Admixture Mapping Using Interval Transmission/Disequilibrium Tests

Family‐based studies of genetic association and linkage play a key role in mapping susceptibility genes of complex human diseases. Recent admixture between genetically differentiated populations can result in high levels of linkage disequilibrium even at far apart loci. This has been capitalized upon to reduce the burden of genotyping in a genomewide association scan. Here the authors describe an alternative approach for admixture mapping. The genome is divided into several non‐overlapping intervals and the information of the markers in the same interval is integrated—the ‘interval transmission/disequilibrium test’ (ITDT). This method requires information in the form of the marker allele frequencies in the founding populations. However, computer simulations show that the performances of ITDT are hardly affected by imprecise allele‐frequency information. Simulations also show that an interval‐by‐interval scan using ITDT perform much better than a conventional marker‐by‐marker scan using TDT, even under conditions where the admixture process occurred over many generations and the individuals in the admixed populations show considerable variation in admixture proportion. Hence the ITDT is a promising new genomewide scan method.     

A family-based association test to detect gene-gene interactions in the presence of linkage

For many complex diseases, quantitative traits contain more information than dichotomous traits. One of the approaches used to analyse these traits in family-based association studies is the quantitative transmission disequilibrium test (QTDT). The QTDT is a regression-based approach that models simultaneously linkage and association. It splits up the association effect in a between- and a within-family genetic component to adjust and test for population stratification and includes a variance components method to model linkage. We extend this approach to detect gene-gene interactions between two unlinked QTLs by adjusting the definition of the between- and within-family component and the variance components included in the model. We simulate data to investigate the influence of the epistasis model, linkage disequilibrium patterns between the markers and the QTLs, and allele frequencies on the power and type I error rates of the approach. Results show that for some of the investigated settings, power gains are obtained in comparison with FAM-MDR. We conclude that our approach shows promising results for candidate-gene studies where too few markers are available to correct for population stratification using standard methods (for example EIGENSTRAT). The proposed method is applied to real-life data on hypertension from the FLEMENGHO study.     

Tests of Association for Quantitative Traits in Nuclear Families Using Principal Components to Correct for Population Stratification

Traditional transmission disequilibrium test (TDT) based methods for genetic association analyses are robust to population stratification at the cost of a substantial loss of power. We here describe a novel method for family-based association studies that corrects for population stratification with the use of an extension of principal component analysis (PCA). Specifically, we adopt PCA on unrelated parents in each family. We then infer principal components for children from those for their parents through a TDT-like strategy. Two test statistics within the variance-components model are proposed for association tests. Simulation results show that the proposed tests have correct type I error rates regardless of population stratification, and have greatly improved power over two popular TDT-based methods: QTDT and FBAT. The application to the Genetic Analysis Workshop 16 (GAW16) data sets attests to the feasibility of the proposed method.     

Distinguishing Population Stratification from Genuine Allelic Effects with Mx: Association of ADH2 with Alcohol Consumption

A universal problem in genetic association studies is to distinguish associations due to genuine effects of the locus under investigation, or linkage disequilibrium with a nearby locus that has a genuine effect, from associations due to population stratification or other artifacts. Fulker et al. (1999) have suggested a test using unselected sib pairs to distinguish these two causes of association. The test is readily implemented within a standard maximum-likelihood framework using the Mx package. The approach is applied to data on ADH2 genotypes and a measure of alcohol consumption from an Australian DZ twin pair sample. Results indicate that the association of the ADH2*2 allele with lower alcohol consumption cannot be explained by simple admixture and that there may be genuine allelic effects of the locus on alcohol consumption. Power calculations are provided to show that these results are plausible for the sample size in this study and consider the effects of genetic architecture and sample structure on required sample sizes for the Fulker et al. test.     

