Gender specific airway gene expression in COPD sub-phenotypes supports a role of mitochondria and of different types of leukocytes.

Chronic obstructive pulmonary disease (COPD) is a destructive inflammatory disease and the genes expressed within the lung are crucial to its pathophysiology. We have determined the RNAseq transcriptome of bronchial brush cells from 312 stringently defined ex-smoker patients. Compared to healthy controls there were for males 40 differentially expressed genes (DEGs) and 73 DEGs for females with only 26 genes shared. The gene ontology (GO) term "response to bacterium" was shared, with several different DEGs contributing in males and females. Strongly upregulated genes TCN1 and CYP1B1 were unique to males and females, respectively. For male emphysema (E)-dominant and airway disease (A)-dominant COPD (defined by computed tomography) the term "response to stress" was found for both sub-phenotypes, but this included distinct up-regulated genes for the E-sub-phenotype (neutrophil-related CSF3R, CXCL1, MNDA) and for the A-sub-phenotype (macrophage-related KLF4, F3, CD36). In E-dominant disease, a cluster of mitochondria-encoded (MT) genes forms a signature, able to identify patients with emphysema features in a confirmation cohort. The MT-CO2 gene is upregulated transcriptionally in bronchial epithelial cells with the copy number essentially unchanged. Both MT-CO2 and the neutrophil chemoattractant CXCL1 are induced by reactive oxygen in bronchial epithelial cells. Of the female DEGs unique for E- and A-dominant COPD, 88% were detected in females only. In E-dominant disease we found a pronounced expression of mast cell-associated DEGs TPSB2, TPSAB1 and CPA3. The differential genes discovered in this study point towards involvement of different types of leukocytes in the E- and A-dominant COPD sub-phenotypes in males and females.

Family-based genome-wide association study of leprosy in Vietnam.

Leprosy is a chronic infectious disease of the skin and peripheral nerves with a strong genetic predisposition. Recent genome-wide approaches have identified numerous common variants associated with leprosy, almost all in the Chinese population. We conducted the first family-based genome-wide association study of leprosy in 622 affected offspring from Vietnam, followed by replication in an independent sample of 1181 leprosy cases and 668 controls of the same ethnic origin. The most significant results were observed within the HLA region, in which six SNPs displayed genome-wide significant associations, all of which were replicated in the independent case/control sample. We investigated the signal in the HLA region in more detail, by conducting a multivariate analysis on the case/control sample of 319 GWAS-suggestive HLA hits for which evidence for replication was obtained. We identified three independently associated SNPs, two located in the HLA class I region (rs1265048: OR = 0.69 [0.58-0.80], combined p-value = 5.53x10-11; and rs114598080: OR = 1.47 [1.46-1.48], combined p-value = 8.77x10-13), and one located in the HLA class II region (rs3187964 (OR = 1.67 [1.55-1.80], combined p-value = 8.35x10-16). We also validated two previously identified risk factors for leprosy: the missense variant rs3764147 in the LACC1 gene (OR = 1.52 [1.41-1.63], combined p-value = 5.06x10-14), and the intergenic variant rs6871626 located close to the IL12B gene (OR = 0.73 [0.61-0.84], combined p-value = 6.44x10-8). These results shed new light on the genetic control of leprosy, by dissecting the influence of HLA SNPs, and validating the independent role of two additional variants in a large Vietnamese sample.

POLR1B and neural crest cell anomalies in Treacher Collins syndrome type 4.

