 O.OOOS26 O.O91377 3.327731 O.OOO902 HT status O.104684 O.OO869 O.329856 12.04586 1.24E-31 Average weekly exercise (hours) -OOO211 O.OOO705 -0.082O7 -298647 O.OO2879 Dependent Variable: BMI In

The model was separately used for samples with minor allele present and samples that are homozygous for the major allele, B = regression coefficient SE = se of the coefficient P = P-value of the test statistic

TABLE 26 Result from BMI per Carbohydrate intake x SNP interaction analysis for RS17491334, VWF gene. SNP P HW MAF CR Alleles CHR Position Gene GeneID class Flanking genes 10K RS1749 1334 3.71E-06 0.393.652 O. 102151 1 AG 12 S9741OS WWF 74SO introl WWF 7450

B1 SE1 t1 P1 n1 B2 SE2 t2 P2 n2 Z.

-OOO242 O.OOO633 -3.82229 O.OOO178 195 O.OOO833 O.OOO3OS 2.726,287 O.OO6535 861 -4.627O6

TABLE 27 Significant P-values for RS1749 1334 in BMI and WHR analyses MARKER MIN P BMI ANALYSIS BMI MIN P WHR ANALYSIS WHR RS1749 1334 O4O79 MEN 0.365562 P BIN VE WOMEN WHR

TABLE 28 P-values for VWF gene in BMI analysis MARKER MIN P BMI ANALYSIS BMI CHR POSITION CLASS GENE GENE ID HWE CONT MAF CR RS7955850 O.OOO2766 MEN 12 6045840 intron VWF 7450 O.857624 O.O74O28 1 RS21681.1 O.OOOS1 OS WOMEN 12 5985.53S introl VWF 7450 O.OOO396 O.346761 O.995 US 2012/0225,047 A1 Sep. 6, 2012 27

TABLE 29 P-values for VWF gene in WHR analysis MARKER MIN P WHR ANALYSIS WHR CHR POSITION CLASS

RS2O58473 0.005923 P VE WOMEN WHR 12 S964187 intron RS21681.1 0.012571 P BIN VE ALL WHR 12 598.5535 intron

