Prospects for association-based fine mapping of a susceptibility gene for a complex disease

The potential of association studies for fine-mapping loci with common disease susceptibility alleles for complex genetic diseases in outbred populations is unclear. For a battery of tightly linked anonymous genetic markers spanning a candidate region centered around a disease locus, simulation methods based on a coalescent process with mutation, recombination, and genetic drift were used to study the spatial distribution of markers with large noncentrality parameters in a case-control study design. Simulations with a disease allele at intermediate frequency, presumably representing an old mutation, tend to exhibit the largest noncentrality parameter values at markers near the disease locus. In contrast, simulations with a disease allele at low frequency, presumably representing a young mutation, often exhibit the largest noncentrality parameter values at markers scattered over the candidate region. In the former case, sample sizes or marker densities sufficient to detect association are likely to lead to useful localization, whereas, in the latter case, localization of the disease locus within the candidate region is much less likely, regardless of the sample size or density of the map. The simulations suggest that for a single marker analysis, the simple strategy of choosing the marker with smallest associated P value to begin a laboratory search for the disease locus performs adequately for a common disease allele.     

Genome-wide association analyses reveal significant loci and strong candidate genes for growth and fatness traits in two pig populations

Background

Recently, genome-wide association studies (GWAS) have been reported on various pig traits. We performed a GWAS to analyze 22 traits related to growth and fatness on two pig populations: a White Duroc × Erhualian F₂ intercross population and a Chinese Sutai half-sib population.

Results

We identified 14 and 39 loci that displayed significant associations with growth and fatness traits at the genome-wide level and chromosome-wide level, respectively. The strongest association was between a 750 kb region on SSC7 (SSC for Sus scrofa) and backfat thickness at the first rib. This region had pleiotropic effects on both fatness and growth traits in F₂ animals and contained a promising candidate gene HMGA1 (high mobility group AT-hook 1). Unexpectedly, population genetic analysis revealed that the allele at this locus that reduces fatness and increases growth is derived from Chinese indigenous pigs and segregates in multiple Chinese breeds. The second strongest association was between the region around 82.85 Mb on SSC4 and average backfat thickness. PLAG1 (pleiomorphic adenoma gene 1), a gene under strong selection in European domestic pigs, is proximal to the top SNP and stands out as a strong candidate gene. On SSC2, a locus that significantly affects fatness traits mapped to the region around the IGF2 (insulin-like growth factor 2) gene but its non-imprinting inheritance excluded IGF2 as a candidate gene. A significant locus was also detected within a recombination cold spot that spans more than 30 Mb on SSCX, which hampered the identification of plausible candidate genes. Notably, no genome-wide significant locus was shared by the two experimental populations; different loci were observed that had both constant and time-specific effects on growth traits at different stages, which illustrates the complex genetic architecture of these traits.

Conclusions

We confirm several previously reported QTL and provide a list of novel loci for porcine growth and fatness traits in two experimental populations with Chinese Taihu and Western pigs as common founders. We showed that distinct loci exist for these traits in the two populations and identified HMGA1 and PLAG1 as strong candidate genes on SSC7 and SSC4, respectively.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0089-5) contains supplementary material, which is available to authorized users.     

Variance Analysis of Immunoglobulin Alleles in Natural Populations of Rabbit (Oryctolagus Cuniculus): The Extensive Interallelic Divergence at the B Locus Could Be the Outcome of Overdominance-Type Selection

Population genetic data are presented which should contribute to evaluation of the hypothesis that the extraordinary evolutionary patterns observed at the b locus of the rabbit immunoglobulin light chain constant region can be the outcome of overdominance-type selection. The analysis of allele correlations in natural populations revealed an excess of heterozygotes of about 10% at the b locus while heterozygote excess was not observed at loci determining the immunoglobulin heavy chain. Data from the published literature, where homozygote advantage was suggested, were reevaluated and found in agreement with data here presented. Gene diversity was evenly distributed among populations and showed similarities with patterns reported for histocompatibility loci. Analysis of genotypic disequilibria revealed strong digenic associations between the leading alleles of heavy and light chain constant region loci in conjunction with trigenic disequilibria corresponding to a preferential association of b locus heterozygosity with the predominant allele of the heavy chain e locus. It is argued that this may indicate compensatory or nonadditive aspects of a putative heterozygosity enhancing mechanism, implying that effects at the light chain might be more pronounced in populations fixed for the heavy chain polymorphism.     