PURPOSE: Treacher Collins syndrome (TCS) is a rare autosomal dominant mandibulofacial dysostosis, with a prevalence of 0.2-1/10,000. Features include bilateral and symmetrical malar and mandibular hypoplasia and facial abnormalities due to abnormal neural crest cell (NCC) migration and differentiation. To date, three genes have been identified: TCOF1, POLR1C, and POLR1D. Despite a large number of patients with a molecular diagnosis, some remain without a known genetic anomaly. METHODS: We performed exome sequencing for four individuals with TCS but who were negative for pathogenic variants in the known causative genes. The effect of the pathogenic variants was investigated in zebrafish. RESULTS: We identified three novel pathogenic variants in POLR1B. Knockdown of polr1b in zebrafish induced an abnormal craniofacial phenotype mimicking TCS that was associated with altered ribosomal gene expression, massive p53-associated cellular apoptosis in the neuroepithelium, and reduced number of NCC derivatives. CONCLUSION: Pathogenic variants in the RNA polymerase I subunit POLR1B might induce massive p53-dependent apoptosis in a restricted neuroepithelium area, altering NCC migration and causing cranioskeletal malformations. We identify POLR1B as a new causative gene responsible for a novel TCS syndrome (TCS4) and establish a novel experimental model in zebrafish to study POLR1B-related TCS.

Familial breast cancer and DNA repair genes: Insights into known and novel susceptibility genes from the GENESIS study, and implications for multigene panel testing.

Pathogenic variants in BRCA1 and BRCA2 only explain the underlying genetic cause of about 10% of hereditary breast and ovarian cancer families. Because of cost-effectiveness, multigene panel testing is often performed even if the clinical utility of testing most of the genes remains questionable. The purpose of our study was to assess the contribution of rare, deleterious-predicted variants in DNA repair genes in familial breast cancer (BC) in a well-characterized and homogeneous population. We analyzed 113 DNA repair genes selected from either an exome sequencing or a candidate gene approach in the GENESIS study, which includes familial BC cases with no BRCA1 or BRCA2 mutation and having a sister with BC (N = 1,207), and general population controls (N = 1,199). Sequencing data were filtered for rare loss-of-function variants (LoF) and likely deleterious missense variants (MV). We confirmed associations between LoF and MV in PALB2, ATM and CHEK2 and BC occurrence. We also identified for the first time associations between FANCI, MAST1, POLH and RTEL1 and BC susceptibility. Unlike other associated genes, carriers of an ATM LoF had a significantly higher risk of developing BC than carriers of an ATM MV (ORLoF = 17.4 vs. ORMV = 1.6; p Het = 0.002). Hence, our approach allowed us to specify BC relative risks associated with deleterious-predicted variants in PALB2, ATM and CHEK2 and to add MAST1, POLH, RTEL1 and FANCI to the list of DNA repair genes possibly involved in BC susceptibility. We also highlight that different types of variants within the same gene can lead to different risk estimates.

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons.

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting ß-cells and neurons and underline the existence of trans­nosology pathways in diabetes and its co-morbidities.

Genomic regulation of type 2 diabetes endophenotypes: Contribution from genetic studies in the Goto-Kakizaki rat.

The inbred Goto-Kakizaki (GK) rat strain is a unique model of spontaneous type 2 diabetes mellitus caused by naturally occurring genetic variants that have been selectively isolated from an outbred colony of Wistar rats. Genetic and genomic studies in experimental crosses and congenic strains of the GK have shed light on the complex etiopathogenesis of diabetes phenotypes in this model. Diabetes-related phenotypes in the GK are under polygenic control and distinct genetic loci regulate glucose tolerance, insulin secretion, ß-cell mass and plasma lipids. Metabolome and transcriptome profiling data in GK crosses and congenics, combined with GK genome resequencing, have resulted in a comprehensive landscape of genomic regulations of metabolism that can disentangle causal relationships between GK variants and diabetes phenotypes. Application of systems biology and systems genetics in the GK has contributed to improve our understanding of the fundamental mechanisms regulating metabolism. The wealth of physiological, genetic and genomic information in this strain makes it one of the most powerful model systems to improve our understanding of genetic regulations of metabolism and for testing therapeutic solutions for diabetes.

Transcriptome Profiling in Rat Inbred Strains and Experimental Cross Reveals Discrepant Genetic Architecture of Genome-Wide Gene Expression.

