Candidate gene identification following linkage: Search for hypertension susceptibility genes

Following up genetic linkage studies to identify the underlying susceptibility gene(s) for complex disease traits is an arduous yet biologically and clinically important task. Complex traits, such as hypertension, are considered polygenic with many genes influencing risk, each with small effects. Chromosome 2 has been consistently identified as a genomic region with genetic linkage evidence suggesting that one or more loci contribute to blood pressure levels and hypertension status. Using combined positional candidate gene methods, the Family Blood Pressure Program has concentrated efforts in investigating this region of chromosome 2 in an effort to identify underlying candidate hypertension susceptibility gene(s). Initial informatics efforts identified the boundaries of the region and the known genes within it. A total of 82 polymorphic sites in eight positional candidate genes were genotyped in a large hypothesis-generating sample consisting of 1640 African Americans, 1339 whites, and 1616 Mexican Americans. To adjust for multiple comparisons, resampling-based false discovery adjustment was applied, extending traditional resampling methods to sibship samples. Following this adjustment for multiple comparisons, SLC4A5, a sodium bicarbonate transporter, was identified as a primary candidate gene for hypertension. Polymorphisms in SLC4A5 were subsequently genotyped and analyzed for validation in two populations of African Americans (N = 461; N = 778) and two of whites (N = 550; N = 967). Again, SNPs within SLC4A5 were significantly associated with blood pressure levels and hypertension status. While not identifying a single causal DNA sequence variation that is significantly associated with blood pressure levels and hypertension status across all samples, the results further implicate SLC4A5 as a candidate hypertension susceptibility gene, validating previous evidence for one or more genes on chromosome 2 that influence hypertension related phenotypes in the population-at-large. The methodology and results reported provide a case study of one approach for following up the results of genetic linkage analyses to identify genes influencing complex traits. ^

CsRAV1: a candidate gene for improving lignocellulosic biomass yield

Fast-growing tree species of Populus spp.,Salix spp. and Eucalyptus spp. are cultivated to produce wood in a short time. Poplars are cultivated with cycles of 15-18 years to obtain saw timber and peeler logs, but when grown as short -rotation coppice(SRC) to produce biomass, planting density increases and rotation is considerably reduced (3-5 years). In this regard, research efforts are focused in the identification of traits and loci that allow the generation of improved SRC biomass-yielding genotypes. Biomass yield is a highly complex trait as it is the combined outcome of many other complex traits, each under separate polygenic control. Among profitable biomass yield-related traits are the amount of sylleptic branching and the length of winter dormancy. In poplar and in a few other Salicaceae species some lateral buds grow out sylleptically, the same season in which they form without the need of an intervening rest period. Sylleptic branching in poplar increases branch number, leaf area and general growth of the tree in its early years, and is a reasonable predictor of coppice yield. On the other hand, the length of winter dormancy determines the extent of the growth period. Our group has characterized the RAV1 gene of Castanea sativa (CsRAV1), encoding a transcription factor of the subfamily RAV (Related to ABI3/VP1). CsRAV1 expression shows a marked seasonal pattern, being higher in autumn and winter both in stems and buds. We generated transgenic lines of the hybrid clone Populus tremulax P. alba INRA 717 1B4 constitutively expressing CsRAV 1. These CsRAV1-expressing poplars develop sylleptic branches only a few weeks after potting. In addition to the sylleptic branching phenotype, these trees show phenological features that could give rise to an extended growth period. We are currently assessing the phenotype and behavior of these transgenic trees in a field trial, and ultimately, we will evaluate the impact on lignocellulosic biomass quality and production.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

Interleukin 9: A candidate gene for asthma

Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of atopy and bronchial hyperresponsiveness. Recent studies localized a major gene for asthma to chromosome 5q31-q33 in humans. Thus, this segment of the genome represents a candidate region for genes that determine susceptibility to bronchial hyperresponsiveness and atopy in animal models. Homologs of candidate genes on human chromosome 5q31-q33 are found in four regions in the mouse genome, two on chromosome 18, and one each on chromosomes 11 and 13. We assessed bronchial responsiveness as a quantitative trait in mice and found it linked to chromosome 13. Interleukin 9 (IL-9) is located in the linked region and was analyzed as a gene candidate. The expression of IL-9 was markedly reduced in bronchial hyporesponsive mice, and the level of expression was determined by sequences within the qualitative trait locus (QTL). These data suggest a role for IL-9 in the complex pathogenesis of bronchial hyperresponsiveness as a risk factor for asthma.

Adaptive evolution of a candidate gene for aging in Drosophila

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.

The μ opiate receptor as a candidate gene for pain: Polymorphisms, variations in expression, nociception, and opiate responses

There are differences between human individuals and between mouse strains in levels of μ opiate receptor (μOR) expression, responses to painful stimuli, and responses to opiate drugs. One of the best candidates for contributing to these differences is variation at the μOR gene locus. Support for this idea comes from analyses of the human and murine μOR genes. Assessments of individual differences in human μOR expression add further support. Studies with mice, including knockout-transgenic, quantitative trait locus, and strain-comparison studies, also strongly support the possibility that μOR gene alleles would be strong candidates for contributing to individual differences in human nociception and opiate drug responses. This paper reviews current analyses of the murine and human μOR genes, their important variants, and correlations between these variants and opiate influences on pain.