MARKER GENE GENE ID HWE CONT MAF CR

RS2O58473 WWF 7450 O.OS7936 O.403146 O.999.173 RS216811 WWF 7450 O.OOO396 O.346761 O.995864

0315 VWF von Willebrand Factor 0326) “Visceral obesity has been associated with an 0316 GeneID: 7450 increased cardiovascular risk. However, the exact mecha 0317 mRNA: NM 000552.3 nisms are not completely clear. In this study we investigated 0318 Genomic sequence: NC 000012.11 the relationship between von Willebrand factor (VWF) and 0319. Official Symbol: VWF visceral adipose tissue (VAT) in a group of 181 overweight 0320 Official Full Name von Willebrand factor and obese premenopausal women visiting the weight man 0321. Also known as VWD; F8VWF agement clinic of a university hospital. von Willebrand factor 0322 VWF Literature Related to Body Mass, Insulin antigen (v.WF: Ag), plasminogen activator inhibitor 1 (PAI-1) Resistance activity, VAT (computed tomography scan), insulin resistance 0323 Meigs JB, Odonnell CJ, Tofler GH, Benjamin EJ, (homeostasis model assessment of insulin resistance), and Fox CS, Lipinska I, Nathan DM, Sullivan L. M. D’Agostino other anthropometric and metabolic parameters were mea RB, Wilson PW. Hemostatic markers of endothelial dysfunc sured. Subjects with VAT in the highest quintile had signifi tion and risk of incident type 2 diabetes: the Framingham cantly higher levels of VWF:Ag (171+/-60 vs 129+/-40%: Offspring Study. Diabetes. 2006 February:55(2):530-7 P=0.001) and PAI-1 (24.7+/-8.5 vs 15.2+/- 12.0 AU/mL: 0324 “Endothelial dysfunction may precededevelopment P<0.001) compared with subjects in the lowest quintile. After of type 2 diabetes. We tested the hypothesis that elevated correction for fat mass and homeostasis model assessment of levels of hemostatic markers of endothelial dysfunction, plas insulin resistance the difference was still significant for VWF: minogen activator inhibitor-1 (PAI-1) antigen, and von Will Ag (P=0.046), but not for PAI-1 (Pd(0.05). Stepwise multiple ebrand factor (VWF) antigen predicted incident diabetes inde regression analysis showed VAT and insulin resistance as pendent of other diabetes risk factors. We followed 2,924 independent determinants of VWF: Ag, whereas waist cir Framingham Offspring Subjects (54% women, mean age 54 cumference, high-density lipoprotein cholesterol, and insulin years) without diabetes at baseline (defined by treatment, resistance were independent determinants of PAI-1 activity. fasting plasma glucose-or-7 or 2-h postchallenge In a Subgroup of 115 patients, we measured high-sensitivity glucose-or=11.1 mmol/l) over 7 years for new cases of dia C-reactive protein and found it to influence the relationship betes (treatment or fasting plasma glucose or 7.0 mmol/l). between VAT and v WF:Ag (r=0.16; P=0.088), whereas the We used a series of regression models to estimate relative relationship with PAI-1 was still significant (r-0.21; P=0. risks for diabetes per interquartile range (IQR) increase in 025). The results from this preliminary study suggest a plau PAI-1 (IQR 16.8 ng/ml) and vWF (IQR 66.8% of control) sible relation between visceral obesity and endothelial acti conditioned on baseline characteristics. Over follow-up, Vation, possibly mediated by low-grade inflammation' there were 153 new cases of diabetes. Age- and sex-adjusted 0327 Garanty-Bogacka B, Syrenicz, M. Syrenicz A, relative risks of diabetes were 1.55 per IQR for PAI-1 (95%CI Gebala A. Walczak M. Relation of acute-phase reaction and 141-1.70) and 1.49 for vWF (1.21-1.85). These effects endothelial activation to insulin resistance and adiposity in remained after further adjustment for diabetes risk factors obese children and adolescents. Neuro Endocrinol Lett. 2005 (including physical activity: HDL cholesterol, triglyceride, October:26(5):473-9. and blood pressure levels; Smoking; parental history of dia 0328 “There is increasing evidence that an ongoing betes; use of alcohol, nonsteroidal anti-inflammatory drugs, cytokine-induced acute-phase response is closely involved in exogenous estrogen, or hypertension therapy; and impaired the pathogenesis of type 2 diabetes and associated complica glucose tolerance), waist