A versatile gene-based test for genome-wide association studies

We have derived a versatile gene-based test for genome-wide association studies (GWAS). Our approach, called VEGAS (versatile gene-based association study), is applicable to all GWAS designs, including family-based GWAS, meta-analyses of GWAS on the basis of summary data, and DNA-pooling-based GWAS, where existing approaches based on permutation are not possible, as well as singleton data, where they are. The test incorporates information from a full set of markers (or a defined subset) within a gene and accounts for linkage disequilibrium between markers by using simulations from the multivariate normal distribution. We show that for an association study using singletons, our approach produces results equivalent to those obtained via permutation in a fraction of the computation time. We demonstrate proof-of-principle by using the gene-based test to replicate several genes known to be associated on the basis of results from a family-based GWAS for height in 11,536 individuals and a DNA-pooling-based GWAS for melanoma in approximately 1300 cases and controls. Our method has the potential to identify novel associated genes; provide a basis for selecting SNPs for replication; and be directly used in network (pathway) approaches that require per-gene association test statistics. We have implemented the approach in both an easy-to-use web interface, which only requires the uploading of markers with their association p-values, and a separate downloadable application.

Common variants in the trichohyalin gene are associated with straight hair in Europeans

Hair morphology is highly differentiated between populations and among people of European ancestry. Whereas hair morphology in East Asian populations has been studied extensively, relatively little is known about the genetics of this trait in Europeans. We performed a genome-wide association scan for hair morphology (straight, wavy, curly) in three Australian samples of European descent. All three samples showed evidence of association implicating the Trichohyalin gene (TCHH), which is expressed in the developing inner root sheath of the hair follicle, and explaining approximately 6% of variance (p=1.5x10(-31)). These variants are at their highest frequency in Northern Europeans, paralleling the distribution of the straight-hair EDAR variant in Asian populations.

Replication of the association of common rs9939609 variant of FTO with increased BMI in an Australian adult twin population but no evidence for gene by environment (G x E) interaction

OBJECTIVE: To further investigate a common variant (rs9939609) in the fat mass- and obesity-associated gene (FTO), which recent genome-wide association studies have shown to be associated with body mass index (BMI) and obesity. DESIGN: We examined the effect of this FTO variant on BMI in 3353 Australian adult male and female twins. RESULTS: The minor A allele of rs9939609 was associated with an increased BMI (P=0.0007). Each additional copy of the A allele was associated with a mean BMI increase of approximately 1.04 kg/m(2) (approximately 3.71 kg). Using variance components decomposition, we estimate that this single-nucleotide polymorphism accounts for approximately 3% of the genetic variance in BMI in our sample (approximately 2% of the total variance). By comparing intrapair variances of monozygotic twins of different genotypes we were able to perform a direct test of gene by environment (G x E) interaction in both sexes and gene by parity (G x P) interaction in women, but no evidence was found for either. CONCLUSIONS: In addition to supporting earlier findings that the rs9939609 variant in the FTO gene is associated with an increased BMI, our results indicate that the associated genetic effect does not interact with environment or parity.

Polymorphisms in the vascular endothelial growth factor gene and the risk of familial endometriosis

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein suspected to be involved in the pathogenesis of endometriosis by establishing a new blood supply to the human exfoliated endometrium. Several transcription factor-binding sites are found in the VEGF 5'-untranslated region and variation within the region increases the transcriptional activity. Six previous studies which tested between one and three single nucleotide polymorphisms (SNPs) in samples comprising 105-215 cases and 100-219 controls have produced conflicting evidence for association between the SNPs in the VEGF region and endometriosis. To further investigate the reported association between VEGF variants and endometriosis, we tested the four VEGF polymorphisms (-2578 A/C, rs699947; -460 T/C, rs833061; +405 G/C, rs2010963 and +936 C/T, rs3025039) in a large Australian sample of 958 familial endometriosis cases and 959 controls. We also conducted a literature-based review of all relevant association studies of these VEGF SNPs in endometriosis and performed a meta-analysis. There was no evidence for association between endometriosis and the VEGF polymorphisms genotyped in our study. Combined association results from a meta-analysis did not provide any evidence for either genotypic or allelic association with endometriosis. Our detailed review and meta-analysis of the VEGF polymorphisms suggests that genotyping assay problems may underlie the previously reported associations between VEGF variants and endometriosis.