To test the impact of genetic heterogeneity on cis- and trans-mediated mechanisms of gene expression regulation, we profiled the transcriptome of adipose tissue in 20 inbred congenic strains derived from diabetic Goto-Kakizaki (GK) rats and Brown-Norway (BN) controls, which contain well-defined blocks (1-183 Mb) of genetic polymorphisms, and in 123 genetically heterogeneous rats of an (GK × BN)F2 offspring. Within each congenic we identified 73-1351 differentially expressed genes (DEGs), only 7.7% of which mapped within the congenic blocks, and which may be regulated in cis The remainder localized outside the blocks, and therefore must be regulated in trans Most trans-regulated genes exhibited approximately twofold expression changes, consistent with monoallelic expression. Altered biological pathways were replicated between congenic strains sharing blocks of genetic polymorphisms, but polymorphisms at different loci also had redundant effects on transcription of common distant genes and pathways. We mapped 2735 expression quantitative trait loci (eQTL) in the F2 cross, including 26% predominantly cis-regulated genes, which validated DEGs in congenic strains. A hotspot of >300 eQTL in a 10 cM region of chromosome 1 was enriched in DEGs in a congenic strain. However, many DEGs among GK, BN and congenic strains did not replicate as eQTL in F2 hybrids, demonstrating distinct mechanisms of gene expression when alleles segregate in an outbred population or are fixed homozygous across the entire genome or in short genomic regions. Our analysis provides conceptual advances in our understanding of the complex architecture of genome expression and pathway regulation, and suggests a prominent impact of epistasis and monoallelic expression on gene transcription.

Aristolochic acid exposure in Romania and implications for renal cell carcinoma.

BACKGROUND: Aristolochic acid (AA) is a nephrotoxicant associated with AA nephropathy (AAN) and upper urothelial tract cancer (UUTC). Whole-genome sequences of 14 Romanian cases of renal cell carcinoma (RCC) recently exhibited mutational signatures consistent with AA exposure, although RCC had not been previously linked with AAN and AA exposure was previously reported only in localised rural areas. METHODS: We performed mass spectrometric measurements of the aristolactam (AL) DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) in nontumour renal tissues of the 14 Romanian RCC cases and 15 cases from 3 other countries. RESULTS: We detected dA-AL-I in the 14 Romanian cases at levels ranging from 0.7 to 27 adducts per 10(8) DNA bases, in line with levels reported in Asian and Balkan populations exposed through herbal remedies or food contamination. The 15 cases from other countries were negative. INTERPRETATION: Although the source of exposure is uncertain and likely different in AAN regions than elsewhere, our results demonstrate that AA exposure in Romania exists outside localised AAN regions and provide further evidence implicating AA in RCC.

ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

The SAM domain of ANKS6 has different interacting partners and mutations can induce different cystic phenotypes.

The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.

Variation in genomic landscape of clear cell renal cell carcinoma across Europe.

The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

Gene-wide analysis detects two new susceptibility genes for Alzheimer's disease.

BACKGROUND: Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls. PRINCIPAL FINDINGS: In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10-6) and 14 (IGHV1-67 p = 7.9×10-8) which indexed novel susceptibility loci. SIGNIFICANCE: The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.

Genetic control of differential acetylation in diabetic rats.

Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats.

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Inhibition of Comt with tolcapone slows progression of polycystic kidney disease in the more severely affected PKD/Mhm (cy/+) substrain of the Hannover Sprague-Dawley rat.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. METHODS: Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. RESULTS: Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. CONCLUSIONS: Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.

Untargeted metabolome quantitative trait locus mapping associates variation in urine glycerate to mutant glycerate kinase.

With successes of genome-wide association studies, molecular phenotyping systems are developed to identify genetically determined disease-associated biomarkers. Genetic studies of the human metabolome are emerging but exclusively apply targeted approaches, which restricts the analysis to a limited number of well-known metabolites. We have developed novel technical and statistical methods for systematic and automated quantification of untargeted NMR spectral data designed to perform robust and accurate quantitative trait locus (QTL) mapping of known and previously unreported molecular compounds of the metabolome. For each spectral peak, six summary statistics were calculated and independently tested for evidence of genetic linkage in a cohort of F2 (129S6xBALB/c) mice. The most significant evidence of linkages were obtained with NMR signals characterizing the glycerate (LOD10-42) at the mutant glycerate kinase locus, which demonstrate the power of metabolomics in quantitative genetics to identify the biological function of genetic variants. These results provide new insights into the resolution of the complex nature of metabolic regulations and novel analytical techniques that maximize the full utilization of metabolomic spectra in human genetics to discover mappable disease-associated biomarkers.