circumference, homeostasis model tions such as dyslipidemia and atherosclerosis. Garanty assessment of insulin resistance, and inflammation (assessed Bogacka et al. (2005) investigated the relationship of inflam by levels of C-reactive protein): the adjusted relative risks mation and endothelial activation with insulin resistance in were 1.18 per IQR for PAI-1 (1.01-1.37) and 1.39 for vWF childhood obesity. Two hundred and eleven (122 boys) obese (1.09-1.77). We conclude that in this community-based children and adolescents were examined. Fasting levels of sample, plasma markers of endothelial dysfunction increased ultra-sensitive C-reactive protein (CRP), fibrinogen (FB), risk of incident diabetes independent of other diabetes risk interleukin-6 (IL-6), interleukin-1beta (IL-1beta), intercellu factors including obesity, insulin resistance, and inflamma lar cell adhesion molecule-1 (ICAM-1), vascular cell adhe tion. sion molecule-1 (VCAM-1), von Willebrand factor (vWF), 0325 Mertens I, Van der Planken M, Corthouts B, Van glucose, insulin, and Hb Alc were determined. Insulin resis Gaal L. F. Is visceral adipose tissue a determinant of von tance was assessed by the homeostasis method. HOMAIR Willebrand factor in overweight and obese premenopausal correlated significantly with all measures of adiposity as well women? Metabolism. 2006 May:55(5):650-5 as with majority of inflammation and endothelial dysfunction US 2012/0225,047 A1 Sep. 6, 2012 28 markers. After adjustment forage, gender, BMI and fat mass, 42) with serum triglyceride levels (P<0.001), age (P<0.01), the correlation with insulin resistance remained significant insulin sensitivity index (P<0.02), and albuminuria levels for CRP, ICAM-1 and von Willebrand factor. There was a (P<0.04). VWF levels were associated (adjusted r2=0.22) trend for association between HOMAIR and IL-6 as well as with albuminuria (P<0.001), fibrinogen levels (P<0.02), and HOMAIR and fibrinogen. Acute-phase reaction and endot BMI (P<0.03). CONCLUSIONS: Compared with hypercho helial activation correlate with insulin resistance in obese lesterolemic patients, type 2 diabetic patients with dyslipi youth. It is possible that the cluster of these pro-atherogenic demia have increased levels of ET-1 and VWF which may factors may contribute to the accelerated atherosclerosis in indicate more pronounced endothelial injury. These findings obese children' appear to be related to components of the insulin resistance 0329 Weyer C, Yudkin JS, Stehouwer CD, Schalkwijk C syndrome. G, Pratley RE, Tataranni PA. Humoral markers of inflam 0333 Summary: The present invention proposes that mation and endothelial dysfunction in relation to adiposity endothelial dysfunction markers, such as VWF, correlate with and in vivo insulin action in Pima Indians. Atherosclerosis. obesity and insulin resistance. What is unclear is whether 2002 March;161(1):233-42. there is any specific metabolic route related to obesity in 0330 “In adults, obesity and IR are associated with higher which VWF could be directly involved or is VWF only a levels of circulating endothelial dysfunction biomarkers such marker of specific metabolic situations. as soluble intercellular adhesion molecule-1 (SICAM-1) and 0334 MS4A2 Membrane-Spanning 4-Domains, Subfam von Willebrand factor (vWF). Weyer et al (2002) measured ily A. Member 2 (Fc Fragment of IgE, High Affinity I, Recep fasting plasma concentrations of the inflammatory markers tor for; Beta Polypeptide) C-reactive protein (CRP), secretory phospholipase A2 0335| Official Symbol: MS4A2 (sPLA2) and soluble intercellular adhesion molecule-1 (sI 0336. Official Full Name: membrane-spanning 4-do CAM-1) and of the endothelial markers E-selectin and von mains, subfamily A, member 2 (Fc fragment of IgE, high Willebrand factor (vWF) in 32 non-diabetic Pima Indians (18 affinity I, receptor for; beta polypeptide) M/14 F, age 27+/- 1 years) in whom percent body fat and 0337 Also known as: APY; IGEL: IGER: ATOPY: insulin-stimulated glucose disposal (M) were assessed by FCERI; IGHER: MS4A1. FCER1B DEXA and a hyperinsulinemic clamp, respectively. CRP, 0338 GeneID: 2206 