Genome-wide Association Studies Identify Genetic Loci Associated with Albuminuria in Diabetes

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and associate with increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes mellitus and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (p=2.4*10(-10)). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. SNPs at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in average UACR per minor allele was 21% for HS6ST1 and 13% for RAB38/CTSC (p=6.3*10(-7) and 5.8*10(-7), respectively). Experiments using streptozotocin-treated diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout vs. control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared to controls. The loci identified here confirm known and highlight novel pathways influencing albuminuria.

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

Risk Prediction for Epithelial Ovarian Cancer in 11 United States-Based Case-Control Studies: Incorporation of Epidemiologic Risk Factors and 17 Confirmed Genetic Loci.

Previously developed models for predicting absolute risk of invasive epithelial ovarian cancer have included a limited number of risk factors and have had low discriminatory power (area under the receiver operating characteristic curve (AUC) < 0.60). Because of this, we developed and internally validated a relative risk prediction model that incorporates 17 established epidemiologic risk factors and 17 genome-wide significant single nucleotide polymorphisms (SNPs) using data from 11 case-control studies in the United States (5,793 cases; 9,512 controls) from the Ovarian Cancer Association Consortium (data accrued from 1992 to 2010). We developed a hierarchical logistic regression model for predicting case-control status that included imputation of missing data. We randomly divided the data into an 80% training sample and used the remaining 20% for model evaluation. The AUC for the full model was 0.664. A reduced model without SNPs performed similarly (AUC = 0.649). Both models performed better than a baseline model that included age and study site only (AUC = 0.563). The best predictive power was obtained in the full model among women younger than 50 years of age (AUC = 0.714); however, the addition of SNPs increased the AUC the most for women older than 50 years of age (AUC = 0.638 vs. 0.616). Adapting this improved model to estimate absolute risk and evaluating it in prospective data sets is warranted.

Evidence for three genetic loci involved in both anorexia nervosa risk and variation of body mass index

The maintenance of normal body weight is disrupted in patients with anorexia nervosa (AN) for prolonged periods of time. Prior to the onset of AN, premorbid body mass index (BMI) spans the entire range from underweight to obese. After recovery, patients have reduced rates of overweight and obesity. As such, loci involved in body weight regulation may also be relevant for AN and vice versa. Our primary analysis comprised a cross-trait analysis of the 1000 single-nucleotide polymorphisms (SNPs) with the lowest P-values in a genome-wide association meta-analysis (GWAMA) of AN (GCAN) for evidence of association in the largest published GWAMA for BMI (GIANT). Subsequently we performed sex-stratified analyses for these 1000 SNPs. Functional ex vivo studies on four genes ensued. Lastly, a look-up of GWAMA-derived BMI-related loci was performed in the AN GWAMA. We detected significant associations (P-values <5 × 10(-5), Bonferroni-corrected P<0.05) for nine SNP alleles at three independent loci. Interestingly, all AN susceptibility alleles were consistently associated with increased BMI. None of the genes (chr. 10: CTBP2, chr. 19: CCNE1, chr. 2: CARF and NBEAL1; the latter is a region with high linkage disequilibrium) nearest to these SNPs has previously been associated with AN or obesity. Sex-stratified analyses revealed that the strongest BMI signal originated predominantly from females (chr. 10 rs1561589; Poverall: 2.47 × 10(-06)/Pfemales: 3.45 × 10(-07)/Pmales: 0.043). Functional ex vivo studies in mice revealed reduced hypothalamic expression of Ctbp2 and Nbeal1 after fasting. Hypothalamic expression of Ctbp2 was increased in diet-induced obese (DIO) mice as compared with age-matched lean controls. We observed no evidence for associations for the look-up of BMI-related loci in the AN GWAMA. A cross-trait analysis of AN and BMI loci revealed variants at three chromosomal loci with potential joint impact. The chromosome 10 locus is particularly promising given that the association with obesity was primarily driven by females. In addition, the detected altered hypothalamic expression patterns of Ctbp2 and Nbeal1 as a result of fasting and DIO implicate these genes in weight regulation.

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci : a conservation alert?

Genetic variation at allozyme and mitochondrial DNA loci was investigated in the Australian lungfish, Neoceratodus forsteri Krefft 1870. Tissue samples for genetic analysis were taken non-lethally from 278 individuals representing two spatially distinct endemic populations (Mary and Burnett rivers), as well as one population thought to be derived from an anthropogenic translocation in the 1890's (Brisbane river). Two of 24 allozyme loci resolved from muscle tissue were polymorphic. Mitochondrial DNA nucleotide sequence diversity estimated across 2,235 base pairs in each of 40 individuals ranged between 0.000423 and 0.001470 per river. Low genetic variation at allozyme and mitochondrial loci could be attributed to population bottlenecks, possibly induced by Pleistocene aridity. Limited genetic differentiation was detected among rivers using nuclear and mitochondrial markers suggesting that admixture may have occurred between the endemic Mary and Burnett populations during periods of low sea level when the drainages may have converged before reaching the ocean. Genetic data was consistent with the explanation that lungfish were introduced to the Brisbane river from the Mary river. Further research using more variable genetic loci is needed before the conservation status of populations can be determined, particularly as anthropogenic demands on lungfish habitat are increasing. In the interim we recommend a management strategy aimed at conserving existing genetic variation within and between rivers.