Association study of the dystrobrevin-binding gene with schizophrenia in Australian and Indian samples

Numerous studies have reported association between variants in the dystrobrevin binding protein 1 (dysbindin) gene (DTNBP1) and schizophrenia. However, the pattern of results is complex and to date, no specific risk marker or haplotype has been consistently identified. The number of single nucleotide polymorphisms (SNPs) tested in these studies has ranged from 5 to 20. We attempted to replicate previous findings by testing 16 SNPs in samples of 41 Australian pedigrees, 194 Australian cases and 180 controls, and 197 Indian pedigrees. No globally significant evidence for association was observed in any sample, despite power calculations indicating sufficient power to replicate several previous findings. Possible explanations for our results include sample differences in background linkage disequilibrium and/or risk allele effect size, the presence of multiple risk alleles upon different haplotypes, or the presence of a single risk allele upon multiple haplotypes. Some previous associations may also represent false positives. Examination of Caucasian HapMap phase II genotype data spanning the DTNBP1 region indicates upwards of 40 SNPs are required to satisfactorily assess all nonredundant variation within DTNBP1 and its potential regulatory regions for association with schizophrenia. More comprehensive studies in multiple samples will be required to determine whether specific DTNBP1 variants function as risk factors for schizophrenia.

Exclusion of angiotensinogen gene in molecular basis of human hypertension: Sibpair linkage and association analyses in Australian anglo-caucasians

Linkage with essential hypertension has been claimed for a microsatellite marker near the angiotensinogen gene (AGT; chromosome 1q42), as has association for the AGT variants M235T, G(-6)A and A(-20)C. To more rigorously evaluate AGT as a candidate gene for hypertension we performed sibpair analysis with multiple microsatellite markers surrounding this locus and using more sophisticated analysis programs. We also performed an association study of the AGT variants in unrelated subjects with a strong family history (two affected parents). For the linkage study, single and multiplex polymerase chain reaction (PCRs) and automated genescan analysis were conducted on DNA from 175 Australian Anglo-Celtic Caucasian hypertensives for the following markers: D1S2880-(2.1 cM)-D1S213-(2.8 cM)-D1S251-(6.5 cM)-AGT-(2.0 cM) -D1S235. Statistical evaluation of genotype data by nonparametric methods resulted in the following scores: Single-point analysis - SPLINK, P > 0.18; APM method, P > 0.25; ASPEX, MLOD < 0.28; SIB-PAIR, P > 0. 24; Multipoint analysis - MAPMAKER/SIBS, MLOD < 0.24; GENEHUNTER, P > 0.35. Exclusion scores of Lod -4.1 to -5.1 were obtained for these markers using MAPMAKER/SIBS for a lambda(s) of 1.6. The association study of G(-6)A, A(-20)C and M235T variants in 111 hypertensives with strong family history and 190 normotensives with no family history showed significant linkage disequilibrium between particular haplotypes, but we could find no association with hypertension. The present study therefore excludes AGT in the etiology of hypertension, at least in the population of Australian Anglo-Celtic Caucasians studied.

G-protein beta3 subunit gene (GNB3) variant in causation of essential hypertension

-Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein beta3 subunit gene (GNB3) induces formation of a splice variant (Gbeta3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (chi2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105+/-7, 109+/-16, and 128+/-28 mm Hg (mean+/-SD) for CC, CT, and TT, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein beta3 subunit gene in the causation of essential hypertension.

Cross-sectional study of a microsatellite marker in the low density lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Mice lacking the serotonin Htr2B receptor gene present an antipsychotic-sensitive schizophrenic-like phenotype

Impulsivity and hyperactivity share common ground with numerous mental disorders, including schizophrenia. Recently, a population-specific serotonin 2B (5-HT2B) receptor stop codon (ie, HTR2B Q20*) was reported to segregate with severely impulsive individuals, whereas 5-HT2B mutant (Htr2B−/−) mice also showed high impulsivity. Interestingly, in the same cohort, early-onset schizophrenia was more prevalent in HTR2B Q*20 carriers. However, the putative role of 5-HT2B receptor in the neurobiology of schizophrenia has never been investigated. We assessed the effects of the genetic and the pharmacological ablation of 5-HT2B receptors in mice subjected to a comprehensive series of behavioral test screenings for schizophrenic-like symptoms and investigated relevant dopaminergic and glutamatergic neurochemical alterations in the cortex and the striatum. Domains related to the positive, negative, and cognitive symptom clusters of schizophrenia were affected in Htr2B−/− mice, as shown by deficits in sensorimotor gating, in selective attention, in social interactions, and in learning and memory processes. In addition, Htr2B−/− mice presented with enhanced locomotor response to the psychostimulants dizocilpine and amphetamine, and with robust alterations in sleep architecture. Moreover, ablation of 5-HT2B receptors induced a region-selective decrease of dopamine and glutamate concentrations in the dorsal striatum. Importantly, selected schizophrenic-like phenotypes and endophenotypes were rescued by chronic haloperidol treatment. We report herein that 5-HT2B receptor deficiency confers a wide spectrum of antipsychotic-sensitive schizophrenic-like behavioral and psychopharmacological phenotypes in mice and provide first evidence for a role of 5-HT2B receptors in the neurobiology of psychotic disorders