sPLA2, and sICAM-1 were all positively correlated with percent body fat (r=0.71, 0.57, and 0.51, all P-0.01). E-se 0339 mRNA: NM 000139.3 lectin and VWF were not correlated with percent body fat, but (0340 Genomic Sequence: NC 000011.9 were negatively correlated with M (r--0.65 and -0.46, both 0341 Summary: The allergic response involves the bind P<0.001) and positively correlated with CRP (r—0,46, and ing of allergen to receptor-bound IgE followed by cell acti 0.33, both P-0.05). These findings indicated that humoral vation and the release of mediators responsible for the mani markers of inflammation increase with increasing adiposity festations of allergy. The IgE-receptor, a tetramer composed in Pima Indians whereas humoral markers of endothelial of an alpha, beta, and 2 disulfide-linked gamma chains, is dysfunction increase primarily in proportion to the degree of found on the Surface of mast cells and basophils. This gene insulin resistance and inflammation. Thus, obesity and insu encodes the beta subunit of the high affinity IgE receptor lin resistance appear to be associated with low-grade inflam which is a member of the membrane-spanning 4A gene fam mation and endothelial dysfunction, respectively, even in an ily. Members of this nascent protein family are characterized by commonstructural features and similar intron/exon splice obesity- and diabetes-prone population with relatively low boundaries and display unique expression patterns among propensity for atherosclerosis.” hematopoietic cells and nonlymphoid tissues. This family 0331 Seligman B. G. Biolo A, Polanczyk CA, Gross J L. member is localized to 1 1 q12, among a cluster of family Clausell N. Increased plasma levels of endothelin 1 and von members. (Entrez) Willebrand factor in patients with type 2 diabetes and dys 0342 Function: Binds to the Fc region of immunoglobu lipidemia. Diabetes Care. 2000 September:23(9): 1395-400 lins epsilon. High affinity receptor. Responsible for initiating 0332 OBJECTIVE: Endothelial markers endothelin 1 the allergic response. Binding of allergen to receptor-bound (ET-1) and von Willebrand factor (VWF) were assessed in IgE leads to cell activation and the release of mediators (such patients with type 2 diabetes and dyslipidemia and in patients as histamine) responsible for the manifestations of allergy. with hypercholesterolemia. RESEARCH DESIGN AND The same receptor also induces the secretion of important METHODS: In this case-control study, plasma ET-and VWF lymphokines (GeneCards) levels were measured by enzyme-linked immunosorbent assay in 35 normoalbuminuric type 2 diabetic patients with 0343 References dyslipidemia (56+/-5 years), in 21 nondiabetic patients with 0344 Donnadieu, E.; Jouvin, M.-H.; Rana, S.; Moffatt, M. hypercholesterolemia (52+/-7 years), and in 19 healthy con F.; Mockford, E. H.; Cookson, W.O.; Kinet, J.-P. :Competing trol subjects (45+/-4 years). All of the individuals were nor functions encoded in the allergy-associated Fc-epsilon-RI motensive and nonsmokers. Urinary albumin was measured beta gene. Immunity 18: 665-674, 2003. by immunoturbidimetry. RESULTS: ET-1 levels were higher (0345 Foister-Hoist, R.; Moises, H. W.; Yang, L.; Fritsch, (P<0.0001) in type 2 diabetic dyslipidemic patients (1.62+/- W.; Weissenbach, J.; Christophers, E. Linkage between atopy 0.73 pg/ml) than in both nondiabetic hypercholesterolemic and the IgE high-affinity receptor gene at 11 q13 in atopic patients (0.91+/-0.73 pg/ml) and control subjects (0.69+/-0. dermatitis families. Hum. Genet. 102:236-239, 1998. 25 pg/ml). VWF levels were significantly increased (P=0.02) 0346 Hill, M. R.; Cookson, W. O. C. M. A new variant of in type 2 diabetic (185.49+/-72.1%) and hypercholester the beta subunit of the high-affinity receptor for immunoglo olemic (163.29--/-50.7%) patients compared with control bulin E (Fc-epsilon-RI-beta E237G): associations with mea subjects (129.70+/-35.2%). In the multiple linear regression Sures of atopy and bronchial hyper-responsiveness. Hum. analysis. ET-1 was significantly associated (adjusted r2–0. Molec. Genet. 5: 959-962, 1996. US 2012/0225,047 A1 Sep. 6, 2012 29