Is there a higher genetic load of susceptibility loci in familial ankylosing spondylitis?

Objective Several genetic risk variants for ankylosing spondylitis (AS) have been identified in genome-wide association studies. Our objective was to examine whether familial AS cases have a higher genetic load of these susceptibility variants. Methods Overall, 502 AS patients were examined, consisting of 312 patients who had first-degree relatives (FDRs) with AS (familial) and 190 patients who had no FDRs with AS or spondylarthritis (sporadic). All patients and affected FDRs fulfilled the modified New York criteria for AS. The patients were recruited from 2 US cohorts (the North American Spondylitis Consortium and the Prospective Study of Outcomes in Ankylosing Spondylitis) and from the UK-Oxford cohort. The frequencies of AS susceptibility loci in IL-23R, IL1R2, ANTXR2, ERAP-1, 2 intergenic regions on chromosomes 2p15 and 21q22, and HLA-B27 status as determined by the tag single-nucleotide polymorphism (SNP) rs4349859 were compared between familial and sporadic cases of AS. Association between SNPs and multiplex status was assessed by logistic regression controlling for sibship size. Results HLA-B27 was significantly more prevalent in familial than sporadic cases of AS (odds ratio 4.44 [95% confidence interval 2.06, 9.55], P = 0.0001). Furthermore, the AS risk allele at chromosome 21q22 intergenic region showed a trend toward higher frequency in the multiplex cases (P = 0.08). The frequency of the other AS risk variants did not differ significantly between familial and sporadic cases, either individually or combined. Conclusion HLA-B27 is more prevalent in familial than sporadic cases of AS, demonstrating higher familial aggregation of AS in patients with HLA-B27 positivity. The frequency of the recently described non-major histocompatibility complex susceptibility loci is not markedly different between the sporadic and familial cases of AS.

Fine mapping and replication of genetic risk Loci in primary sclerosing cholangitis

Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10 -6, OR A vs G = 1.28, 95% CI (1.16-1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10 -4, OR A vs G = 1.20, 95% CI (1.09-1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10 -4, OR A vs T = 1.21, 95% CI (1.09-1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10 -12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10 -8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association. © Informa Healthcare.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Novel genetic susceptibility loci for diabetic end-stage renal disease identified through robust naive Bayes classification

Aims/hypothesis: Diabetic nephropathy is a major diabetic complication, and diabetes is the leading cause of end-stage renal disease (ESRD). Family studies suggest a hereditary component for diabetic nephropathy. However, only a few genes have been associated with diabetic nephropathy or ESRD in diabetic patients. Our aim was to detect novel genetic variants associated with diabetic nephropathy and ESRD. Methods: We exploited a novel algorithm, ‘Bag of Naive Bayes’, whose marker selection strategy is complementary to that of conventional genome-wide association models based on univariate association tests. The analysis was performed on a genome-wide association study of 3,464 patients with type 1 diabetes from the Finnish Diabetic Nephropathy (FinnDiane) Study and subsequently replicated with 4,263 type 1 diabetes patients from the Steno Diabetes Centre, the All Ireland-Warren 3-Genetics of Kidneys in Diabetes UK collection (UK–Republic of Ireland) and the Genetics of Kidneys in Diabetes US Study (GoKinD US). Results: Five genetic loci (WNT4/ZBTB40-rs12137135, RGMA/MCTP2-rs17709344, MAPRE1P2-rs1670754, SEMA6D/SLC24A5-rs12917114 and SIK1-rs2838302) were associated with ESRD in the FinnDiane study. An association between ESRD and rs17709344, tagging the previously identified rs12437854 and located between the RGMA and MCTP2 genes, was replicated in independent case–control cohorts. rs12917114 near SEMA6D was associated with ESRD in the replication cohorts under the genotypic model (p < 0.05), and rs12137135 upstream of WNT4 was associated with ESRD in Steno. Conclusions/interpretation: This study supports the previously identified findings on the RGMA/MCTP2 region and suggests novel susceptibility loci for ESRD. This highlights the importance of applying complementary statistical methods to detect novel genetic variants in diabetic nephropathy and, in general, in complex diseases.

