Marker-marker Linkage Disequilibrium Research Articles

Evaluation of an approximation method for assessment of overall significance of multiple‐dependent tests in a genomewide association study

We describe implementation of a set-based method to assess the significance of findings from genomewide association study data. Our method, implemented in PLINK, is based on theoretical approximation of Fisher's statistics such that the combination of P-vales at a gene or across a pathway is carried out in a manner that accounts for the correlation structure, or linkage disequilibrium, between single nucleotide polymorphisms. We compare our method to a permutation-based product of P-values approach and show a typical correlation in excess of 0.98 for a number of comparisons. The method gives Type I error rates that are less than or equal to the corresponding nominal significance levels, making it robust to the effects of false positives. We show that in broadly similar populations, reference data sets of markers are an appropriate substrate for deriving marker-marker linkage disequilibrium (LD), negating the need to access individual level genotypes, greatly facilitating its generic applicability. We show that the method is thus robust to LD-associated bias and has equivalent performance to permutation-based methods, with a significantly shorter runtime. This is particularly relevant at a time of increasing public availability of significantly larger genetic data sets and should go a long way to assist in the rapid analysis of these data sets.

A Focal Epilepsy and Intellectual Disability Syndrome Is Due to a Mutation in TBC1D24

We characterized an autosomal-recessive syndrome of focal epilepsy, dysarthria, and mild to moderate intellectual disability in a consanguineous Arab-Israeli family associated with subtle cortical thickening. We used multipoint linkage analysis to map the causative mutation to a 3.2 Mb interval within 16p13.3 with a LOD score of 3.86. The linked interval contained 160 genes, many of which were considered to be plausible candidates to harbor the disease-causing mutation. To interrogate the interval in an efficient and unbiased manner, we used targeted sequence enrichment and massively parallel sequencing. By prioritizing unique variants that affected protein translation, a pathogenic mutation was identified in TBC1D24 (p.F251L), a gene of unknown function. It is a member of a large gene family encoding TBC domain proteins with predicted function as Rab GTPase activators. We show that TBC1D24 is expressed early in mouse brain and that TBC1D24 protein is a potent modulator of primary axonal arborization and specification in neuronal cells, consistent with the phenotypic abnormality described.

Genomewide linkage scan of schizophrenia in a large multicenter pedigree sample using single nucleotide polymorphisms.

A genomewide linkage scan was carried out in eight clinical samples of informative schizophrenia families. After all quality control checks, the analysis of 707 European-ancestry families included 1,615 affected and 1,602 unaffected genotyped individuals, and the analysis of all 807 families included 1900 affected and 1839 unaffected individuals. Multipoint linkage analysis with correction for marker-marker linkage disequilibrium was carried out with 5,861 single nucleotide polymorphisms (SNPs; Illumina 4.0 linkage map). Suggestive evidence for linkage (European families) was observed on chromosomes 8p21, 8q24.1, 9q34 and 12q24.1 in non-parametric and/or parametric analyses. In a logistic regression allele-sharing analysis of linkage allowing for intersite heterogeneity, genomewide significant evidence for linkage was observed on chromosome 10p12. Significant heterogeneity was also observed on chromosome 22q11.1. Evidence for linkage across family sets and analyses was most consistent on chromosome 8p21, with a one-lod support interval that does not include the candidate gene NRG1, suggesting that one or more other susceptibility loci might exist in the region. In this era of genomewide association and deep resequencing studies, consensus linkage regions deserve continued attention, given that linkage signals can be produced by many types of genomic variation, including any combination of multiple common or rare SNPs or copy number variants in a region.

Linkage disequilibrium compared between five populations of domestic sheep

BackgroundThe success of genome-wide scans depends on the strength and magnitude of linkage disequilibrium (LD) present within the populations under investigation. High density SNP arrays are currently in development for the sheep genome, however little is known about the behaviour of LD in this livestock species. This study examined the behaviour of LD within five sheep populations using two LD metrics, D' and x2'. Four economically important Australian sheep flocks, three pure breeds (White Faced Suffolk, Poll Dorset, Merino) and a crossbred population (Merino × Border Leicester), along with an inbred Australian Merino museum flock were analysed.ResultsShort range LD (0 – 5 cM) was observed in all five populations, however the persistence with increasing distance and magnitude of LD varied considerably between populations. Average LD (x2') for markers spaced up to 20 cM exceeded the non-syntenic average within the White Faced Suffolk, Poll Dorset and Macarthur Merino. LD decayed faster within the Merino and Merino × Border Leicester, with LD below or consistent with observed background levels. Using marker-marker LD as a guide to the behaviour of marker-QTL LD, estimates of minimum marker spacing were made. For a 95% probability of detecting QTL, a microsatellite marker would be required every 0.1 – 2.5 centimorgans, depending on the population used.ConclusionSheep populations were selected which were inbred (Macarthur Merino), highly heterogeneous (Merino) or intermediate between these two extremes. This facilitated analysis and comparison of LD (x2') between populations. The strength and magnitude of LD was found to differ markedly between breeds and aligned closely with both observed levels of genetic diversity and expectations based on breed history. This confirmed that breed specific information is likely to be important for genome wide selection and during the design of successful genome scans where tens of thousands of markers will be required.