Distribution of ancestral chromosomal segments in admixed genomes and its implications for inferring population history and admixture mapping

The ancestral chromosomal segments in admixed genomes are of significant importance for both population history inference and admixture mapping, because they essentially provide the basic information for tracking genetic events. However, the distributions of the lengths of ancestral chromosomal segments (LACS) under some admixture models remain poorly understood. Here we introduced a theoretical framework on the distribution of LACS in two representative admixture models, that is, hybrid isolation (HI) model and gradual admixture (GA) model. Although the distribution of LACS in the GA model differs from that in the HI model, we demonstrated that the mean LACS in the HI model is approximately half of that in the GA model if both admixture proportion and admixture time in the two models are identical. We showed that the theoretical framework greatly facilitated the inference and understanding of population admixture history by analyzing African-American and Mexican empirical data. In addition, we found the peak of association signatures in the HI model was much narrower and sharper than that in the GA model, indicating that the identification of putative causal allele in the HI model is more efficient than that in the GA model. Thus admixture mapping with case-only data would be a reasonable and economical choice in the HI model due to the weak background noise. However, according to our previous studies, many populations are likely to be gradually admixed and have pretty high background linkage disequilibrium. Therefore, we suggest using a case-control approach rather than a case-only approach to conduct admixture mapping to retain the statistics power in recently admixed populations.     

Analysis of chromosome 6q in Basque families with type 1 diabetes. GEPV-N. Basque-Navarre Endocrinology and Paediatric Group

Over the last few years several studies of linkage between non-HLA loci and type 1 diabetes mellitus have mapped several putative susceptibility genes on chromosome 6q; in fact, positive evidence of linkage and/or association of IDDM5 (6q25), IDDM8 (6q27) and IDDM15 (6q21) with type 1 diabetes has been reported. We have studied these loci in diabetic families of Basque origin, a genetically homogeneous population, to avoid artifactual association results due to admixture within the sample analysed. Statistical analyses of linkage were performed using a transmission disequilibrium test (TDT). We could not confirm linkage for IDDM5, IDDM8 and IDDM15 in our population, possibly due to population-specific differences in genetic susceptibility and/or environmental triggering factors to type 1 diabetes.     

Linkage and association analysis of CACNG3 in childhood absence epilepsy

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy characterised by absence seizures manifested by transitory loss of awareness with 2.5-4 Hz spike-wave complexes on ictal EEG. A genetic component to aetiology is established but the mechanism of inheritance and the genes involved are not fully defined. Available evidence suggests that genes encoding brain expressed voltage-gated calcium channels, including CACNG3 on chromosome 16p12-p13.1, may represent susceptibility loci for CAE. The aim of this work was to further evaluate CACNG3 as a susceptibility locus by linkage and association analysis. Assuming locus heterogeneity, a significant HLOD score (HLOD = 3.54, alpha = 0.62) was obtained for markers encompassing CACNG3 in 65 nuclear families with a proband with CAE. The maximum non-parametric linkage score was 2.87 (P < 0.002). Re-sequencing of the coding exons in 59 patients did not identify any putative causal variants. A linkage disequilibrium (LD) map of CACNG3 was constructed using 23 single nucleotide polymorphisms (SNPs). Transmission disequilibrium was sought using individual SNPs and SNP-based haplotypes with the pedigree disequilibrium test in 217 CAE trios and the 65 nuclear pedigrees. Evidence for transmission disequilibrium (P < or = 0.01) was found for SNPs within a approximately 35 kb region of high LD encompassing the 5'UTR, exon 1 and part of intron 1 of CACNG3. Re-sequencing of this interval was undertaken in 24 affected individuals. Seventy-two variants were identified: 45 upstream; two 5'UTR; and 25 intronic SNPs. No coding sequence variants were identified, although four variants are predicted to affect exonic splicing. This evidence supports CACNG3 as a susceptibility locus in a subset of CAE patients.     