Using Linkage Analysis to Detect Gene-Gene Interaction by Stratifying Family Data on Known Disease,or Disease-Associated,Alleles

Detecting gene-gene interaction in complex diseases is a major challenge for common disease genetics. Most interaction detection approaches use disease-marker associations and such methods have low power and unknown reliability in real data. We developed and tested a powerful linkage-analysis-based gene-gene interaction detection strategy based on conditioning the family data on a known disease-causing allele or disease-associated marker allele. We computer-generated multipoint linkage data for a disease caused by two epistatically interacting loci (A and B). We examined several two-locus epistatic inheritance models: dominant-dominant, dominant-recessive, recessive-dominant, recessive-recessive. At one of the loci (A), there was a known disease-related allele. We stratified the family data on the presence of this allele, eliminating family members who were without it. This elimination step has the effect of raising the “penetrance” at the second locus (B). We then calculated the lod score at the second locus (B) and compared the pre- and post-stratification lod scores at B. A positive difference indicated interaction. We also examined if it was possible to detect interaction with locus B based on a disease-marker association (instead of an identified disease allele) at locus A. We also tested whether the presence of genetic heterogeneity would generate false positive evidence of interaction. The power to detect interaction for a known disease allele was 60–90%. The probability of false positives, based on heterogeneity, was low. Decreasing linkage disequilibrium between the disease and marker at locus A decreased the likelihood of detecting interaction. The allele frequency of the associated marker made little difference to the power.     

An Evaluation of Association between a Novel Hippocampal Biology Related SNP (rs7294919) and Schizophrenia

Recent genetic analyses have implicated several candidate susceptibility variants for schizophrenia. The single nucleotide polymorphism (SNP) rs7294919 is likely a schizophrenia-susceptibility variant according to its significant association with hippocampal volume, hippocampus function, and cognitive performance as well as the nominal association with schizophrenia. However, all previous analyses were conducted only in Europeans, and whether rs7294919 is associated with schizophrenia in other populations are yet to be tested. Here, we conducted a case-control analysis of rs7294919 with schizophrenia in six independent Chinese (N = 3) and Japanese (N = 3) samples, including a total of 7,352 cases and 10,824 controls. The results of our association analysis were not able to confirm the association of rs7294919 with schizophrenia (p = 0.51 in total samples, odds ratio = 1.02 for allele[C]). The absence of rs7294919’s association in Chinese and Japanese suggest a potential genetic heterogeneity in the susceptibility of schizophrenia on this locus and also demonstrate the difficulties in replicating associations of schizophrenia across different ethnic populations.     

KIAA1462, A Coronary Artery Disease Associated Gene,Is a Candidate Gene for Late Onset Alzheimer Disease in APOE Carriers

Alzheimer disease (AD) is a devastating neurodegenerative disease affecting more than five million Americans. In this study, we have used updated genetic linkage data from chromosome 10 in combination with expression data from serial analysis of gene expression to choose a new set of thirteen candidate genes for genetic analysis in late onset Alzheimer disease (LOAD). Results in this study identify the KIAA1462 locus as a candidate locus for LOAD in APOE4 carriers. Two genes exist at this locus, KIAA1462, a gene associated with coronary artery disease, and “rokimi”, encoding an untranslated spliced RNA The genetic architecture at this locus suggests that the gene product important in this association is either “rokimi”, or a different isoform of KIAA1462 than the isoform that is important in cardiovascular disease. Expression data suggests that isoform f of KIAA1462 is a more attractive candidate for association with LOAD in APOE4 carriers than “rokimi” which had no detectable expression in brain.     

Identification of a role for the ARHGEF3 gene in postmenopausal osteoporosis

Osteoporosis is a common and debilitating bone disease characterized by low bone mineral density (BMD), a highly heritable and polygenic trait. Genome-wide linkage studies have identified 3p14-p21 as a quantitative trait locus for BMD. The ARHGEF3 gene is situated within this region and was identified as a strong positional candidate. The aim of this study was to evaluate the role of variation in ARHGEF3 on bone density in women. Sequence variation within ARHGEF3 was analyzed with 17 single-nucleotide polymorphisms (SNPs) in a discovery cohort of 769 female sibs. Significant associations were found with family-based association tests between five SNPs and various measures of age-adjusted BMD (p = 0.0007-0.041) with rs7646054 showing maximal association. Analysis of the data with QPDTPHASE suggested that the more common G allele at rs7646054 is associated with decreased age-adjusted BMD. Significant associations were also demonstrated between 3-SNP haplotypes and age-adjusted spine and femoral-neck BMD (p = 0.002 and 0.003, respectively). rs7646054 was then genotyped in a replication cohort, and significant associations with hip and spine BMD were confirmed (p = 0.003-0.038), as well as an association with fracture rate (p = 0.02). Again, the G allele was associated with a decrease in age-adjusted BMD at each site studied. In conclusion, genetic variation in ARHGEF3 plays a role in the determination of bone density in Caucasian women. This data implicates the RhoGTPase-RhoGEF pathway in osteoporosis.     