Multipoint Approximations of Identity-by-Descent Probabilities for Accurate Linkage Analysis of Distantly Related Individuals

We propose an analytical approximation method for the estimation of multipoint identity by descent (IBD) probabilities in pedigrees containing a moderate number of distantly related individuals. We show that in large pedigrees where cases are related through untyped ancestors only, it is possible to formulate the hidden Markov model of the Lander-Green algorithm in terms of the IBD configurations of the cases. We use a first-order Markov approximation to model the changes in this IBD-configuration variable along the chromosome. In simulated and real data sets, we demonstrate that estimates of parametric and nonparametric linkage statistics based on the first-order Markov approximation are accurate. The computation time is exponential in the number of cases instead of in the number of meioses separating the cases. We have implemented our approach in the computer program ALADIN (accurate linkage analysis of distantly related individuals). ALADIN can be applied to general pedigrees and marker types and has the ability to model marker-marker linkage disequilibrium with a clustered-markers approach. Using ALADIN is straightforward: It requires no parameters to be specified and accepts standard input files.

Genetics of Recurrent Early-Onset Major Depression (GenRED): Significant Linkage on Chromosome 15q25-q26 After Fine Mapping With Single Nucleotide Polymorphism Markers

The authors studied a dense map of single nucleotide polymorphism (SNP) DNA markers on chromosome 15q25-q26 to maximize the informativeness of genetic linkage analyses in a region where they previously reported suggestive evidence for linkage of recurrent early-onset major depressive disorder. In 631 European-ancestry families with multiple cases of recurrent early-onset major depressive disorder, 88 SNPs were genotyped, and multipoint allele-sharing linkage analyses were carried out. Marker-marker linkage disequilibrium was minimized, and a simulation study with founder haplotypes from these families suggested that linkage scores were not inflated by linkage disequilibrium. The dense SNP map increased the information content of the analysis from around 0.7 to over 0.9. The maximum evidence for linkage was the Z likelihood ratio score statistic of Kong and Cox (Z(LR))=4.69 at 109.8 cM. The exact p value was below the genomewide significance threshold. By contrast, in the genome scan with microsatellite markers at 9 cM spacing, the maximum Z(LR) for European-ancestry families was 3.43 (106.53 cM). It was estimated that the linked locus or loci in this region might account for a 20% or less populationwide increase in risk to siblings of cases. This region has produced modestly positive evidence for linkage to depression and related traits in other studies. These results suggest that DNA sequence variations in one or more genes in the 15q25-q26 region can increase susceptibility to major depression and that efforts are warranted to identify these genes.

The Affected-/Discordant-Sib-Pair Design Can Guarantee Validity of Multipoint Model-Free Linkage Analysis of Incomplete Pedigrees When There Is Marker-Marker Disequilibrium

Genomewide linkage studies are tending toward the use of single-nucleotide polymorphisms (SNPs) as the markers of choice. However, linkage disequilibrium (LD) between tightly linked SNPs violates the fundamental assumption of linkage equilibrium (LE) between markers that underlies most multipoint calculation algorithms currently available, and this leads to inflated affected-relative-pair allele-sharing statistics when founders' multilocus genotypes are unknown. In this study, we investigate the impact that the degree of LD, marker allele frequency, and association type have on estimating the probabilities of sharing alleles identical by descent in multipoint calculations and hence on type I error rates of different sib-pair linkage approaches that assume LE. We show that marker-marker LD does not inflate type I error rates of affected sib pair (ASP) statistics in the whole parameter space, and that, in any case, discordant sib pairs (DSPs) can be used to control for marker-marker LD in ASPs. We advocate the ASP/DSP design with appropriate sib-pair statistics that test the difference in allele sharing between ASPs and DSPs.

Relative Contribution of Genetic and Nongenetic Modifiers to Intestinal Obstruction in Cystic Fibrosis