0347 Hizawa, N.; Yamaguchi, E.; Furuya, K. Ohnuma, 0364 Agiant novel gene undergoing extensive alternative N.: Kodama, N.; Kojima, J.: Ohe, M.: Kawakami, Y. Asso splicing is severed by a Cornelia de Lange-associated trans ciation between high serum total IgE levels and D11S97 on location breakpoint at 3q26.3. Hum. Genet. 115: 139-148, chromosome 11q13 in Japanese subjects. J. Med. Genet. 32: 2004. 363-369, 1995. 0365 All publications, patents, patent applications, Gene 0348 Kuster, H.; Zhang, L.; Brini, A.T.: MacGlashan, D. IDs, and accession numbers for nucleic acid or amino acid W. J.; Kinet, J.-P. The gene and cDNA for the human high sequences cited herein are hereby incorporated by reference affinity immunoglobulin E receptor beta chain and expression in their entirety for all purposes to the same extent as if each of the complete human receptor. J. Biol. Chem. 267: 12782 individual publication, patent, patent application, nucleic 12787, 1992. acid or amino acid sequence were specifically and individu 0349 Sandford, A. J.; Shirakawa, T.; Moffatt, M. F.; ally indicated to be so incorporated by reference (i.e. as if the Daniels, S. E.; Ra, C.; Faux, J. A.; Young, R. P.; Nakamura, Y.: publications and sequences were disclosed as Such in the Lathrop, G. M.; Cookson, W. O. C.M.; Hopkin, J. M. Locali specification). sation of atopy and beta Subunit of high-affinity IgE receptor 1-80. (canceled) (FCER1) on chromosome 11q. Lancet 341: 332-334, 1993. 81. A method for risk assessment, diagnosis or prognosis of 0350 Shirakawa, T.: Li, A.; Dubowitz, M.: Dekker, J.W.; obesity or type 2 diabetes (T2D) in a mammalian subject Shaw, A. E.; Faux, J. A.; Ra, C.; Cookson, W. O. C. M.; comprising: Hopkin, J. M. Association between atopy and variants of the a) providing a biological sample taken from the Subject; beta subunit of the high-affinity immunoglobulin E receptor. b) detecting one or more T2D and/or obesity associated Nature Genet. 7: 125-130, 1994. genetic markers in said sample, wherein the genetic 0351. Traherne, J. A.: Hill, M. R.: Hysi, P.; D'Amato, M.: markers are related to SUCLA2 gene, and; Broxholme, J.; Mott, R.; Moffatt, M. F.; Cookson, W. O. C. c) comparing the genetic marker data from the Subject to M. LD mapping of maternally and non-maternally derived genetic marker data from healthy and diseased people to alleles and atopy in Fc-epsilon-RI-beta. Hum. Molec. Genet. make risk assessment, diagnosis or prognosis of obesity 12: 2577-2585, 2003. Or T2D. 0352 NAALADL2, N-Acetylated Alpha-Linked Acidic 82. The method according to claim 81, wherein the genetic Dipeptidase-Like 2 marker is SNP marker RS12873870 in SUCLA2 gene. 0353 GeneID: 254827 83. A test kit for risk assessment, diagnosis or prognosis of 0354 mRNA: NM 207015.2 obesity or T2D comprising: 0355 Genomic Sequence: NC 000003.11 a) reagents, materials and protocols for assessing type and/ 0356. Official Symbol: NAALADL2 or level of one or more T2D and/or obesity phenotype 0357 Official Full Name: N-acetylated alpha-linked associated genetic markers in a biological sample, acidic dipeptidase-like 2 wherein the genetic markers are related to SUCLA2 gene, and; 0358 Function: not known b) instructions and Software for comparing the genetic 0359 Domain Descriptions: PA hNAALADL2 like: marker data from a Subject to genetic marker data from Protease-associated domain containing proteins like human healthy and diseased people to make risk assessment, N-acetylated alpha-linked acidic dipeptidase-like 2 protein diagnosis or prognosis of obesity or T2D. (hNAALADL2). This group contains various PA domain 84. The kit according to claim 83, wherein the genetic containing proteins similar to hNAALADL2. The function of marker is SNP marker RS12873870 in SUCLA2 gene. hNAALADL2 is unknown. This gene has been mapped to a 85. A method for screening agents for preventing or treat chromosomal region associated with Cornelia de Lange Syn ing obesity or T2D in a mammal comprising determining the drome. The significance of the PA domain to hNAALADL2 effect of an agent either on a metabolic pathway related to a has not been ascertained. It may be a protein-protein interac polypeptide or a RNA molecule encoded by SUCLA2 gene in tion domain. At peptidase active sites, the PA domain may living cells; wherein an agent altering activity of the meta participate in Substrate binding and/or promoting conforma bolic pathway is considered useful in prevention or treatment tional changes, which influence the stability and accessibility of obesity or T2D. of the site to substrate. 86. Method for monitoring a risk of an individual to 0360 TFR dimer; Transferrin receptor-like dimerisation become obese comprising a step of measuring the urinary domain. This domain is involved in dimerisation of the trans excretion of a Krebs cycle metabolite dependent on SUCLA2 ferrin receptor as shown in its crystal structure. gene activity, wherein the metabolite of the SUCLA2 gene is 0361 M20 dimer Super-family; Peptidase dimerisation urinary methylmalonic acid, Succinate (), fuma domain. This domain consists of 4 beta Strands and two alpha rate (fumaric acid) or Succinyl-CoA synthetase activity. helices which make up the dimerisation surface of members 87. A method for risk assessment, diagnosis or prognosis of of the M20 family of peptidases. This family includes a range obesity, type 2 diabetes (T2D) or a T2D related condition in a of Zinc metallopeptidases belonging to several families in the mammalian Subject comprising: peptidase classification. Family M20 are Glutamate carbox a) providing a biological sample taken from the Subject; ypeptidases. Peptidase family M25 contains X-His dipepti b) detecting one or more T2D and/or obesity or related dases. phenotype associated biomarkers in said sample, 0362 References wherein the biomarkers are related to one or more genes 0363 1. Tonkin, E.T.: Smith, M.: Eichhorn, P.; Jones, S.; selected from the group consisting of SUCLA2, KLF4, Imamwerdi, B.: Lindsay, S.; Jackson, M.; Wang, T.-J.: Ire MS4A2, ANO5, NELL1, DNAH11, RNF216, VGLL3, land, M.; Burn, J.; Krantz, I. D.; Carr, P.; Strachan, T.: CDKAL1, VWF, NAALADL2, HSL, PLIN1, and US 2012/0225,047 A1 Sep. 6, 2012 30