Asthma is associated with single-nucleotide polymorphisms in ADAM33

BACKGROUND : The ADAM33 gene has recently been associated with asthma and bronchial hyper-reactivity. It codes for a disintegrin and metalloproteinase that triggers intra- and extracellular signalling by protein shedding. OBJECTIVE : We examined whether polymorphisms in ADAM33 are associated with asthma and related traits in two German populations. METHODS : We genotyped 15 intragenic single-nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of allele-specific primer extension products. The transmission disequilibrium test was used for association analysis in the German asthma family study. Additionally, we tested for association of these SNPs in a case-control sample from the European Community Respiratory Health Study using Armitage's trend test. RESULTS : In both studies, we found SNPs that were significantly associated with asthma and related traits. In the family study, significant associations were observed for the SNPs F+1, ST+4 and ST+5 (with the lowest P-value for F+1, P=0.005). Remarkably, this association is seen even in the absence of linkage with two microsatellite markers from a previous genome scan either 3.1 million bases (Mb) up- or 5.6 Mb downstream. In the case-control study, SNP ST+7 (P=0.008) was significantly associated with asthma. Some of these SNPs overlapped with those found to be associated with elevated total IgE levels and bronchial hyper-responsiveness. CONCLUSION : This study replicates the recently published association between asthma and ADAM33 gene variants. However, most of the associated SNPs were at non-identical positions in the German, UK and US samples. As linkage disequilibrium is high among the tested SNPs, and there is no known functional polymorphism, either not-tested variants in ADAM33, unknown regulatory elements or a gene in close proximity is responsible for this association.     

Randomization tests of disease-marker associations

A powerful test for population association of a disease with alleles at a bi-allelic marker locus is the transmission/disequilibrium test ( TDT ). A generalization of the test to multi-allelic marker locus is proposed which utilizes the maximal association of individual alleles with the disease, given by the maximum TDT statistic, TDT _(max). To overcome the multiple testing problem encountered when using the maximal association to test the null hypothesis of no disease-marker association, a randomization procedure is developed. An investigation of the power of the test suggests that the randomization procedure performs almost as well as a recently proposed likelihood based test of linkage disequilibrium. The advantage of the new test is that it can be applied sequentially, based on a one-sided version of the TDT statistic, for investigating patterns of association of several individual alleles with the disease.     

Identification of polymorphisms within Disrupted in Schizophrenia 1 and Disrupted in Schizophrenia 2, and an investigation of their association with schizophrenia and bipolar affective disorder

We have undertaken a search for polymorphic sequence variation within Disrupted in Schizophrenia 1 and Disrupted in Schizophrenia 2 (DISC1 and DISC2), which are both novel genes that span a translocation breakpoint strongly associated with schizophrenia and related psychoses in a large Scottish family. A scan of the coding sequence, intron/exon boundaries, and part of the 5' and 3' untranslated regions of DISC1, plus 2.7 kb at the 3' end of DISC2, has revealed a novel microsatellite and 15 novel single nucleotide polymorphisms (SNPs). We have tracked the inheritance of four of the SNPs through multiply affected families, and carried out case-control association studies using the microsatellite and four common SNPs on populations of patients with schizophrenia or bipolar affective disorder versus normal control subjects. Neither co-segregation with disease status nor significant association was detected; however, we could not detect linkage disequilibrium between all these markers in the control population, arguing that an even greater density of informative markers is required to test rigorously for association in this genomic region.     

Admixture estimates for Churuguara,a Venezuelan town in the State of Falcón

Background:?The present Venezuelan population is the admixture product of Amerindians, Europeans and Africans, a process which was not homogeneous over the country. Blood groups, STRs and VNTRs, specifically D1S80, have been used successfully in admixture studies, but few have been made in Venezuela.

Aim:?This study aims to estimate the admixture components of Churuguara, Venezuela, and to evaluate the genetic relationship of this population with other Venezuelan as well as worldwide populations through principal component analysis and the study of dendrograms based on genetic distances.

Subjects and methods:?Gene frequencies of blood groups ABO and Rh (only anti D), of STRs VWA, F13A01, FES/FPS and VNTR D1S80 were studied in a sample of 60 individuals born in Churuguara, a Venezuelan town of admixed ancestry in the State of Falcón. Admixture was estimated with Chakraborty's gene identity method, and Nei's standard genetic distance was used to build two dendrograms with the neighbour-joining approach, one based on the three STRs and the other based only on D1S80. Principal component analyses with the gene frequencies of these markers were also performed.

Results:?The frequency of allele ABO*O was 0.788, of ABO*A was 0.187 and of RH*D was 0.74. D1S80 showed 16 different alleles with a heterozygosity of 0.880, whilst the three STRs showed only eight different alleles and heterozygosities between 0.733 and 0.797. The estimates of admixture obtained in this analysis were 52.5% for the Spanish parental group, 27.6% for the African and 19.9% for the Amerindian. Comparison of Churuguara with other Latin American populations shows that its African component is not as high as that observed in Colombian Choco, but it is higher than that observed in other samples from Colombia, Chile and Maracaibo (Venezuela).