Disequilibrium mapping of a quantitative-trait locus in an expanding population.

Linkage disequilibrium (LD) mapping can be successful if there is strong nonrandom association between marker alleles and an allele affecting a trait of interest. The principles of LD mapping of dichotomous traits are well understood, but less is known about LD mapping of a quantitative-trait locus (QTL). It is shown in this report that selective genotyping can increase the power to detect and map a rare allele of large effect at a QTL. Two statistical tests of the association between an allele and a quantitative character are proposed. These tests are approximately independent, so information from them can be combined. Analytic theory is developed to show that these two tests are effective in detecting the presence of a low-frequency allele with a relatively large effect on the character when the QTL is either already a candidate locus or closely linked to a marker locus that is in strong LD with the QTL. The latter situation is expected in a rapidly growing population in which the allele of large effect was present initially in one copy. Therefore, the proposed tests are useful under the same conditions as those for successful LD mapping of a dichotomous trait or disease. Simulations show that, for detection of the presence of a QTL, these tests are more powerful than a simple t-test. The tests also provide a basis for defining a measure of association, gamma, between a low-frequency allele at a putative QTL and a low-frequency allele at a marker locus.     

Genome-Wide Association Study Identifies a Novel Canine Glaucoma Locus

Glaucoma is an optic neuropathy and one of the leading causes of blindness. Its hereditary forms are classified into primary closed-angle (PCAG), primary open-angle (POAG) and primary congenital glaucoma (PCG). Although many loci have been mapped in human, only a few genes have been identified that are associated with the development of glaucoma and the genetic basis of the disease remains poorly understood. Glaucoma has also been described in many dog breeds, including Dandie Dinmont Terriers (DDT) in which it is a late-onset (>7 years) disease. We designed clinical and genetic studies to better define the clinical features of glaucoma in the DDT and to identify the genetic cause. Clinical diagnosis was based on ophthalmic examinations of the affected dogs and 18 additionally investigated unaffected DDTs. We collected DNA from over 400 DTTs and a genome wide association study was performed in a cohort of 23 affected and 23 controls, followed by a fine mapping, a replication study and candidate gene sequencing. The clinical study suggested that ocular abnormalities including abnormal iridocorneal angles and pectinate ligament dysplasia are common (50% and 72%, respectively) in the breed and the disease resembles human PCAG. The genetic study identified a novel 9.5 Mb locus on canine chromosome 8 including the 1.6 Mb best associated region (p = 1.63×10⁻¹⁰, OR = 32 for homozygosity). Mutation screening in five candidate genes did not reveal any causative variants. This study indicates that although ocular abnormalities are common in DDTs, the genetic risk for glaucoma is conferred by a novel locus on CFA8. The canine locus shares synteny to a region in human chromosome 14q, which harbors several loci associated with POAG and PCG. Our study reveals a new locus for canine glaucoma and ongoing molecular studies will likely help to understand the genetic etiology of the disease.     

IL2RA Genetic Heterogeneity in Multiple Sclerosis and Type 1 Diabetes Susceptibility and Soluble Interleukin-2 Receptor Production

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report “allelic heterogeneity” at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.     

SNP association studies in Alzheimer's disease highlight problems for complex disease analysis.

Genetic linkage and association analyses are two distinct approaches to understanding the genetic etiology of complex disease. Association analysis has become particularly popular in recent times, but the true utility of the strategy remains uncertain. To try to gain better insight into the relevant issues, we have used genetic association analysis to explore the etiology of Alzheimer's disease. Our empirical findings supplement the theoretical debate, illustrating the general doubtfulness of previous positive findings and the limited ability of typical association studies based on candidate genes to discern true medium-sized signals from false positives. Improvements in genotyping technologies and increasing the number of SNPs tested, without sophisticated allowance for all other issues, could simply lead to an unmanageable overload of false-positive signals, themselves obscuring true disease associations.     