An investigation of the 5-HT2C receptor gene as a migraine candidate gene

Migraine is a common complex disorder, currently classified into two main subtypes, migraine with aura (MA) and migraine without aura (MO). The strong preponderance of females to males suggests an X-linked genetic component. Recent studies have identified an X chromosomal susceptibility region (Xq24-q28) in two typical migraine pedigrees. This region harbours a potential candidate gene for the disorder, the serotonin receptor 2C (5-HT2C) gene. This study involved a linkage and association approach to investigate two single nucleotide variants in the 5-HT2C gene. In addition, exonic coding regions of the 5-HT2C gene were also sequenced for mutations in X-linked migraine pedigrees. Results of this study did not detect any linkage or association, and no disease causing mutations were identified. Hence, results for this study do not support a significant role of the 5-HT2C gene in migraine predisposition. (C) 2003 Wiley-Liss, Inc.

Association analyses pointed the TIPARP as a potential candidate gene influencing residual feed intake variation in Nelore cattle.

2016

Candidate disease gene prediction using Gentrepid: application to a genome-wide association study on coronary artery disease

Current single-locus-based analyses and candidate disease gene prediction methodologies used in genome-wide association studies (GWAS) do not capitalize on the wealth of the underlying genetic data, nor functional data available from molecular biology. Here, we analyzed GWAS data from the Wellcome Trust Case Control Consortium (WTCCC) on coronary artery disease (CAD). Gentrepid uses a multiple-locus-based approach, drawing on protein pathway- or domain-based data to make predictions. Known disease genes may be used as additional information (seeded method) or predictions can be based entirely on GWAS single nucleotide polymorphisms (SNPs) (ab initio method). We looked in detail at specific predictions made by Gentrepid for CAD and compared these with known genetic data and the scientific literature. Gentrepid was able to extract known disease genes from the candidate search space and predict plausible novel disease genes from both known and novel WTCCC-implicated loci. The disease gene candidates are consistent with known biological information. The results demonstrate that this computational approach is feasible and a valuable discovery tool for geneticists.

Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey.

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white-born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white-born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid-coloured controls. To the authors' knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys.

Association between polymorphisms in the progesterone receptor gene and endometriosis

The progesterone receptor (PR) is a candidate gene for the development of endometriosis, a complex disease with strong hormonal features, common in women of reproductive age. We typed the 306 base pair Alu insertion (AluIns) polymorphism in intron G of PR in 101 individuals, estimated linkage disequilibrium (LD) between five single-nucleotide polymorphisms (SNPs) across the PR locus in 980 Australian triads (endometriosis case and two parents) and used transmission disequilibrium testing (TDT) for association with endometriosis. The five SNPs showed strong pairwise LD, and the AluIns was highly correlated with proximal SNPs rs1042839 (Δ2 = 0.877, D9 = 1.00, P < 0.0001) and rs500760 (Δ2 = 0.438, D9 = 0.942, P < 0.0001). TDT showed weak evidence of allelic association between endometriosis and rs500760 (P = 0.027) but not in the expected direction. We identified a common susceptibility haplotype GGGCA across the five SNPs (P = 0.0167) in the whole sample, but likelihood ratio testing of haplotype transmission and non-transmission of the AluIns and flanking SNPs showed no significant pattern. Further, analysis of our results pooled with those from two previous studies suggested that neither the T2 allele of the AluIns nor the T1/T2 genotype was associated with endometriosis.

Variants in the human potassium channel gene (KCNN3) are associated with migraine in a high risk genetic isolate

The calcium-activated potassium ion channel gene (KCNN3) is located in the vicinity of the familial hemiplegic migraine type 2 locus on chromosome 1q21.3. This gene is expressed in the central nervous system and plays a role in neural excitability. Previous association studies have provided some, although not conclusive, evidence for involvement of this gene in migraine susceptibility. To elucidate KCNN3 involvement in migraine, we performed gene-wide SNP genotyping in a high-risk genetic isolate from Norfolk Island, a population descended from a small number of eighteenth century Isle of Man ‘Bounty Mutineer’ and Tahitian founders. Phenotype information was available for 377 individuals who are related through the single, well-defined Norfolk pedigree (96 were affected: 64 MA, 32 MO). A total of 85 SNPs spanning the KCNN3 gene were genotyped in a sub-sample of 285 related individuals (76 affected), all core members of the extensive Norfolk Island ‘Bounty Mutineer’ genealogy. All genotyping was performed using the Illumina BeadArray platform. The analysis was performed using the statistical program SOLAR v4.0.6 assuming an additive model of allelic effect adjusted for the effects of age and sex. Haplotype analysis was undertaken using the program HAPLOVIEW v4.0. A total of four intronic SNPs in the KCNN3 gene displayed significant association (P < 0.05) with migraine. Two SNPs, rs73532286 and rs6426929, separated by approximately 0.1 kb, displayed complete LD (r 2 = 1.00, D′ = 1.00, D′ 95% CI = 0.96–1.00). In all cases, the minor allele led to a decrease in migraine risk (beta coefficient = 0.286–0.315), suggesting that common gene variants confer an increased risk of migraine in the Norfolk pedigree. This effect may be explained by founder effect in this genetic isolate. This study provides evidence for association of variants in the KCNN3 ion channel gene with migraine susceptibility in the Norfolk genetic isolate with the rarer allelic variants conferring a possible protective role. This the first comprehensive analysis of this potential candidate gene in migraine and also the first study that has utilised the unique Norfolk Island large pedigree isolate to implicate a specific migraine gene. Studies of additional variants in KCNN3 in the Norfolk pedigree are now required (e.g. polyglutamine variants) and further analyses in other population data sets are required to clarify the association of the KCNN3 gene and migraine risk in the general outbred population.