Multilocus Tests Research Articles

Association of the vitamin D metabolism gene CYP27B1 with type 1 diabetes.

Epidemiological studies have linked vitamin D deficiency with the susceptibility to type 1 diabetes. Higher levels of the active metabolite 1 alpha,25-dihydroxyvitamin D (1 alpha,25(OH)(2)D) could protect from immune destruction of the pancreatic beta-cells. 1 alpha,25(OH)(2)D is derived from its precursor 25-hydroxyvitamin D by the enzyme 1 alpha-hydroxylase encoded by the CYP27B1 gene and is inactivated by 24-hydroxylase encoded by the CYP24A1 gene. Our aim was to study the association between the CYP27B1 and CYP24A1 gene polymorphisms and type 1 diabetes. We studied 7,854 patients with type 1 diabetes, 8,758 control subjects from the U.K., and 2,774 affected families. We studied four CYP27B1 variants, including common polymorphisms -1260C>A (rs10877012) and +2838T>C (rs4646536) and 16 tag polymorphisms in the CYP24A1 gene. We found evidence of association with type 1 diabetes for CYP27B1 -1260 and +2838 polymorphisms, which are in perfect linkage disequilibrium. The common C allele of CYP27B1 -1260 was associated with an increased disease risk in the case-control analysis (odds ratio for the C/C genotype 1.22, P = 9.6 x 10(-4)) and in the fully independent collection of families (relative risk for the C/C genotype 1.33, P = 3.9 x 10(-3)). The combined P value for an association with type 1 diabetes was 3.8 x 10(-6). For the CYP24A1 gene, we found no evidence of association with type 1 diabetes (multilocus test, P = 0.23). The present data provide evidence that common inherited variation in the vitamin D metabolism affects susceptibility to type 1 diabetes.

Association of the interleukin‐2 receptor alpha (IL‐2Rα)/CD25 gene region with Graves’ disease using a multilocus test and tag SNPs

A small number of immune response genes have been consistently associated with the common autoimmune conditions. Recently, a linkage disequilibrium (LD) mapping approach, using tag single nucleotide polymorphisms (SNPs), identified genetic association between type 1 diabetes (T1D) and the interleukin-2 receptor alpha (IL-2Ralpha)/CD25 gene region on chromosome 10p15. Because certain autoimmune diseases, such as autoimmune thyroid disease (AITD) and T1D cluster together in certain families, we sought to determine if the TID-associated CD25 region was also associated with Graves' disease (GD). We performed a case-control association study of 20 tag SNPs. 1896 GD patients were collected from seven major centres in the UK and 1822 geographically matched controls from the 1958 British Birth Cohort. The 20 tag SNPs were analysed using a multilocus test to identify an association between GD and the CD25 region. Odds ratios (ORs) were calculated for the tag SNPs, allowing a comparison with previous results for T1D. RESULTS The multilocus test provided statistical evidence of an association between GD and the CD25 region (P = 4.5 x 10(-4)), with the pattern of association of the 20 tag SNPs similar to that found in T1D. CONCLUSIONS Association with GD, as well as that previously reported with T1D, suggests that the CD25 region is acting as a general susceptibility locus for autoimmune disease, and is consistent with a major role for the IL-2-receptor pathway in the development and function of T cells in the control of autoimmunity.

Powerful Multilocus Tests of Genetic Association in the Presence of Gene-Gene and Gene-Environment Interactions

In modern genetic epidemiology studies, the association between the disease and a genomic region, such as a candidate gene, is often investigated using multiple SNPs. We propose a multilocus test of genetic association that can account for genetic effects that might be modified by variants in other genes or by environmental factors. We consider use of the venerable and parsimonious Tukey's 1-degree-of-freedom model of interaction, which is natural when individual SNPs within a gene are associated with disease through a common biological mechanism; in contrast, many standard regression models are designed as if each SNP has unique functional significance. On the basis of Tukey's model, we propose a novel but computationally simple generalized test of association that can simultaneously capture both the main effects of the variants within a genomic region and their interactions with the variants in another region or with an environmental exposure. We compared performance of our method with that of two standard tests of association, one ignoring gene-gene/gene-environment interactions and the other based on a saturated model of interactions. We demonstrate major power advantages of our method both in analysis of data from a case-control study of the association between colorectal adenoma and DNA variants in the NAT2 genomic region, which are well known to be related to a common biological phenotype, and under different models of gene-gene interactions with use of simulated data.