Broad-ranging natural metabotype variation drives physiological plasticity in healthy control inbred rat strains.

Maintaining homeostasis in higher organisms involves a complex interplay of multiple ubiquitous and organ-specific molecular mechanisms that can be characterized using functional genomics technologies such as transcriptomics, proteomics, and metabonomics and dissected out through genetic investigations in healthy and diseased individuals. We characterized the genomic, metabolic, and physiological divergence of several inbred rat strains--Brown Norway, Lewis, Wistar Kyoto, Fisher (F344)--frequently used as healthy controls in genetic studies of the cardiometabolic syndrome. Hierarchical clustering of (1)H NMR-based metabolic profiles (n = 20 for urine, n = 16 for plasma) identified metabolic phenotype (metabotype) divergence patterns similar to the phylogenetic variability based on single nucleotide polymorphisms. However, the observed urinary metabotype variation exceeded that explainable by genetic polymorphisms. To understand further this natural variation, we used an integrative, knowledge-based network biology metabolic pathway analysis approach, coined Metabolite-Set Enrichment Analysis (MSEA). MSEA reveals that homeostasis and physiological plasticity can be achieved despite widespread divergences in glucose, lipid, amino acid, and energy metabolism in the host, together with different gut microbiota contributions suggestive of strain-specific transgenomic interactions. This work illustrates the concept of natural metabolomic variation, leading to physiologically stable albeit diverse strategies within the range of normality, all of which are highly relevant to animal model physiology, genetical genomics, and patient stratification in personalized healthcare.

Transgenic overexpression of Anks6(p.R823W) causes polycystic kidney disease in rats.

The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6((p.R823W))) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6((p.R823W)) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6((p.R823W)) cDNA in the renal tubular epithelium. The transgenic Anks6((p.R823W)) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6((p.R823W)) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.

Functional annotations of diabetes nephropathy susceptibility loci through analysis of genome-wide renal gene expression in rat models of diabetes mellitus.

BACKGROUND: Hyperglycaemia in diabetes mellitus (DM) alters gene expression regulation in various organs and contributes to long term vascular and renal complications. We aimed to generate novel renal genome-wide gene transcription data in rat models of diabetes in order to test the responsiveness to hyperglycaemia and renal structural changes of positional candidate genes at selected diabetic nephropathy (DN) susceptibility loci. METHODS: Both Affymetrix and Illumina technologies were used to identify significant quantitative changes in the abundance of over 15,000 transcripts in kidney of models of spontaneous (genetically determined) mild hyperglycaemia and insulin resistance (Goto-Kakizaki-GK) and experimentally induced severe hyperglycaemia (Wistar-Kyoto-WKY rats injected with streptozotocin [STZ]). RESULTS: Different patterns of transcription regulation in the two rat models of diabetes likely underlie the roles of genetic variants and hyperglycaemia severity. The impact of prolonged hyperglycaemia on gene expression changes was more profound in STZ-WKY rats than in GK rats and involved largely different sets of genes. These included genes already tested in genetic studies of DN and a large number of protein coding sequences of unknown function which can be considered as functional and, when they map to DN loci, positional candidates for DN. Further expression analysis of rat orthologs of human DN positional candidate genes provided functional annotations of known and novel genes that are responsive to hyperglycaemia and may contribute to renal functional and/or structural alterations. CONCLUSION: Combining transcriptomics in animal models and comparative genomics provides important information to improve functional annotations of disease susceptibility loci in humans and experimental support for testing candidate genes in human genetics.