Single nucleotide polymorphism and linkage disequilibrium within the TCR alpha/delta locus

Much attention is being given to the identification of common disease genes through whole-genome linkage disequilibrium (LD) screens with single nucleotide polymorphisms (SNPs). Simulation studies have suggested that useful LD is unlikely to extend beyond 3 kb, and that > 500,000 SNPs may be needed for comprehensive coverage of the genome. The TCR alpha/delta locus on chromosome 14q contains many V, J and D segments that combine with constant domains to produce either an alpha or a delta chain of the T cell receptor. Multiple SNPs have been recognized within the V segments, and it has been suggested that variation within the locus may modify the course of autoimmune and allergic diseases. We have examined LD within an 850 kb section of the TCR alpha/delta locus on chromosome 14q by typing 24 V gene segment SNPs and two microsatellites. One hundred and fifty-nine nuclear and extended families were genotyped in order to derive haplotypes, and the pair-wise LD between SNPs was investigated in 600 haplotypes from unrelated individuals (the parents). The mean extent of useful LD was much greater than suggested by simulations: significant LD was relatively common at 250 kb and was detectable beyond 500 kb. The mean extent of LD was twice as far between alleles of low frequency than between common alleles. The distribution of LD was highly irregular and concentrated in three distinct islands. The results differ from those obtained by simulation, and if they are typical of other genomic regions, suggest that the minimum number of markers necessary for comprehensive LD mapping may be reduced by at least an order of magnitude.     

Comparative study of the haplotype structure and linkage disequilibrium of chromosome 1p36.2 region in the Korean and Japanese populations

The patterns of linkage disequilibrium (LD) in the human genome provide important information for disease gene mapping. LDs may vary depending on chromosomal regions and populations. We have compared LD and haplotypes defined by SNPs in the chromosome 1p36.2 region of the Korean and Japanese populations. Fifty-eight SNPs in about 418 kb ranging from tumor necrosis factor receptor 2 (TNFR2:TNFRSF1B) to procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) gene were examined in 96 healthy Koreans and Japanese each by direct sequencing and fluorescence correlation spectroscopy combined with the PCR-sequence specific primer method (PCR-SSP-FCS), respectively. Upon pair-wise LD analysis, a total of 25 and 16 out of 58 SNPs greater than MAF 10% were included in LD blocks, encompassing almost 81 kb and 55 kb in total, in Koreans and Japanese, respectively. Both similarities and differences were observed in LD strength and haplotype frequencies between the populations. Considerable similarities were observed in the telomeric region where a long-range block of approximately 80 kb including three genes was found to have strong LDs in both Koreans and Japanese. Significant difference in LD strength was present near the TNFR2 region between the Japanese and Korean populations.     

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.     

Simulation studies of detection of a complex disease in a partially isolated population.

Simulation studies were undertaken with POPGEN, a new population simulation program, to explore strategies for detecting loci underlying rare and common disorders in a small population that has been partially isolated for 10 generations. Haplotype-sharing analysis (HSA) and non-parametric linkage analysis (NPL) were applied to the simulated haplotype and pedigree data for 100 cases, 100 controls, and an average of 28 multiplex pedigrees from cases' families, for a 2-5 cM map of markers. When identity by descent (IBD) status was known (using unique founder marker allele designations assigned during simulation), a linkage disequilibrium (LD) signal could be detected under disease-generating models predicting relative risk to sibs of 11.8 (high-RR) or 2.67 (mod-RR). Detection was more difficult when marker alleles were down-coded to resemble microsatellites (heterozygosities 0.75-0.80). False-positive peaks on nondisease chromosomes were uncommon. NPL analysis was more powerful than HSA at this marker density using down-coded alleles and assuming availability of all affected relatives. LD mapping of common disorders is likely to require denser maps of highly polymorphic markers to approximate full IBD information. LD and linkage mapping provide independent information, and strategies that combine these two methods could be useful in studies of small isolated populations.     