Commentary: Chronic SSRI stimulation of astrocytic 5-HT2B receptors change multiple gene expressions/editings and metabolism of glutamate, glucose and glycogen: A potential paradigm shift

Working on the serotonin (5-hydroxytryptamine, 5-HT) 5-HT2B receptor since several years, we have read with high interest the review by Hertz et al. (2015). Previous studies from our group demonstrated that a direct injection in mouse raphe nucleus of the 5-HT2B agonist BW723C86 has the ability to increase extracellular levels of serotonin, which can be blocked by the selective 5-HT2B receptor antagonist RS127445 (Doly et al., 2008, 2009). We also reported that an acute injection of paroxetine 2 mg/kg in mice knocked out for the 5-HT2B receptor gene or in wild type mice injected with RS127445 (0.5 mg/kg) triggers a strong reduction in extracellular accumulation of 5-HT in hippocampus (Diaz et al., 2012). Following these observations, we showed that acute and chronic BW723C86 injection (3 mg/kg) can mimic the fluoxetine (3 mg/kg) and paroxetine (1 mg/kg) behavioral and biochemical antidepressant effects in mice (Diaz and Maroteaux, 2011; Diaz et al., 2012)...

Next-generation sequencing: A frameshift in skeletal dysplasia gene discovery

In the last decade, huge breakthroughs in genetics - driven by new technology and different statistical approaches - have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis. © International Osteoporosis Foundation and National Osteoporosis Foundation 2013.

Pheo-type: A diagnostic gene-expression assay for the classification of pheochromocytoma and paraganglioma

Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Genetic and epigenetic variants in the MTHFR gene are not associated with non-Hodgkin lymphoma

The methylenetetrahydrofolate reductase (MTHFR) gene codes for the MTHFR enzyme which plays a key role in the pathway of folate and methionine metabolism. Polymorphisms of genes in this pathway affect its regulation and have been linked to lymphoma. In this study we examined whether we could detect an association between two common non-synonomous MTHFR polymorphisms, 677C>T (rs1801133) and 1298A>C (rs1801131), and susceptibility to non-Hodgkin lymphoma (NHL) in an Australian case-control cohort. We found no significant differences between genotype or allele frequencies for either polymorphisms between lymphoma cases and controls. We also explored whether epigenetic modification of MTHFR, specifically DNA methylation of a CpG island in the MTHFR promoter region, is associated with NHL using blood samples from patients. No difference in methylation levels was detected between the case and control samples suggesting that although hypermethylation of MTHFR has been reported in tumour tissues, particularly in the diffuse large B-cell lymphoma subtype of NHL, methylation of this MTHFR promoter CpG island is not a suitable epigenetic biomarker for NHL diagnosis or prognosis in peripheral blood samples. Further studies into epigenetic variants could focus on genes that are robustly associated with NHL susceptibility.

Evaluation of a 7-gene genetic profile for athletic endurance phenotype in Ironman championship triathletes

Polygenic profiling has been proposed for elite endurance performance, using an additive model determining the proportion of optimal alleles in endurance athletes. To investigate this model’s utility for elite triathletes, we genotyped seven polymorphisms previously associated with an endurance polygenic profile (ACE Ins/Del, ACTN3 Arg577Ter, AMPD1 Gln12Ter, CKMM 1170bp/985+185bp, HFE His63Asp, GDF8 Lys153Arg and PPARGC1A Gly482Ser) in a cohort of 196 elite athletes who participated in the 2008 Kona Ironman championship triathlon. Mean performance time (PT) was not significantly different in individual marker analysis. Age, sex, and continent of origin had a significant influence on PT and were adjusted for. Only the AMPD1 endurance-optimal Gln allele was found to be significantly associated with an improvement in PT (model p=5.79 x 10-17, AMPD1 genotype p=0.01). Individual genotypes were combined into a total genotype score (TGS); TGS distribution ranged from 28.6 to 92.9, concordant with prior studies in endurance athletes (mean±SD: 60.75±12.95). TGS distribution was shifted toward higher TGS in the top 10% of athletes, though the mean TGS was not significantly different (p=0.164) and not significantly associated with PT even when adjusted for age, sex, and origin. Receiver operating characteristic curve analysis determined that TGS alone could not significantly predict athlete finishing time with discriminating sensitivity and specificity for three outcomes (less than median PT, less than mean PT, or in the top 10%), though models with the age, sex, continent of origin, and either TGS or AMPD1 genotype could. These results suggest three things: that more sophisticated genetic models may be necessary to accurately predict athlete finishing time in endurance events; that non-genetic factors such as training are hugely influential and should be included in genetic analyses to prevent confounding; and that large collaborations may be necessary to obtain sufficient sample sizes for powerful and complex analyses of endurance performance.