Fine mapping of the psoriasis susceptibility locus PSORS1 supports HLA-C as the susceptibility gene in the Han Chinese population

PSORS1 (psoriasis susceptibility gene 1) is a major susceptibility locus for psoriasis. Several fine-mapping studies have highlighted a 300-kb candidate region of PSORS1 where multiple biologically plausible candidate genes were suggested. The most recent study has indicated HLA-Cw6 as the primary PSORS1 risk allele within the candidate region in a Caucasian population. In this study, a family-based association analysis of the PSORS1 locus was performed by analyzing 10 polymorphic microsatellite markers from the PSORS1 region as well as HLA-B, HLA-C and CDSN loci in 163 Chinese families of psoriasis. Five marker loci show strong evidence (P<10⁻³), and one marker locus shows weak evidence (P=0.04) for association. The haplotype cluster analysis showed that all the risk haplotypes are Cw6 positive and share a 369-kb region of homologous marker alleles which carries all the risk alleles, including HLA-Cw6 and CDSN*TTC, identified in this study. The recombinant haplotype analysis of the HLA-Cw6 and CDSN*TTC alleles in 228 Chinese families showed that the HLA-Cw6⁻/CDSN*TTC⁺ recombinant haplotype is clearly not associated with risk for psoriasis (TNT=29:57, p=0.0025) in a Chinese population, suggesting that the CDSN*TTC allele itself does not confer risk without the presence of the HLA-Cw6 allele. The further exclusion analysis of the non-risk HLA-Cw6⁻/CDSN*TTC⁺ recombinant haplotypes with common recombination breakpoints has allowed us to refine the location of PSORS1 to a small candidate region. Finally, we performed a conditional linkage analysis and showed that the HLA-Cw6 is a major risk allele but does not explain the full linkage evidence of the PSORS1 locus in a Chinese population. By performing a series of family-based association analyses of haplotypes as well as an exclusion analysis of recombinant haplotypes, we were able to refine the PSORS1 gene to a small critical region where HLA-C is a strong candidate to be the PSORS1 susceptibility gene.     

Antagonistic pleiotropy,mutation accumulation,and human genetic disease

The antagonistic pleiotropy theory of senescence is the most convincing theoretical explanation of the existence of aging. As yet, no locus or allele has been identified in a wild population with the features predicted by the pleiotropic theory. Human genetic diseases offer the opportunity to identify potentially pleiotropic alleles/loci. Four human genetic diseases—Huntington's disease, idiopathic hemochromatosis, myotonic dystrophy, and Alzheimer's disease—may exhibit pleiotropic effects and further study of these diseases might result in the identification of pleiotropic genes causing aging. Inability to find an early life selective benefit associated with these disease-causing alleles would favor the major alternative genetic explanation for aging, the mutation accumulation theory.     

The critical region for Behçet disease in the human major histocompatibility complex is reduced to a 46-kb segment centromeric of HLA-B, by association analysis using refined microsatellite mapping

The HLA-B51 allele is known to be associated with Behçet disease. Recently, we found a higher risk for Behçet disease in the MICA gene, 46 kb centromeric of HLA-B, by investigation of GCT repetitive polymorphism within exon 5 of MICA. The pathogenic gene causing Behçet disease, however, has remained uncertain. Here, eight polymorphic microsatellite markers, distributed over a 900-kb region surrounding the HLA-B locus, were subjected to association analysis for Behçet disease. Statistical studies of associated alleles detected on each microsatellite locus showed that the pathogenic gene for Behçet disease is most likely found within a 46-kb segment between the MICA and HLA-B genes. The results of this mapping study, and the results of an earlier study of ours, suggest that MICA is a strong candidate gene for the development of Behçet disease.     

Transmission-disequilibrium tests for quantitative traits.

The transmission-disequilibrium test (TDT) of Spielman et al. is a family-based linkage-disequilibrium test that offers a powerful way to test for linkage between alleles and phenotypes that is either causal (i.e., the marker locus is the disease/trait allele) or due to linkage disequilibrium. The TDT is equivalent to a randomized experiment and, therefore, is resistant to confounding. When the marker is extremely close to the disease locus or is the disease locus itself, tests such as the TDT can be far more powerful than conventional linkage tests. To date, the TDT and most other family-based association tests have been applied only to dichotomous traits. This paper develops five TDT-type tests for use with quantitative traits. These tests accommodate either unselected sampling or sampling based on selection of phenotypically extreme offspring. Power calculations are provided and show that, when a candidate gene is available (1) these TDT-type tests are at least an order of magnitude more efficient than two common sib-pair tests of linkage; (2) extreme sampling results in substantial increases in power; and (3) if the most extreme 20% of the phenotypic distribution is selectively sampled, across a wide variety of plausible genetic models, quantitative-trait loci explaining as little as 5% of the phenotypic variation can be detected at the .0001 alpha level with <300 observations.     