Haplotypic relationship between SNP and microsatellite markers at the NOS2A locus in two populations

The density of genetic markers required for successful association mapping of complex diseases depends on linkage disequilibrium (LD) between non-functional markers and functional variants. The haplotypic relationship between stable markers and potentially unstable but highly informative markers (e.g. microsatellites) indicates that LD might be maintained over considerable genetic distance in non-African populations, supporting the use of such 'mixed marker haplotypes' in LD-based mapping, and allowing inferences to be drawn about human origins. We investigated sequence variation in the proximal 2.6 kb of the inducible nitric oxide synthase (NOS2A) promoter and the relationship between SNP haplotypes and a pentanucleotide microsatellite (the 'NOS2A(-2.6) microsatellite') in Gambians and UK Caucasians. UK Caucasians exhibited a subset of sequence diversity observed in Gambians, sharing four of 11 SNPs and a similar haplotypic structure. Five SNPs were found in the sequence of interspersed repetitive DNA elements. In both populations, there was dramatic loss of LD between SNP haplotypes and microsatellite alleles across a very short physical distance, suggesting a high intrinsic mutation rate of the NOS2A(-2.6) microsatellite, the SNP haplotypes are relatively ancient, or that this was a region of frequent recombination. Understanding locus- and population-specific LD is essential when designing and interpreting genetic association studies.     

The impact of SNP density on fine-scale patterns of linkage disequilibrium

Linkage disequilibrium (LD) is a measure of the degree of association between alleles in a population. The detection of disease-causing variants by association with neighbouring single nucleotide polymorphisms (SNPs) depends on the existence of strong LD between them. Previous studies have indicated that the extent of LD is highly variable in different chromosome regions and different populations, demonstrating the importance of genome-wide accurate measurement of LD at high resolution throughout the human genome. A uniform feature of these studies has been the inability to detect LD in regions of low marker density. To investigate the dependence of LD patterns on marker selection we performed a high-resolution study in African-American, Asian and UK Caucasian populations. We selected over 5000 SNPs with an average spacing of approximately 1 SNP per 2 kb after validating ca 12 000 SNPs derived from a dense SNP collection (1 SNP per 0.3 kb on average). Applications of different statistical methods of LD assessment highlight similar areas of high and low LD. However, at high resolution, features such as overall sequence coverage in LD blocks and block boundaries vary substantially with respect to marker density. Model-based linkage disequilibrium unit (LDU) maps appear robust to marker density and consistently influenced by marker allele frequency. The results suggest that very dense marker sets will be required to yield stable views of fine-scale LD in the human genome.     

Origin of the prevalent SFTPB indel g.1549C > GAA (121ins2) mutation causing surfactant protein B (SP-B) deficiency

The SFTPB gene indel g.1549C > GAA (121ins2) accounts for about 2/3 of the mutant alleles underlying complete surfactant protein B deficiency. It is unclear, however, whether its prevalence is due to recurrent mutation or a founder effect. The underlying mutational mechanism was therefore sought through the analysis of local DNA sequence complexity. A relatively complex two-step process was proposed: the first step involving slipped mispairing mediated by a direct repeat and generating an AGAA micro-insertion, the second step involving hairpin loop resolution resulting in a CA micro-deletion. The possibility of a founder effect was then assessed by typing 8 intragenic SNPs in 17 independent 121ins2 chromosomes from 10 probands, with parental non-121ins2 chromosomes serving as controls. The 121ins2 chromosomes were assigned to three discrete haplotypes, whilst control chromosomes were distributed between 10 of the 11 observed parental haplotypes. The 121ins2 mutation was in strong and significant linkage disequilibrium (LD) with the tightly linked marker g.1580T/C (|D'| = 1; P approximately 0.024), although only moderate LD was found with the rest of the locus (|D'| approximately 0.54; P approximately 0.136). Data on haplotype structure and the locus LD pattern, obtained from 81 independent Western-European chromosomes, were consistent with the three mutation-bearing haplotypes having originated from a common ancestor by recombination. Interestingly, all families harboring the 121ins2 indel had ancestors from a region of Northwestern Europe populated by Frankish/Saxon migration. Taken together, these data are consistent with the view that an indel mutation occurred on a relatively common SFTPB haplotype and now accounts for the majority of (and possibly all) extant 121ins2 chromosomes.     

