Evaluation of linkage disequilibrium measures between multi-allelic markers as predictors of linkage disequilibrium between single nucleotide polymorphisms

Effectiveness of marker-assisted selection (MAS) and quantitative trait locus (QTL) mapping using population-wide linkage disequilibrium (LD) between markers and QTLs depends on the extent of LD and how it declines with distance between markers and QTLs in a population. Marker-QTL LD can be predicted from LD between markers. Our previous work evaluated LD measures between multi-allelic markers as predictors of usable LD of multi-allelic markers with QTLs. Since single nucleotide polymorphisms (SNPs) are the current marker of choice for high-density genotyping and LD-mapping of QTLs, the objective of this study was to use LD between multi-allelic markers to predict LD among biallelic SNPs or between SNPs and QTLs. Observable LD between multi-allelic markers was evaluated using nine measures. These included two pooled and standardized measures of LD between pairs of alleles at two markers based on Lewontin's LD measure, two pooled measures of squared correlations between alleles, one standardized measure using Hardy-Weinberg heterozygosities, and four measures based on the chi-square statistic for testing for association between alleles at two loci. The standardized chi-square measure that best predicted usable LD between multi-allelic markers and QTLs, based on our previous work, overestimated usable SNP-SNP or SNP-QTL LD. Instead, three other measures were found to be good predictors of usable SNP-SNP or SNP-QTL LD when LD is generated by drift. Therefore, the LD measure between multi-allelic markers that is best for predicting usable LD in a population depends on the type of markers (i.e. multi-allelic or biallelic) that will eventually be used for QTL mapping or MAS.     

Haplotypes at ATM identify coding-sequence variation and indicate a region of extensive linkage disequilibrium

Genetic variation in the human population may lead to functional variants of genes that contribute to risk for common chronic diseases such as cancer. In an effort to detect such possible predisposing variants, we constructed haplotypes for a candidate gene and tested their efficacy in association studies. We developed haplotypes consisting of 14 biallelic neutral-sequence variants that span 142 kb of the ATM locus. ATM is the gene responsible for the autosomal recessive disease ataxia-telangiectasia (AT). These ATM noncoding single-nucleotide polymorphisms (SNPs) were genotyped in nine CEPH families (89 individuals) and in 260 DNA samples from four different ethnic origins. Analysis of these data with an expectation-maximization algorithm revealed 22 haplotypes at this locus, with three major haplotypes having frequencies > or = .10. Tests for recombination and linkage disequilibrium (LD) show reduced recombination and extensive LD at the ATM locus, in all four ethnic groups studied. The most striking example was found in the study population of European ancestry, in which no evidence for recombination could be discerned. The potential of ATM haplotypes for detection of genetic variants through association studies was tested by analysis of 84 individuals carrying one of three ATM coding SNPs. Each coding SNP was detected by association with an ATM haplotype. We demonstrate that association studies with haplotypes for candidate genes have significant potential for the detection of genetic backgrounds that contribute to disease.     

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.     

Multilocus sequence typing (MLST) of Entamoeba histolytica identifies kerp2 as a genetic marker associated with disease outcomes

Amoebiasis caused by protozoan parasite Entamoeba histolytica has diverse infection outcomes. The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needs to be explored. Genome information of infecting strains from endemic areas throughout the world is essential to explore this relation. Comparative genetics between E. histolytica populations from different disease outcomes have been studied to identify potential genetic markers having single nucleotide polymorphisms (SNPs) significantly associated with specific clinical outcome. Coding and non-coding regions have significantly different rates of polymorphism. Non-synonymous base substitutions were significantly more frequent than synonymous within coding loci. Both synonymous and non-synonymous SNPs within lysine- and glutamic acid rich protein 2 (kerp2) locus were significantly associated with disease outcomes. An incomplete linkage disequilibrium (LD) value with potential recombination events and significant population differentiation (F_ST) value have also been identified at kerp2 locus within the study population. Presence of disease specific SNPs, potential recombination events, and significant F_ST value at kerp2 locus indicate that kerp2 gene and its gene product are under constant selection pressure exerted by host on parasite and could also be a potential determinant of disease outcome of E. histolytica infection. Furthermore, E. histolytica isolated from asymptomatic carriers are phylogenetically closer to those causing liver abscess in human and exhibit potential inter-population recombination among them. Individuals with persistent asymptomatic E. histolytica infection may be under high risk of developing amoebic liver abscess formation in future and detailed investigation of asymptomatic individuals from endemic areas should be always required.     