Conclusions:?Results of the admixture analysis are consistent with those obtained with two dendrograms and principal component analyses, suggesting that the strong initial Amerindian component of 500 years ago has been diluted by the continuous flow of European genes, mainly Spanish, to this region.     

Pathway analysis with next-generation sequencing data

Although pathway analysis methods have been developed and successfully applied to association studies of common variants, the statistical methods for pathway-based association analysis of rare variants have not been well developed. Many investigators observed highly inflated false-positive rates and low power in pathway-based tests of association of rare variants. The inflated false-positive rates and low true-positive rates of the current methods are mainly due to their lack of ability to account for gametic phase disequilibrium. To overcome these serious limitations, we develop a novel statistic that is based on the smoothed functional principal component analysis (SFPCA) for pathway association tests with next-generation sequencing data. The developed statistic has the ability to capture position-level variant information and account for gametic phase disequilibrium. By intensive simulations, we demonstrate that the SFPCA-based statistic for testing pathway association with either rare or common or both rare and common variants has the correct type 1 error rates. Also the power of the SFPCA-based statistic and 22 additional existing statistics are evaluated. We found that the SFPCA-based statistic has a much higher power than other existing statistics in all the scenarios considered. To further evaluate its performance, the SFPCA-based statistic is applied to pathway analysis of exome sequencing data in the early-onset myocardial infarction (EOMI) project. We identify three pathways significantly associated with EOMI after the Bonferroni correction. In addition, our preliminary results show that the SFPCA-based statistic has much smaller P-values to identify pathway association than other existing methods.     

Admixture estimates based on ABO, Rh and nine STRs in two Venezuelan regions

BACKGROUND: The Venezuelan population is the product of Native American, African and European admixture. Few admixture studies have been made in Venezuela using short tandem repeats (STRs). AIM: The study estimated the contribution of each parental group in two Venezuelan regions: the Northern-Central and the Central-Western Regions. SUBJECTS AND METHODS: Frequencies for ABO and Rh were estimated by maximum likelihood, and by direct count for nine STRs, for 211 individuals. Admixture was estimated using Chakraborty's gene identity method. Neighbour-joining dendrograms were obtained with Nei's DS distance calculated between the two regions, the parental populations and other Venezuelan and Latin American populations. A principal component analysis (PCA) was also performed. RESULTS: For the Northern-Central Region, the estimate of admixture was 37.7% for the European component, 37.7% for the African and 24.6% for the Native American. For the Central-Western region, the estimate of admixture was 58.5% for the European, 16.5% for the African and 25.0% for the Native American component. CONCLUSIONS: (i) All systems were in Hardy-Weinberg equilibrium, except the Rh blood group of the Central-Western Region; (ii) the European contribution is high in both groups; (iii) in the dendrogram and PCA, the studied populations appear close to other admixed populations, and their relative position with regard to the three parental populations coincides with the admixture analysis.     

Admixture estimates for Churuguara, a Venezuelan town in the State of Falcón

BACKGROUND: The present Venezuelan population is the admixture product of Amerindians, Europeans and Africans, a process which was not homogeneous over the country. Blood groups, STRs and VNTRs, specifically D1S80, have been used successfully in admixture studies, but few have been made in Venezuela. AIM: This study aims to estimate the admixture components of Churuguara, Venezuela, and to evaluate the genetic relationship of this population with other Venezuelan as well as worldwide populations through principal component analysis and the study of dendrograms based on genetic distances. SUBJECTS AND METHODS: Gene frequencies of blood groups ABO and Rh (only anti D), of STRs VWA, F13A01, FES/FPS and VNTR D1S80 were studied in a sample of 60 individuals born in Churuguara, a Venezuelan town of admixed ancestry in the State of Falc6n. Admixture was estimated with Chakraborty's gene identity method, and Nei's standard genetic distance was used to build two dendrograms with the neighbour-joining approach, one based on the three STRs and the other based only on D1S80. Principal component analyses with the gene frequencies of these markers were also performed. RESULTS: The frequency of allele ABO*O was 0.788, of ABO*A was 0.187 and of RH*D was 0.74. D1S80 showed 16 different alleles with a heterozygosity of 0.880, whilst the three STRs showed only eight different alleles and heterozygosities between 0.733 and 0.797. The estimates of admixture obtained in this analysis were 52.5% for the Spanish parental group, 27.6% for the African and 19.9% for the Amerindian. Comparison of Churuguara with other Latin American populations shows that its African component is not as high as that observed in Colombian Choco, but it is higher than that observed in other samples from Colombia, Chile and Maracaibo (Venezuela). CONCLUSIONS: Results of the admixture analysis are consistent with those obtained with two dendrograms and principal component analyses, suggesting that the strong initial Amerindian component of 500 years ago has been diluted by the continuous flow of European genes, mainly Spanish, to this region.     