Extent of linkage disequilibrium in a Sardinian sub-isolate: sampling and methodological considerations

The extent of linkage disequilibrium (LD) is an important factor when designing experiments for mapping disease or trait loci using LD mapping methods. It depends on the population history and hence is a characteristic of each population. Here, we have assessed the extent of LD in a sub-isolate of the general Sardinian population (775 members of one village) using 22 polymorphic markers on chromosome 19. We found high levels of disequilibrium that extended to 8 cM, when based on D', and 11 cM when based on the significance level of the allelic association. The fact that conclusions based on both methods are similar suggests that the estimates are quite robust. We have also shown, through a simple resampling technique, that small sample sizes can overestimate both the mean value of D' and its variance up to a factor of about 2 and 16, respectively, when the number of diplotypes (the pair of haplotypes that compose the genotype) decreased from 186 to 26. We evaluated the effect on D' of the depth of the pedigree available when using phased founders, and compared the estimates with those obtained when using unphased founders, and also the effect of grouping alleles on the value of D' and the significance level. Owing to the high sampling variance of LD, we recommend the use of at least 200 unrelated individuals when characterizing the extent of LD.     

Association between variants in IDE-KIF11-HHEX and plasma amyloid β levels

Genetic linkage and association studies in late-onset Alzheimer’s disease (LOAD) or its endophenotypes have pointed to several regions on chromosome 10q, among these the ∼ 250 kb linkage disequilibrium (LD) block harboring the genes IDE, KIF1, and HHEX. We explored the association between variants in the genomic region harboring the IDE-KIF11-HHEX complex with plasma Aβ40 and Aβ42 levels in a case-control cohort of Caribbean Hispanics. First, we performed single marker linear regression analysis relating the individual single nucleotide polymorphisms (SNPs) with plasma Aβ40 and Aβ42 levels. Then we performed 3-SNP sliding window haplotype analyses, correcting all analyses for multiple testing. Out of 32 SNPs in this region, 3 SNPs in IDE (rs2421943, rs12264682, rs11187060) were associated with plasma Aβ40 or Aβ42 levels in single marker and haplotype analyses after correction for multiple testing. All these SNPs lie within the same LD block, and are in LD with the previously reported haplotypes. Our findings provide support for an association in the IDE region on chromosome 10q with Aβ40 and 42 levels.     

应用连锁不平衡图谱的关联研究分析方法

目的提出一种对高通量单核苷酸多态位点(single nucleotide polymorphism,SNP)关联研究的数据分析方法。方法在160名上海地区中国人中进行754个SNP的基因型检测,分别构建病例组和对照组的连锁不平衡(linkage disequilibrium,LD)图谱,通过比较两组间染色体区域LD图谱随物理距离的变化趋势寻找与疾病相关的位点,并与传统LD分析以及SNP单点、单倍型分析进行比较。结果LD图谱的分析能判断出两组间LD存在差异的染色体区域,并且该区域SNP等位基因频率和单倍型频率在两组间分布存在统计学差异或差异趋势。结论可应用该方法对高通量SNP的关联研究进行数据分析。     

Combined high resolution linkage and association mapping of quantitative trait loci

In this paper, we investigate variance component models of both linkage analysis and high resolution linkage disequilibrium (LD) mapping for quantitative trait loci (QTL). The models are based on both family pedigree and population data. We consider likelihoods which utilize flanking marker information, and carry out an analysis of model building and parameter estimations. The likelihoods jointly include recombination fractions, LD coefficients, the average allele substitution effect and allele dominant effect as parameters. Hence, the model simultaneously takes care of the linkage, LD or association and the effects of the putative trait locus. The models clearly demonstrate that linkage analysis and LD mapping are complementary, not exclusive, methods for QTL mapping. By power calculations and comparisons, we show the advantages of the proposed method: (1) population data can provide information for LD mapping, and family pedigree data can provide information for both linkage analysis and LD mapping; (2) using family pedigree data and a sparse marker map, one may investigate the prior suggestive linkage between trait locus and markers to obtain low resolution of the trait loci, because linkage analysis can locate a broad candidate region; (3) with the prior knowledge of suggestive linkage from linkage analysis, both population and family pedigree data can be used simultaneously in high resolution LD mapping based on a dense marker map, since LD mapping can increase the resolution for candidate regions; (4) models of high resolution LD mappings using two flanking markers have higher power than that of models of using only one marker in the analysis; (5) excluding the dominant variance from the analysis when it does exist would lose power; (6) by performing linkage interval mappings, one may get higher power than by using only one marker in the analysis.     