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1alpha

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1alpha) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1alpha was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

A genetic analysis of serotonergic biosynthetic and metabolic enzymes in migraine using a DNA pooling approach

Migraine is a common debilitating primary headache disorder with significant mental, physical and social health implications. The brain neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) is involved in nociceptive pathways and has been implicated in the pathophysiology of migraine. With few genetic studies investigating biosynthetic and metabolic enzymes governing the rate of 5-HT activity and their relationship to migraine, it was the objective of this study to assess genetic variants within the human tryptophan hydroxylase (TPH), amino acid decarboxylase (AADC) and monoamine oxidase A (MAOA) genes in migraine susceptibility. This objective was undertaken using a high-throughput DNA pooling experimental design, which proved to be a very accurate, sensitive and specific method of estimating allele frequencies for single nucleotide polymorphism, insertion deletion and variable number tandem repeat loci. Application of DNA pooling to a wide array of genetic loci provides greater scope in the assessment of population-based genetic association study designs. Despite the application of this high-throughput genotyping method, negative results from the two-stage DNA pooling design used to screen loci within the TPH, AADC and MAOA genes did not support their role in migraine susceptibility.

Scanning the genome for essential hypertension loci

1. Essential hypertension occurs in people with an underlying genetic predisposition who subject themselves to adverse environmental influences. The number of genes involved is unknown, as is the extent to which each contributes to final blood pressure and the severity of the disease. 2. In the past, studies of potential candidate genes have been performed by association (case-control) analysis of unrelated individuals or linkage (pedigree or sibpair) analysis of families. These studies have resulted in several positive findings but, as one may expect, also an enormous number of negative results. 3. In order to uncover the major genetic loci for essential hypertension, it is proposed that scanning the genome systematically in 100- 200 affected sibships should prove successful. 4. This involves genotyping sets of hypertensive sibships to determine their complement of several hundred microsatellite polymorphisms. Those that are highly informative, by having a high heterozygosity, are most suitable. Also, the markers need to be spaced sufficiently evenly across the genome so as to ensure adequate coverage. 5. Tests are performed to determine increased segregation of alleles of each marker with hypertension. The analytical tools involve specialized statistical programs that can detect such differences. Non- parametric multipoint analysis is an appropriate approach. 6. In this way, loci for essential hypertension are beginning to emerge.

Genetic susceptibility to complex traits : moving towards informed analysis of whole-genome screens

Susceptibility to complex traits, by definition, involves aetiological polymorphisms at multiple genetic loci combined with variable contributions by environmental factors. However, the approaches taken to identifying genetic loci implicated in susceptibility to complex traits frequently overlooks the compounding contribution of multiple loci in favour of highlighting a single gene solely responsible for predisposition. It is only in a small minority of cases that this has resulted in clear disease heritability associated with polymorphisms in a single gene. More often, this approach has led to an accumulation of single-gene associations with minor contributions to disease susceptibility. As the genomic era advances and genome-wide screens become higher in resolution and throughput, the need for simultaneous consideration of multiple loci is becoming more important. With special reference to non-Hodgkin’s lymphoma (NHL), this chapter will overview the current progress made in elucidating genetic polymorphisms associated with disease susceptibility. We also present novel data from a high-resolution single nucleotide polymorphism (SNP) microarray screen for susceptibility loci that are involved in NHL. Using an ‘informed approach’, the findings are highlighted within the context of cellular pathways, and provide insight and new ideas for methods of analysis for genome-wide screens for susceptibility.

A genome-wide association study identifies five loci influencing facial morphology in Europeans

Inter-individual variation in facial shape is one of the most noticeable phenotypes in humans, and it is clearly under genetic regulation; however, almost nothing is known about the genetic basis of normal human facial morphology. We therefore conducted a genome-wide association study for facial shape phenotypes in multiple discovery and replication cohorts, considering almost ten thousand individuals of European descent from several countries. Phenotyping of facial shape features was based on landmark data obtained from three-dimensional head magnetic resonance images (MRIs) and two-dimensional portrait images. We identified five independent genetic loci associated with different facial phenotypes, suggesting the involvement of five candidate genes-PRDM16, PAX3, TP63, C5orf50, and COL17A1-in the determination of the human face. Three of them have been implicated previously in vertebrate craniofacial development and disease, and the remaining two genes potentially represent novel players in the molecular networks governing facial development. Our finding at PAX3 influencing the position of the nasion replicates a recent GWAS of facial features. In addition to the reported GWA findings, we established links between common DNA variants previously associated with NSCL/P at 2p21, 8q24, 13q31, and 17q22 and normal facial-shape variations based on a candidate gene approach. Overall our study implies that DNA variants in genes essential for craniofacial development contribute with relatively small effect size to the spectrum of normal variation in human facial morphology. This observation has important consequences for future studies aiming to identify more genes involved in the human facial morphology, as well as for potential applications of DNA prediction of facial shape such as in future forensic applications.