Neonatal intestinal obstruction (meconium ileus [MI]) occurs in 15% of patients with cystic fibrosis (CF). Our aim was to determine the relative contribution of genetic and nongenetic modifiers to the development of this major complication of CF. A total of 65 monozygous twin pairs, 23 dizygous twin/triplet sets, and 349 sets of siblings with CF were analyzed for MI status, significant covariates, and genome-wide linkage. Specific mutations in the CF transmembrane conductance regulator (CFTR), the gene responsible for CF, correlated with MI, indicating a role for CFTR genotype. Monozygous twins showed substantially greater concordance for MI than dizygous twins and siblings (P = 1 x 10(-5)), showing that modifier genes independent of CFTR contribute substantially to this trait. Regression analysis revealed that MI was correlated with distal intestinal obstruction syndrome (P = 8 x 10(-4)). Unlike MI, concordance analysis indicated that the risk for development of distal intestinal obstruction syndrome in CF patients is caused primarily by nongenetic factors. Regions of suggestive linkage (logarithm of the odds of linkage >2.0) for modifier genes that cause MI (chromosomes 4q35.1, 8p23.1, and 11q25) or protect from MI (chromosomes 20p11.22 and 21q22.3) were identified by genome-wide analyses. These analyses did not support the existence of a major modifier gene on chromosome 19 in a region previously linked to MI. The CFTR gene along with 2 or more modifier genes are the major determinants of intestinal obstruction in newborn CF patients, whereas intestinal obstruction in older CF patients is caused primarily by nongenetic factors.

The effect of linkage disequilibrium on linkage analysis of incomplete pedigrees

Dense SNP maps can be highly informative for linkage studies. But when parental genotypes are missing, multipoint linkage scores can be inflated in regions with substantial marker-marker linkage disequilibrium (LD). Such regions were observed in the Affymetrix SNP genotypes for the Genetic Analysis Workshop 14 (GAW14) Collaborative Study on the Genetics of Alcoholism (COGA) dataset, providing an opportunity to test a novel simulation strategy for studying this problem. First, an inheritance vector (with or without linkage present) is simulated for each replicate, i.e., locations of recombinations and transmission of parental chromosomes are determined for each meiosis. Then, two sets of founder haplotypes are superimposed onto the inheritance vector: one set that is inferred from the actual data and which contains the pattern of LD; and one set created by randomly selecting parental alleles based on the known allele frequencies, with no correlation (LD) between markers. Applying this strategy to a map of 176 SNPs (66 Mb of chromosome 7) for 100 replicates of 116 sibling pairs, significant inflation of multipoint linkage scores was observed in regions of high LD when parental genotypes were set to missing, with no linkage present. Similar inflation was observed in analyses of the COGA data for these affected sib pairs with parental genotypes set to missing, but not after reducing the marker map until r2 between any pair of markers was ≤ 0.05. Additional simulation studies of affected sib pairs assuming uniform LD throughout a marker map demonstrated inflation of significance levels at r2 values greater than 0.05. When genotypes are available only from two affected siblings in many families in a sample, trimming SNP maps to limit r2 to 0–0.05 for all marker pairs will prevent inflation of linkage scores without sacrificing substantial linkage information. Simulation studies on the observed pedigree structures and map can also be used to determine the effect of LD on a particular study.

An exploration of sex-specific linkage disequilibrium on chromosome X in Caucasians from the COGA study

This paper explores the decay of linkage disequilibrium (LD) on the autosomes and chromosome X. The extent of marker-marker LD is important for both linkage and association studies. The analysis of the Caucasian sample from the Collaborative Study on the Genetics of Alcoholism study revealed the expected negative relationship between the magnitude of the marker-marker LD and distance (cM), with the male and female subgroups exhibiting similar patterns of LD. The observed extent of LD in females was less across the pseudoautosomal markers relative to the heterosomal region of chromosome X. Marked differences in LD patterns were also observed between chromosomes X and the 22 autosomes in both males and females.

Power and Sample Size Calculations for Genetic Case/Control Studies Using Gene-Centric SNP Maps: Application to Human Chromosomes 6, 21, and 22 in Three Populations

Power and sample size calculations are critical parts of any research design for genetic association. We present a method that utilizes haplotype frequency information and average marker-marker linkage disequilibrium on SNPs typed in and around all genes on a chromosome. The test statistic used is the classic likelihood ratio test applied to haplotypes in case/control populations. Haplotype frequencies are computed through specification of genetic model parameters. Power is determined by computation of the test’s non-centrality parameter. Power per gene is computed as a weighted average of the power assuming each haplotype is associated with the trait. We apply our method to genotype data from dense SNP maps across three entire chromosomes (6, 21, and 22) for three different human populations (African-American, Caucasian, Chinese), three different models of disease (additive, dominant, and multiplicative) and two trait allele frequencies (rare, common). We perform a regression analysis using these factors, average marker-marker disequilibrium, and the haplotype diversity across the gene region to determine which factors most significantly affect average power for a gene in our data. Also, as a ‘proof of principle’ calculation, we perform power and sample size calculations for all genes within 100 kb of the PSORS1 locus (chromosome 6) for a previously published association study of psoriasis. Results of our regression analysis indicate that four highly significant factors that determine average power to detect association are: disease model, average marker-marker disequilibrium, haplotype diversity, and the trait allele frequency. These findings may have important implications for the design of well-powered candidate gene association studies. Our power and sample size calculations for the PSORS1 gene appear consistent with published findings, namely that there is substantial power (>0.99) for most genes within 100 kb of the PSORS1 locus at the 0.01 significance level.

Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families.

In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German non-CF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed delta F508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.