Population haplotype analysis and evolutionary relations of the COL2A1 gene

We have determined the allele frequencies and pairwise linkage disequilibria of restriction fragment length polymorphisms (RFLPs) distributed over the entire COL2A1 gene (spanning 23.6 kb) in a population of unrelated Dutch Caucasians. Pairwise linkage disequilibrium analysis of RFLP sites between exon 5B and 51 indicated a high degree of partly positive (the rare alleles of both loci are associated) and partly negative (the rare allele is associated with the common allele) linkage disequilibrium.
The high degree of linkage disequilibrium enabled the assignment of 13 out of 128 possible haplotypes with 7 RFLPs. An evolutionary tree of these haplotypes was derived using a minimum spanning tree approach, indicating at least two ancestral haplotypes. Our data indicate that disease related population studies involving the COL2A1 gene should include a minimum of 4 RFLPs (D9, A9, H33, P51) to obtain 98% of possible haplotypes occurring.     

Family-based association study of schizophrenia with 444 markers and analysis of a new susceptibility locus mapped to 11q13.3.

Family-based linkage disequilibrium (LD) mapping has been suggested as a powerful and practical alternative to linkage analysis. We have performed a genome-wide LD survey of susceptibility loci for schizophrenia in a Japanese population. We first typed 119 schizophrenic pedigrees (357 individuals) using 444 microsatellite markers, and analyzed the data using the pedigree disequilibrium test. This analysis revealed 14 markers demonstrating significant transmission distortion. To corroborate these findings, the statistical methods were changed to the extended transmission disequilibrium test (ETDT), using 80 independent complete trios (schizophrenic proband and both parents), with 68 derived from initial pedigrees and 12 newly recruited trios. ETDT supported two markers for continued association, D11S987 on 11q13.3 (P = 0.00009) and D16S423 on 16p13.3 (P = 0.002). We scrutinized the most significant genomic locus on 11q11-13 by adding 26 new markers for analysis. Results of three-marker haplotype analysis in the region showed evidence of association with schizophrenia (most significant haplotype P = 0.0005, global P = 0.022). Although the present study may have missed other potential genomic intervals because of the sparse mapping density, we hope that it has identified promising anchor points for further studies to identify risk-conferring genes for schizophrenia in the Japanese population. In addition, we provide useful information on genomic LD structures in Japanese populations, which can be used for LD mapping of complex diseases.     

Positive association of the <Emphasis Type="Italic">FTSJ1</Emphasis> gene polymorphisms with nonsyndromic X-linked mental retardation in young Chinese male subjects

To investigate the possible genetic association of nonsyndromic X-linked mental retardation (NS-XLMR) with FTSJ1 gene polymorphisms, a case-control association study was performed focusing on the Chinese Han population in the Qinba mountain region. Three common single nucleotide polymorphisms (SNPs) (rs2268954, rs2070991, rs5905692) in the gene were selected and genotyped using the polymerase chain reaction single-strand confirmation polymorphism (PCR-SSCP) method. Pairwise linkage disequilibrium (LD) analysis showed that the three SNPs were in strong LD (all D' > 0.8). There were significant differences between cases and controls in allele frequency distribution of rs2268954 (P = 0.036), rs2070991 (P = 0.043), and rs5905692 (P = 0.014) and in the distributions of common haplotypes combined by these SNPs (global P = 0.01236) in male subjects. In female subjects, however, no positive results were found. Our results suggest a positive association between the genetic variants of the FTSJ1 gene and NS-XLMR in young male subjects in the Chinese Han population in the Qinba region.     

Distinct linkage disequilibrium (LD) runs of single nucleotide polymorphisms and microsatellite markers; implications for use of mixed marker haplotypes in LD-based mapping

It has been suggested that the haplotypic relationship between microsatellite markers and single nucleotide polymorphisms (SNPs) is of considerable importance, as microsatellite markers can potentially be incorporated into haplotypes containing SNPs to increase marker density across a region of interest. However, SNPs and microsatellite markers have different mutation rates and durations, and it is conceivable that the linkage disequilibrium (LD) patterns between the genetic markers may considerably differ. We assessed the LD patterns using 1,661 SNPs and 65 microsatellite markers along chromosome 22 and investigated whether common patterns of LD between the two genetic markers are deduced from the results. The results demonstrated that the patterns of LD among microsatellite markers varied considerably and the LD runs of SNPs and microsatellite markers showed distinct patterns. Microsatellite markers have a much higher mutation rate and the evolution of microsatellite markers is a more complex process which has distinct mutation properties from those of SNPs. We consider that these might contribute to the different LD patterns between the two genetic markers. Therefore, it would seem inadvisable to make assumptions about persistence of LD across even a relatively small genetic distance among microsatellite markers and to construct mixed marker haplotypes/LD maps employing microsatellite markers.     