A Bayesian Heterogeneous Analysis of Variance Approach to Inferring Recent Selective Sweeps

The distribution of microsatellite allele sizes in populations aids in understanding the genetic diversity of species and the evolutionary history of recent selective sweeps. We propose a heterogeneous Bayesian analysis of variance model for inferring loci involved in recent selective sweeps by analyzing the distribution of allele sizes at multiple loci in multiple populations. Our model is shown to be consistent with a multilocus test statistic, ln RV, proposed for identifying microsatellite loci involved in recent selective sweeps. Our methodology differs in that it accepts original allele size data rather than summary statistics and allows the incorporation of prior knowledge about allele frequencies using a hierarchical prior distribution consisting of log normal and gamma probability distributions. Interesting features of the model are its ability to simultaneously analyze allele size data for any number of populations and to cope with the presence of any number of selected loci. The utility of the method is illustrated by application to two sets of microsatellite allele size data for a group of West African Anopheles gambiae populations. The results are consistent with the suppressed-recombination model of speciation, and additional candidate loci on chromosomes 2 (079 and 175) and 3 (088) are discovered that escaped former analysis.

Genomic islands of differentiation between house mouse subspecies

Understanding the genes that contribute to reproductive isolation is essential to understanding speciation, but isolating such genes has proven very difficult. In this study I apply a multilocus test statistic to >10,000 SNP markers assayed in wild-derived inbred strains of house mice to identify genomic regions of elevated differentiation between two subspecies of house mice, Mus musculus musculus and M. m. domesticus. Differentiation was high through approximately 90% of the X chromosome. In addition, eight regions of high differentiation were identified on the autosomes, totaling 7.5% of the autosomal genome. Regions of high differentiation were confirmed by direct sequencing of samples collected from the wild. Some regions of elevated differentiation have an overrepresentation of genes with host-pathogen interactions and olfaction. The most strongly differentiated region on the X has previously been shown to fail to introgress across a hybrid zone between the two subspecies. This survey indicates autosomal regions that should also be examined for differential introgression across the hybrid zone, as containing potential genes causing hybrid unfitness.

Genetic diversity, reproductive biology, and speciation in the entomopathogenic fungusBeauveria bassiana(Balsamo) Vuillemin

Beauveria bassiana, a mitosporic fungus used for the biological control of many insect species, is recognized as a "species complex" comprising genetically diverse lineages. Being predominantly asexual, mating tests cannot be applied to delimit species in this species complex. Genetic tests offer an indirect means of identifying species among isolates. To this end, molecular genetic analysis of a sample of B. bassiana isolates with 2 subsamples, 1 representing a worldwide collection and another from a localized epizootic population was carried out. DNA markers generated through AFLPs (amplified fragment length polymorphisms) and SSCPs (single-strand conformation poly morphisms) and nucleotide sequence data of different allelic forms of 3 genes (large and small subunits of rRNA and beta-tubulin) were evaluated. The B. bassiana isolates from the worldwide sample showed 11% overall similarity and no closely clustered groups. Phylogenetic trees generated from the AFLP and SSCP data of this sample resolved the different isolates into distinct phylogenetic lineages. In the epizootic B. bassiana population, prevalence of recombination was evident from random association of alleles in multilocus tests and lack of phylogenetic concordance among 3 gene genealogies. Thus, the worldwide sample of B. bassiana exhibits a predominantly clonal structure, hinting at species divergence leading to cryptic speciation with recombination being customary among isolates sharing a close ecological niche.