Characterisation of single nucleotide polymorphisms in sugarcane ESTs

Commercial sugarcane cultivars (Saccharum spp. hybrids) are both polyploid and aneuploid with chromosome numbers in excess of 100; these chromosomes can be assigned to 8 homology groups. To determine the utility of single nucleotide polymorphisms (SNPs) as a means of improving our understanding of the complex sugarcane genome, we developed markers to a suite of SNPs identified in a list of sugarcane ESTs. Analysis of 69 EST contigs showed a median of 9 SNPs per EST and an average of 1 SNP per 50 bp of coding sequence. The quantitative presence of each base at 58 SNP loci within 19 contiguous sequence sets was accurately and reliably determined for 9 sugarcane genotypes, including both commercial cultivars and ancestral species, through the use of quantitative light emission technology in pyrophosphate sequencing. Across the 9 genotypes tested, 47 SNP loci were polymorphic and 11 monomorphic. Base frequency at individual SNP loci was found to vary approximately twofold between Australian sugarcane cultivars and more widely between cultivars and wild species. Base quantity was shown to segregate as expected in the IJ76-514 × Q165 sugarcane mapping population, indicating that SNPs that occur on one or two sugarcane chromosomes have the potential to be mapped. The use of SNP base frequencies from five of the developed markers was able to clearly distinguish all genotypes in the population. The use of SNP base frequencies from a further six markers within an EST contig was able to help establish the likely copy number of the locus in two genotypes tested. This is the first instance of a technology that has been able to provide an insight into the copy number of a specific gene locus in hybrid sugarcane. The identification of specific and numerous haplotypes/alleles present in a genotype by pyrophosphate sequencing or alternative techniques ultimately will provide the basis for identifying associations between specific alleles and phenotype and between allele dosage and phenotype in sugarcane.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The posterior probability of linkage allowing for linkage disequilibrium and a new estimate of disequilibrium between a trait and a marker

The posterior probability of linkage (PPL) statistic has been developed as a method for the rigorous accumulation of evidence for or against linkage allowing for both intra- and inter-sample heterogeneity. To date, the method has assumed linkage equilibrium between alleles at the trait locus and the marker locus. We now generalize the PPL to allow for linkage disequilibrium (LD), by incorporating variable phase probabilities into the underlying linkage likelihood. This enables us to recover the marginal posterior density of the recombination fraction, integrating out nuisance parameters of the trait model, including the locus heterogeneity (admixture) parameter, as well as a vector of LD parameters. The marginal posterior density can then be updated across data subsets or new data as they become available, while allowing parameters of the trait model to vary between data sets. The method applies immediately to general pedigree structures and to markers with multiple alleles. In the case of SNPs, the likelihood is parameterized in terms of the standard single LD parameter D'; and it therefore affords a mechanism for estimation of D' between the marker and the trait, again, without fixing the parameters of the trait model and allowing for updating across data sets. It is even possible to allow for a different associated allele in different populations, while accumulating information regarding the strength of LD. While a computationally efficient implementation for multi-allelic markers is still in progress, we have implemented a version of this new LD-PPL for SNPs and evaluated its performance in nuclear families. Our simulations show that LD-PPLs tend to be larger than PPLs (stronger evidence in favor of linkage/LD) with increased LD level, under a variety of generating models; while in the absence of linkage and LD, LD-PPLs tend to be smaller than PPLs (stronger evidence against linkage). The estimate of D' also behaves well even in relatively small, heterogeneous samples.     

Distinct Genetic Loci Control Plasma HIV-RNA and Cellular HIV-DNA Levels in HIV-1 Infection: The ANRS Genome Wide Association 01 Study

Previous studies of the HIV-1 disease have shown that HLA and Chemokine receptor genetic variants influence disease progression and early viral load. We performed a Genome Wide Association study in a cohort of 605 HIV-1-infected seroconverters for detection of novel genetic factors that influence plasma HIV-RNA and cellular HIV-DNA levels. Most of the SNPs strongly associated with HIV-RNA levels were localised in the 6p21 major histocompatibility complex (MHC) region and were in the vicinity of class I and III genes. Moreover, protective alleles for four disease-associated SNPs in the MHC locus (rs2395029, rs13199524, rs12198173 and rs3093662) were strikingly over-represented among forty-five Long Term HIV controllers. Furthermore, we show that the HIV-DNA levels (reflecting the HIV reservoir) are associated with the same four SNPs, but also with two additional SNPs on chromosome 17 (rs6503919; intergenic region flanked by the DDX40 and YPEL2 genes) and chromosome 8 (rs2575735; within the Syndecan 2 gene). Our data provide evidence that the MHC controls both HIV replication and HIV reservoir. They also indicate that two additional genomic loci may influence the HIV reservoir.     