Subregional localization of the chromosome 21 loci D21S24 and D21S26 using physical mapping techniques

Summary We were able to refine the chromosomal position of two existing marker loci, using an extended chromosome 21 somatic cell hybrid panel. The locus D21S26 mapped in the region 21q11.2–q21.1, and the locus D21S24 in 21q22.1–q22.2. Physical and genetic analysis indicated that D21S26 is tightly linked to D21S13 and D21S16, two markers previously linked to familial Alzheimer's disease.     

Multiple Alleles for Tuber Shape in Diploid Potato Detected by Qualitative and Quantitative Genetic Analysis Using Rflps

Tuber shape in potato is commonly regarded as displaying continuous variation, yet at the diploid level phenotypes can be discerned visually, having round or long tubers. Inheritance of qualitative tuber shape can be explained by a single locus Ro, round being dominant to long. With restriction fragment length polymorphisms (RFLPs) the Ro locus was mapped on chromosome 10. Tuber shape was also studied as a quantitative trait, using the length/width ratio as trait value. The estimated broad sense heritability was h(2) = 0.80. The morphologically mapped Ro locus explained 75% of the genetic variation, indicating the presence of a major quantitative trait locus (QTL) at the Ro locus and minor genetic factors. RFLP alleles linked with Ro alleles were used to divide the progeny into four genotypic classes: Ro( &) Ro( &) : Ro( &) ro : roRo( &) : roro = 1 : 1 : 1 : 1. The recessive ro allele is identical by descent in both parents. The significantly different effects (P = 0.0157) of the non-identical alleles Ro( &) and Ro( &) provided evidence for multiallelism at the Ro locus. Linkage mapping of the Ro locus was compared with QTL mapping. Only those markers which are polymorphic in both parents allow accurate QTL mapping when genetic factors segregate from both parents. This finding applies to QTL mapping in all outbreeders without homozygous inbred strains.     

Joint modeling of linkage and association: identifying SNPs responsible for a linkage signal

Once genetic linkage has been identified for a complex disease, the next step is often association analysis, in which single-nucleotide polymorphisms (SNPs) within the linkage region are genotyped and tested for association with the disease. If a SNP shows evidence of association, it is useful to know whether the linkage result can be explained, in part or in full, by the candidate SNP. We propose a novel approach that quantifies the degree of linkage disequilibrium (LD) between the candidate SNP and the putative disease locus through joint modeling of linkage and association. We describe a simple likelihood of the marker data conditional on the trait data for a sample of affected sib pairs, with disease penetrances and disease-SNP haplotype frequencies as parameters. We estimate model parameters by maximum likelihood and propose two likelihood-ratio tests to characterize the relationship of the candidate SNP and the disease locus. The first test assesses whether the candidate SNP and the disease locus are in linkage equilibrium so that the SNP plays no causal role in the linkage signal. The second test assesses whether the candidate SNP and the disease locus are in complete LD so that the SNP or a marker in complete LD with it may account fully for the linkage signal. Our method also yields a genetic model that includes parameter estimates for disease-SNP haplotype frequencies and the degree of disease-SNP LD. Our method provides a new tool for detecting linkage and association and can be extended to study designs that include unaffected family members.     

Substantial Linkage Disequilibrium Across the Dihydrolipoyl Succinyltransferase Gene Region Without Alzheimer's Disease Association

Association of the candidate gene DLST with late-onset Alzheimer's disease (LOAD) risk has been suggested on the basis of case-control studies. This gene, located on chromosome 14q24.3, encodes a subunit of a mitochondrial component known to be defective in AD, the alpha-ketoglutarate dehydrogenase complex. Positive reports have correlated different DLST alleles with LOAD, whereas other groups have failed to find any significant association. We therefore reexamined the association of DLST and LOAD in a more ethnically homogeneous series using three additional single nucleotide polymorphisms (SNP) located within or closely flanking either end of the DLST gene. Pairwise analysis of these SNPs indicated there was strong linkage disequilibrium across the DLST locus. Analysis of complex genotypes or haplotypes based upon all five SNP loci failed to identify a LOAD risk allele, suggesting that further studies of DLST in relation to AD are not warranted.