Ranks of Genuine Associations in Whole-Genome Scans

With the recent advances in high-throughput genotyping techniques, it is now possible to perform whole-genome association studies to fine map causal polymorphisms underlying important traits that influence susceptibility to human diseases and efficacy of drugs. Once a genome scan is completed the results can be sorted by the association statistic value. What is the probability that true positives will be encountered among the first most associated markers? When a particular polymorphism is found associated with the trait, there is a chance that it represents either a "true" or a "false" association (TA vs. FA). Setting appropriate significance thresholds has been considered to provide assurance of sufficient odds that the associations found to be significant are genuine. However, the problem with genome scans involving thousands of markers is that the statistic values of FAs can reach quite extreme magnitudes. In such situations, the distributions corresponding to TAs and the most extreme FAs become comparable and significance thresholds tend to penalize TAs and FAs in a similar fashion. When sorting between true and false associations, the "typical" place (i.e., rank) of TAs among the most significant outcomes becomes important, ordered by the association statistic value. The distribution of ranks that we study here allows calculation of several useful quantities. In particular, it gives the number of most significant markers needed for a follow-up study to guarantee that a true association is included with certain probability. This can be calculated conditionally on having applied a multiple-testing correction. Effects of multilocus (e.g., haplotype association) tests and impact of linkage disequilibrium on the distribution of ranks associated with TAs are evaluated and can be taken into account.

Localization of a Type 1 Diabetes Locus in the IL2RA/CD25 Region by Use of Tag Single-Nucleotide Polymorphisms

As part of an ongoing search for genes associated with type 1 diabetes (T1D), a common autoimmune disease, we tested the biological candidate gene IL2RA (CD25), which encodes a subunit (IL-2R alpha) of the high-affinity interleukin-2 (IL-2) receptor complex. We employed a tag single-nucleotide polymorphism (tag SNP) approach in large T1D sample collections consisting of 7,457 cases and controls and 725 multiplex families. Tag SNPs were analyzed using a multilocus test to provide a regional test for association. We found strong statistical evidence in the case-control collection (P=6.5x10(-8)) for a T1D locus in the CD25 region of chromosome 10p15 and replicated the association in the family collection (P=7.3x10(-3); combined P=1.3x10(-10)). These results illustrate the utility of tag SNPs in a chromosome-regional test of disease association and justify future fine mapping of the causal variant in the region.

Rapid Simulation of P Values for Product Methods and Multiple-Testing Adjustment in Association Studies

A major aim of association studies is the identification of polymorphisms (usually SNPs) associated with a trait. Tests of association may be based on individual SNPs or on sets of neighboring SNPs, by use of (for example) a product P value method or Hotelling's T test. Linkage disequilibrium, the nonindependence of SNPs in physical proximity, causes problems for all these tests. First, multiple-testing correction for individual-SNP tests or for multilocus tests either leads to conservative P values (if Bonferroni correction is used) or is computationally expensive (if permutation is used). Second, calculation of product P values usually requires permutation. Here, we present the direct simulation approach (DSA), a method that accurately approximates P values obtained by permutation but is much faster. It may be used whenever tests are based on score statistics--for example, with Armitage's trend test or its multivariate analogue. The DSA can be used with binary, continuous, or count traits and allows adjustment for covariates. We demonstrate the accuracy of the DSA on real and simulated data and illustrate how it might be used in the analysis of a whole-genome association study.

Genetic structure of northwestern Spanish brown trout (Salmo trutta L.) populations, differences between microsatellite and allozyme loci

Genetic variation in nine wild brown trout (Salmo trutta L.) populations was studied by means of allozyme and microsatellite markers. All brown trout populations were clearly separated into two clusters that represented the Sil and Duero basins. Although both markers revealed a strong genetic differentiation between basins, microsatellite loci resulted much more accurate when population structure at the intrabasin level was analysed. Also pairwise multilocus FST estimates and assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. The analysis of both markers provides new insights in defining the conservation units at this local area and confirms the existence of a recognized sub-lineage in the Duero basin. The management implications of these findings are discussed and changes in trout release activity are recommended to avoid mixing of trout gene pools mainly in the Sil basin.