Multipoint identity-by-descent prediction using dense markers to map quantitative trait loci and estimate effective population size

A novel multipoint method, based on an approximate coalescence approach, to analyze multiple linked markers is presented. Unlike other approximate coalescence methods, it considers all markers simultaneously but only two haplotypes at a time. We demonstrate the use of this method for linkage disequilibrium (LD) mapping of QTL and estimation of effective population size. The method estimates identity-by-descent (IBD) probabilities between pairs of marker haplotypes. Both LD and combined linkage and LD mapping rely on such IBD probabilities. The method is approximate in that it considers only the information on a pair of haplotypes, whereas a full modeling of the coalescence process would simultaneously consider all haplotypes. However, full coalescence modeling is computationally feasible only for few linked markers. Using simulations of the coalescence process, the method is shown to give almost unbiased estimates of the effective population size. Compared to direct marker and haplotype association analyses, IBD-based QTL mapping showed clearly a higher power to detect a QTL and a more realistic confidence interval for its position. The modeling of LD could be extended to estimate other LD-related parameters such as recombination rates.     

Isolates and their potential use in complex gene mapping efforts

Linkage disequilibrium (LD), detectable with microsatellites in disease alleles over wide genetic intervals in population isolates, has facilitated mapping and positional cloning of numerous disease genes. We, among others, have shown that the LD intervals reach up to 1 Mb in general alleles of young subisolates, and that this feature most probably offers an avenue for the initial locus positioning for complex traits. Development of efficient SNP genotyping and characterization of haploblock structure of the human genome have introduced new prospects to LD-based fine mapping and haplotype-association studies. Encouraging associations have been reported for several complex diseases. Final breakthroughs in mapping of complex disease loci have emerged on large pedigrees in population isolates. Conversely, ignoring genealogical makeup of the study population seems to disclose false negative and false positive associations, directing resources down the drain.     

Estimating the age of alleles by use of intraallelic variability.

A method is presented for estimating the age of an allele by use of its frequency and the extent of variation among different copies. The method uses the joint distribution of the number of copies in a population sample and the coalescence times of the intraallelic gene genealogy conditioned on the number of copies. The linear birth-death process is used to approximate the dynamics of a rare allele in a finite population. A maximum-likelihood estimate of the age of the allele is obtained by Monte Carlo integration over the coalescence times. The method is applied to two alleles at the cystic fibrosis (CFTR) locus, deltaF508 and G542X, for which intraallelic variability at three intronic microsatellite loci has been examined. Our results indicate that G542X is somewhat older than deltaF508. Although absolute estimates depend on the mutation rates at the microsatellite loci, our results support the hypothesis that deltaF508 arose < 500 generations (approximately 10,000 years) ago.     

Polymorphisms in cinnamoyl CoA reductase (CCR) are associated with variation in microfibril angle in Eucalyptus spp

Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of marker-trait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees.     

Single-nucleotide polymorphisms and haplotype LD analysis of the 29-kb IGF2 region on chromosome 11p15.5 in the Korean population

OBJECTIVE: We investigated sequence variations of the 29-kb insulin-like growth factor 2 (IGF2) region in human chromosome region 11p15.5 in the Korean population. This region consists of IGF2, insulin-like growth factor 2 antisense (IGF2AS), and the insulin gene, all important candidate genes for various diseases, including cancer, obesity, diabetes, and coronary disease. While single nucleotide polymorphisms (SNPs) have been identified for this region and used in association studies, ethnic differences in genetic variation at this site have not been addressed. To date, SNPs for the entire 29-kb region in the Korean population have not been reported. METHODS: We surveyed a population of 108 Koreans for SNPs in the 29-kb IGF2 region. RESULTS: We identified 62 SNPs, consisting of 6 SNPs in the promoter region, 17 in the untranslated region, 19 in introns, and 20 in the intergenic region. We also analyzed linkage disequilibrium (LD) patterns and haplotypes using 36 high-frequency (> 5%)SNPs and found a well-defined LD block spanning about 13 kb that includes 8 kb of the IGF2AS gene, with two hot-spot regions flanking the LD block. CONCLUSION: These SNPs may be useful as genetic markers in disease association studies in the Korean population.     