A panel of ancestry informative markers for estimating individual biogeographical ancestry and admixture from four continents: utility and applications

Autosomal ancestry informative markers (AIMs) are useful for inferring individual biogeographical ancestry (I-BGA) and admixture. Ancestry estimates obtained from Y and mtDNA are useful for reconstructing population expansions and migrations in our recent past but individual genomic admixture estimates are useful to test for association of admixture with phenotypes, as covariate in association studies to control for stratification and, in forensics, to estimate certain overt phenotypes from ancestry. We have developed a panel of 176 autosomal AIMs that can effectively distinguish I-BGA and admixture proportions from four continental ancestral populations: Europeans, West Africans, Indigenous Americans, and East Asians. We present allele frequencies for these AIMs in all four ancestral populations and use them to assess the global apportionment of I-BGA and admixture diversity among some extant populations. We observed patterns of apportionment similar to those described previously using sex and autosomal markers, such as European admixture for African Americans (14.3%) and Mexicans (43.2%), European (65.5%) and East Asian affiliation (27%) for South Asians, and low levels of African admixture (2.8-10.8%) mirroring the distribution of Y E3b haplogroups among various Eurasian populations. Using simulation studies and pedigree analysis we show that I-BGA estimates obtained using this panel and a four-population model has a high degree of precision (average root mean square error [RMSE]=0.026). Using ancestry-phenotype associations we demonstrate that a large and informative AIM panel such as this can help reduce false-positive and false-negative associations between phenotypes and admixture proportions, which may result when using a smaller panel of less informative AIMs.     

Lack of association between the G protein beta3 subunit gene and essential hypertension in Chinese: a case-control and a family-based study

A C825T polymorphism of the gene encoding the G protein ₃ subunit (GNB3) is associated with enhanced G protein activity and increased intracellular signal transduction. The 825T allele has been implicated in the development of hypertension in some ethnic groups, especially in whites. Studies in Asians and blacks are more controversial, and little information is available on this polymorphism in the susceptibility to hypertension in the Chinese population. Furthermore, the inconsistency between studies may be due to genetic heterogeneity of the population selected and/or the lack of statistical power. We investigated the relationship of this polymorphism with hypertension in two independent northern Chinese populations using both a case-control and a family-based study design. The GNB3 C825T polymorphism was determined by polymerase chain reaction and restriction enzyme digestion. In the case-control study which included 585 hypertensive case subjects and 580 normotensive control subjects there was no significant association between the polymorphism and hypertension status or blood pressure levels. The lack of association was confirmed by the results obtained in 181 hypertensive families using both transmission disequilibrium test and sib transmission disequilibrium test. No preferential transmission was observed for the GNB3 825T allele to the affected subjects. Furthermore, there was no significant association between the polymorphism and body mass index in the case-control study. Therefore our work does not provide evidence in favor of GNB3 C825T being a candidate gene for conferring genetic susceptibility to hypertension or obesity in northern Chinese population.Abbreviations TDT Transmission/disequilibrium test     

Genetic Variation at the Chromosome 16 Chemokine Gene Cluster: Development of a Strategy for Association Studies in Complex Disease