Gene flow and differences among local populations of the land snail Arianta arbustorum (Linnaeus, 1758) (Pulmonata: Helicidae)

The genetic structure of nine populations of Arianta arbustorum (Linnaeus, 1758) in South Poland was studied by means of allozyme electrophoresis on cellulose acetate gel. The aim of the study was to answer the following questions: (1) is there (unlike in mtDNA haplotypes) any geographic variation in allozymes among local populations of A. arbustorum?; (2) what are the levels and pattern of gene flow?; (3) are the subpopulations really panmictic units? To answer the questions six enzyme systems, coded by eight loci (Hbdh, Iddh, Idh-1, Idh-2, Mpi, Pgdh, Pgm-1, Pgm-2), were assayed. Neither Fisher’s method (significant for four pairs of loci) nor Ohta’s D-statistics applied to study linkage disequilibrium indicated close linkage caused by population subdivision. Mean number of alleles per locus was 1.98, mean expected heterozygosity 0.337. Multilocus test showed a heterozygote deficiency for all populations but one; multipopulation test showed the same for all loci, though many loci were at Hardy-Weinberg equilibrium for individual populations. f values were high, values moderate. Inbreeding, Wahlund’s effect (mostly caused by the occurrence of several generations) and selection were considered the main sources of the observed heterozygote deficits. Mean value of was 0.270 and the resulting estimate of gene flow Nm=0.676. Pairwise Nm’s were low (almost 5 in one case, more than 2 in two cases, and more than 1 in four cases, out of 36 comparisons) fairly differentiated and not always consistent with biological reality. Neither pairwise values, nor Cavalli-Sforza and Edwards arc and unbiased Nei genetic distances were statistically significantly associated with geographic distances among populations. Multidimensional scaling on , as well as UPGMA clustering and neighbor joining trees computed on all the three parameters, showed no geographic pattern; correspondence analysis on allele frequencies did not show such a pattern, either. It is hypothesized that the present differentiation is a combined result of the following: (1) time (the snail has probably inhabited the studied area since before the latest glaciation); (2) the low levels of migration; (3) the immigrants’ poor chance of winning in the competition against the autochthons; (4) selection; (5) new mutations (possibly).

DOES SIZE MATTER? FITNESS-RELATED FACTORS IN STEELHEAD TROUT DETERMINED BY GENETIC PARENTAGE ASSIGNMENT

Estimates of reproductive success and the factors that affect it can be inferred from observations of behavior or data on life history traits (e.g., dominance hierarchies, clutch size). Genetic parentage analysis, however, can precisely determine the number of offspring attributable to individual parents. We coupled multilocus microsatellite genotype-based parentage assignment exclusion testing with morphological and behavioral data to test hypotheses concerning the effects of specific traits on fitness of naturally spawning steelhead trout (Oncorhynchus mykiss). With eight microsatellite loci (mean heterozygosity = 89%), we examined two parental generations (n1 = 198, n2 = 226) and their offspring as smolts (age 2: n1 = 365, n2 = 285) and as adults (n1 = 81, n2 = 77) to determine the effects of size, reproductive timing, and origin (hatchery or wild) on reproductive success. The mating system was complex; monogamy, polygamy, polyandry, polygyny, and polygynandry were all observed. Males had a greater var...

Detecting the footprint of positive selection in a european population of Drosophila melanogaster: multilocus pattern of variation and distance to coding regions.