PRKACA or said biomarkers are related to one or more ecule encoded by a T2D and/or obesity associated gene polypeptides encoded by said genes, and; selected from the group consisting of SUCLA2, KLF4. c) comparing the biomarker data from the Subject to biom MS4A2, ANO5, NELL1, DNAH11, RNF216, VGLL3, arker data from healthy and diseased people to make risk CDKAL1, VWF, NAALADL2, HSL, PLIN1, and PRKACA assessment, diagnosis or prognosis of obesity, T2D or a in living cells; wherein an agent altering activity of a meta T2D related condition. bolic pathway is considered useful in prevention or treatment of obesity, T2D or a T2D related condition. 88. The method according to claim 87, wherein said obe 93. The method according to claim 92, wherein said agent sity or T2D related condition comprises glucose intolerance, is administered to a model system or organism, and wherein insulin resistance, metabolic syndrome, obesity, a microvas an agent altering or modulating expression, biological activ cular complication Such as retinopathy, nephropathy or neu ity or function of a T2D and/or obesity associated gene ropathy, or a macrovascular complication Such as coronary selected from the group consisting of SUCLA2, KLF4. heart disease, cerebrovascular disease, congestive heart fail MS4A2, ANO5, NELL1, DNAH11, RNF216, VGLL3, ure, claudication or other clinical manifestation ofatheroscle CDKAL1, VWF, NAALADL2, HSL, PLIN1, and PRKACA rosis or arteriosclerosis. or it's encoded polypeptide is considered useful in prevention 89. The method according to claim 87, wherein at least one or treatment of obesity, T2D or a T2D related condition. biomarker is a metabolite of a polypeptide encoded by a gene 94. Method for monitoring energy efficiency, energy con selected from the group consisting of SUCLA2, KLF4. Sumption and physical activity of an individual or a risk of an MS4A2, ANO5, NELL1, DNAH11, RNF216, VGLL3, individual to become obese comprising a step of measuring CDKAL1, VWF, NAALADL2, HSL, PLIN1, and PRKACA. the urinary excretion of a Krebs cycle metabolite dependent 90. The method according to claim 89, wherein the on SUCLA2 gene activity. metabolite of gene SUCLA2 is selected from the group con 95. The method according to claim 94, wherein said sisting of plasma, serum or blood cell or urinary methylma metabolite is methylmalonate or methylcitrate. lonic acid. Succinate (Succinic acid) and fumarate (fumaric 96. Method for monitoring or assessing energy intolerance acid). or energy efficiency of an individual comprising a step of 91. A test kit for risk assessment, diagnosis or prognosis of calculating the ratio of body mass index, BMI, and/or waist obesity, T2D or a T2D related condition comprising: hip ratio, WHR, to dietary energy intake, wherein high ratio a) reagents, materials and protocols for assessing type and/ of BMI and/or WHR to dietary energy intake denotes energy or level of one or more T2D and/or obesity phenotype intolerance, i.e. BMI/WHR tends to rise easier or at a lower associated biomarkers in a biological sample, wherein energy intake levels. the biomarkers are related to one or more genes selected 97. Recombinant HSL or analogs of HSL for use in the from the group consisting of SUCLA2, KLF4, MS4A2, treatment of obesity, type 2 diabetes (T2D) or a T2D related ANO5, NELL1, DNAH11, RNF216, VGLL3, condition. CDKAL1, VWF, NAALADL2, HSL, PLIN1, and 98. Recombinant perilipin A or analogs of perilipin A or PRKACA or said biomarkers are related to one or more cAMP-dependent protein kinase for use in the treatment of polypeptides encoded by said genes, and; obesity, type 2 diabetes (T2D) or a T2D related condition. b) instructions and software for comparing the biomarker 99. Method for treatment of obesity, type 2 diabetes (T2D) data from a subject to biomarker data from healthy and or a T2D related condition, wherein a pharmaceutically effec diseased people to make risk assessment, diagnosis or tive amount of recombinant HSL, analogs of HSL, recombi prognosis of obesity, T2D or a T2D related condition. nant perilipin A, analogs of perilipin A or cAMP-dependent 92. A method for Screening agents for preventing or treat protein kinase is administered to a patient in need of Such ing obesity, T2D or a T2D related condition in a mammal treatment. comprising determining the effect of an agent either on a metabolic pathway related to a polypeptide or a RNA mol