The chemokine gene cluster [CCL22, CX3CL1, CCL17] (previously known as [SCYA22, SCYD1, SCYA17]) is a candidate locus for one of the susceptibility genes for inflammatory bowel disease that are located in the peri‐centromeric region of chromosome 16. Screening for sequence variation at this locus led to the detection of 14 single nucleotide polymorphisms (SNPs). An efficient experimental and computational approach was developed to estimate allele frequencies and pairwise linkage disequilibrium relationships between SNPs at this locus, and to test them for association with inflammatory bowel disease. The 12 common SNPs were assigned to 5 distinct linkage disequilibrium groups. Genotyping of one SNP from each linkage disequilibrium group in a large cohort of families with inflammatory bowel disease did not provide convincing evidence of association with either Crohn's disease or ulcerative colitis. We describe an efficient experimental design from SNP screening to association testing. This strategy can be used to test candidate genes for involvement in susceptibility to complex disease.     

A Robust Linkage Analysis Method Using Combined Allele Sharing and Transmission Disequilibrium Information from Case-Parent Tetrad Families

There are two types of linkage information, the allele sharing information and transmission disequilibrium information, that can be extracted from case-parent tetrad families for deriving statistics for test of linkage. By extracting allele sharing information the mean test can be derived. By extracting transmission disequilibrium information the transmission/disequilibrium test (TDT) can be derived. The power performances of the two tests are different with respect to the extent of linkage disequilibrium. The TDT is more powerful than the mean test when the extent of linkage disequilibrium is moderate to strong, but the mean test has better power performance than the TDT when linkage disequilibrium is weak. Some previous studies have proposed several post-combination analysis methods, which combine the two tests after they are derived using the two types of linkage information respectively, to yield robust test statistics against the interference of linkage disequilibrium. Instead of adopting the post-combination approach, in this paper we investigate the approach of using the pre-combination idea to yield robust statistics for test of linkage. The pre-combination methods combine the two types of linkage information first, and then use the combined information to construct robust test statistics. Simulation studies are conducted to compare the power performances of the proposed pre-combination tests with those of the existing post-combination methods.     

A linkage disequilibrium study of bipolar disorder and microsatellite markers on 22q13

Bipolar disorder is a major psychiatric disorder characterized by extreme mood states that alternate between mania and depression. Family, twin, and adoption studies indicate a genetic component to the disease, but the etiology is suspected to be complex, with multiple genes contributing to an increased susceptibility to the disorder. We have previously reported a genome scan in which a genome-wide maximum LOD score indicated evidence of linkage at the marker D22S278 at 22q13. This area is of particular interest since it is also implicated in schizophrenia, and thus may harbor a susceptibility gene common to both disorders. In our further efforts to fine map this region, we examined 10 microsatellite markers spanning an interval of 2.3 MB in a set of 142 parent-proband triads. Linkage disequilibrium to illness was tested using the Transmission Disequilibrium Test. Haplotypes were determined and marker-to-marker linkage disequilibrium across the region was examined. D22S281 and D22S685 yielded suggestive evidence of linkage disequilibrium to bipolar disorder (empirical values of 0.023 and 0.036, respectively), but a marker-to-marker analysis indicates that a higher density screen is needed to adequately analyze this region.     

A logistic regression based extension of the TDT for continuous and categorical traits

The transmission disequilibrium test (TDT), designed as a test of linkage in the presence of association (i.e. linkage disequilibrium), has received considerable attention in the recent statistical genetics literature due to its advantages over other within-family analytic methods. One limitation of the conventional TDT is its application solely to linkage disequilibrium between a genetic marker and a single categorical trait (e.g. presence or absence of a disease). In this paper, we present an extension of the TDT using logistic regression to examine the relation between a candidate gene or genetic marker and one or more continuous or categorical explanatory variables. This logistic regression extension of the TDT possesses all of the desirable features of the conventional TDT, as well as many advantages associated with traditional regression analysis. We describe the model and its properties, as well as a number of its possible applications, and apply it to examine linkage disequilibrium between the dopamine receptor D2 gene (DRD2) and symptoms of childhood attention deficit hyperactivity disorder (ADHD). We also briefly compare the logistic regression TDT to other quantitative TDTs that have been proposed in the literature, and highlight the advantages of a regression-based approach for examining the relation between a candidate gene and one or more continuous or categorical traits. Given its features, we regard the logistic regression extension of the TDT as a flexible new data analytic method with extensive potential applications to problems in medical, psychiatric, and behavioral genetics.