The effects on nucleotide variation of adaptations to temperate habitats and of the possible bottleneck associated with the origin of European populations of Drosophila melanogaster should be detectable in DNA sequences given the short time elapsed relative to the species population size. We surveyed nucleotide variation in 109 fragments distributed across the X chromosome in a European population of D. melanogaster to detect the footprint of positive selection. Fragments were located primarily in large noncoding regions. Multilocus tests based on Tajima's D statistic revealed a significant departure from neutral expectations in a stationary panmictic population, with an important contribution from both positive and negative D values. A positive relationship between Tajima's D values and distance to coding region was detected, with a comparative excess of significantly negative D values in the subset of fragments closer to coding regions. Also, there was a significant heterogeneity in the polymorphism to divergence ratio, with 12 fragments contributing 42% to the test statistic. Moreover, these fragments were comparatively closer to coding regions. These findings would imply positive selection events, and thus selective sweeps, during the species expansion to Europe.

A quick and simple method for detecting subjects with abnormal genetic background in case-control samples.

It is important that case-control samples be drawn from a genetically homogeneous population in order to avoid artefactual false positive results and to enhance power to detect disease mutations and markers in linkage disequilibrium with them. Tests which simply compare overall marker allele frequencies between cases and controls will fail to identify a relatively small number of subjects drawn from a different genetic background who could usefully be discarded from the sample. Such subjects can be identified using multilocus tests, but previously described tests have been unnecessarily complex and cumbersome for this simple application. We describe a straightforward test, implemented in the CHECKHET program, which uses a measure of genetic difference and permutation procedures to rapidly identify such subjects using genotypes from multiple unlinked markers. It seems to perform reasonably well on simulated data, and with real data appears to identify two abnormal subjects within a case-control sample. We recommend that such tests be routinely applied to case-control samples once sufficient numbers of markers have been genotyped within them.

A microsatellite-based multilocus screen for the identification of local selective sweeps.

With the availability of completely sequenced genomes, multilocus scans of natural variability have become a feasible approach for the identification of genomic regions subjected to natural and artificial selection. Here, I introduce a new multilocus test statistic, ln RV, which is based on the ratio of observed variances in repeat number at a set of microsatellite loci in two groups of populations. The distribution of ln RV values captures demographic history of the populations as well as variation in microsatellite mutation among loci. Given that microsatellite loci associated with a recent selective sweep differ from the remainder of the genome, they are expected to fall outside of the distribution of neutral ln RV values. The ln RV test statistic is applied to a data set of 94 loci typed in eight non-African and two African human populations.

Conservation genetics of harbour porpoises, Phocoena phocoena, in eastern and central North Atlantic

We examined polymorphism at 12 microsatelliteloci in 807 harbour porpoises , Phocoenaphocoena, collected from throughout thecentral and eastern North Atlantic to theBaltic Sea. Multilocus tests for allelefrequency differences, assignment tests,population structure estimates (FST) andgenetic distance measures (DLR andDC) all indicate six geneticallydifferentiated populations/sub-populationsafter pooling sub-samples within regions.Harbour porpoises from West Greenland, theNorwegian Westcoast, Ireland, the British NorthSea, the Danish North Sea and the inland watersof Denmark (IDW) are all geneticallydistinguishable from each other. A sample ofharbour porpoises collected off the Dutch coast(mainly during winter) was geneticallyheterogeneous and likely comprised a mixture ofindividuals of diverse origin. A mixed stockanalysis indicated that most of the individualsin this sample (∼77%) were likely migrantsfrom the British and Danish North Sea.

Affected sibpair linkage tests for multiple linked susceptibility genes.

Genome-wide searches for susceptibility genes using pairs of affected siblings are being undertaken to dissect out individual polygenes that contribute to human multifactorial disease. Efficient identity-by-descent (IBD)-based sibpair linkage tests are available that test individual markers or maps of linked markers for linkage to a single putative susceptibility gene. In order to assess the support for linkage to a second putative susceptibility gene that happens to map close to an established susceptibility gene, it is necessary to use a method that correctly allows for the IBD distortion that directly results from the linkage between the two genes. A maximum likelihood-based, multilocus linkage test is proposed, which accounts for this interdependency and evaluates the support for an interaction between constituent susceptibility genes. The size and power of a test for a second linked susceptibility gene is investigated by simulation studies.