Genetics of sputum gene expression in chronic obstructive pulmonary disease

Previous expression quantitative trait loci (eQTL) studies have performed genetic association studies for gene expression, but most of these studies examined lymphoblastoid cell lines from non-diseased individuals. We examined the genetics of gene expression in a relevant disease tissue from chronic obstructive pulmonary disease (COPD) patients to identify functional effects of known susceptibility genes and to find novel disease genes. By combining gene expression profiling on induced sputum samples from 131 COPD cases from the ECLIPSE Study with genomewide single nucleotide polymorphism (SNP) data, we found 4315 significant cis-eQTL SNP-probe set associations (3309 unique SNPs). The 3309 SNPs were tested for association with COPD in a genomewide association study (GWAS) dataset, which included 2940 COPD cases and 1380 controls. Adjusting for 3309 tests (p<1.5e-5), the two SNPs which were significantly associated with COPD were located in two separate genes in a known COPD locus on chromosome 15: CHRNA5 and IREB2. Detailed analysis of chromosome 15 demonstrated additional eQTLs for IREB2 mapping to that gene. eQTL SNPs for CHRNA5 mapped to multiple linkage disequilibrium (LD) bins. The eQTLs for IREB2 and CHRNA5 were not in LD. Seventy-four additional eQTL SNPs were associated with COPD at p<0.01. These were genotyped in two COPD populations, finding replicated associations with a SNP in PSORS1C1, in the HLA-C region on chromosome 6. Integrative analysis of GWAS and gene expression data from relevant tissue from diseased subjects has located potential functional variants in two known COPD genes and has identified a novel COPD susceptibility locus.     

Polymorphisms in a desaturase 2 ortholog associate with cuticular hydrocarbon and male mating success variation in a natural population of Drosophila serrata

Elucidating the nature of genetic variation underlying both sexually selected traits and the fitness components of sexual selection is essential to understanding the broader consequences of sexual selection as an evolutionary process. To date, there have been relatively few attempts to connect the genetic variance in sexually selected traits with segregating DNA sequence polymorphisms. We set out to address this in a well‐characterized sexual selection system – the cuticular hydrocarbons (CHCs) of Drosophila serrata – using an indirect association study design that allowed simultaneous estimation of the genetic variance in CHCs, sexual fitness and single nucleotide polymorphism (SNP) effects in an outbred population. We cloned and sequenced an ortholog of the D. melanogaster desaturase 2 gene, previously shown to affect CHC biosynthesis in D. melanogaster, and associated 36 SNPs with minor allele frequencies > 0.02 with variance in CHCs and sexual fitness. Three SNPs had significant multivariate associations with CHC phenotype (q‐value < 0.05). At these loci, minor alleles had multivariate effects on CHCs that were weakly associated with the multivariate direction of sexual selection operating on these traits. Two of these SNPs had pleiotropic associations with male mating success, suggesting these variants may underlie responses to sexual selection due to this locus. There were 15 significant male mating success associations (q‐value < 0.1), and interestingly, we detected a nonrandom pattern in the relationship between allele frequency and direction of effect on male mating success. The minor‐frequency allele usually reduced male mating success, suggesting a positive association between male mating success and total fitness at this locus.     

Genome-wide distribution of linkage disequilibrium in the population of Palau and its implications for gene flow in Remote Oceania

Linkage disequilibrium (LD) between alleles on the same human chromosome results from various evolutionary processes and is thus telling about the history of populations. Recently, LD has garnered substantial interest for its value to map and fine-map disease genes. We examine the distribution of LD between short tandem repeat alleles on autosomes and sex chromosomes in the Remote Oceanic population of Palau to evaluate whether the data are consistent with a recent hypothesis about the origins of genetic variation in Palau, specifically that the population experienced extensive male-biased gene flow following initial settlement. Consistent with evolutionary theory based on effective population size, LD between X-linked alleles is stochastically greater than LD between autosomal alleles, however, small but detectable LD occurs for autosomal markers separated by substantial distances. By contrast, while Y-linked alleles experience only one-third the effective population size of X-linked alleles, their mean value for pairwise LD is only slightly larger than X-linked alleles. For a small population known to experience at least two extreme bottlenecks, 56 six-locus Y haplotypes exhibit remarkable diversity (0.96), comparable to Y diversity of Europeans, however, autosomal and X-linked markers display significantly less diversity, as measured by heterozygosity (4.1% less). Palauan Y haplotypes also fall into distinct clusters, again unlike that of Europe. We argue these data are consistent with waves of male-biased gene flow.