Further evidence for role of a promoter variant in the TNFRSF6 gene in Alzheimer disease

The tumor necrosis factor receptor superfamily, member 6 gene (TNFRSF6) is situated on chromosome 10q, near the region indicated in several AD linkage studies. In a previous study a significant association was found between a promoter variant within the TNFRSF6 gene and Alzheimer disease (AD). To further investigate the TNFRSF6 region, 34 SNPs within 180 kb were first genotyped in an exploratory set of 121 early-onset dementia cases and 152 controls in order to establish the extent of linkage disequilibrium (LD) and the major haplotypes in the region. This analysis showed that the LD was clustered in two large blocks within each of which a limited number of haplotypes represented most of the diversity. Haplotype tagging markers were chosen and genotyped in an additional 204 late onset AD cases and 177 controls. Tests of association were performed for single markers for case/control status and for a quantitative measure of cognitive ability [Mini-Mental State Examination (MMSE) scores] within cases only. The previously associated marker, located in the promoter of TNFRSF6, now gave significant association with cognitive status in the Scottish early-onset dementia samples (p = 0.005) with the strongest signals being evident in the APOE-e4 carrier subgroup, thus providing a second independent positive result for this marker. The same marker was also significantly associated with cognitive performance in the Swedish APOE-e4 carriers (p = 0.014), providing a third independent signal. Association analysis was also performed using the major haplotypes in the region, employing both case/control status and MMSE scores. The haplotype analysis did not give further significant findings. These results together with previous data suggest that a promoter marker in TNFRSF6 plays a moderate but demonstrable role in AD etiology.     

Linkage disequilibrium analysis of -1516, -574 and 4259 single nucleotide polymorphisms in TIM-3 gene]

OBJECTIVE: To investigate the frequencies of -1516,-574 and 4259 single nucleotide polymorphisms (SNPs) of T cells immunoglobulin mucin -3(TIM-3) gene in Hubei population and address the question whether they are in linkage disequilibrium(LD) . METHODS: Genotypes and allele frequencies of TIM-3 gene were examined by allele-specific polymerase chain reaction (AS-PCR) methods in 147 healthy Hubei Han individuals. Hardy-Weinberg equilibrium and Two-point LD analyses and haplotype frequencies were evaluated with Arlequin v3.1 software. RESULTS: The allele frequencies of the 3 SNPs were in agreement with Hardy-Weinberg equilibrium. Minor allelic frequencies of TIM-3 -1516G/T,-574T/G and 4259G/T were 8.5%,1.0% and 2.0%,respectively. The dominant haplotypes comprising the three loci were G-G-G(2.0%),G-G-T(88.4%), T-G-T(8.5%) and G-T-T(1.0%). LD analyses revealed that all of the coefficient of linkage disequilibrium (D') were 1. CONCLUSION: The -1516,-574 and 4259 loci of TIM-3 gene are in complete linkage disequilibrium. Our study has provided population genetic data on TIM-3 gene in Chinese Hubei Han population and a basis for searching immune-mediated disease-related TIM-3 haplotype.     

Haplotype and linkage disequilibrium analysis of the CRMP1 and EVC genes

In this report, we present the haplotype and linkage disequilibrium (LD) pattern in the Collapsin Response Mediator Protein 1 (CRMP1) and Ellis-van Creveld syndrome (EVC) gene region. We genotyped eight different single nucleotide polymorphisms (SNPs) in the CRMP1 and EVC genes in 90 control individuals of diverse ethnicity. The minor allele frequencies ranged from 3.3-49.4%, with most having a frequency >25%. A total of 37 haplotypes were derived from these eight polymorphisms, with only one haplotype having a frequency >10%. Pairwise LD analysis showed a weak but significant LD between markers located about 243 kb apart in this region. The LD was significant between markers spaced about 208 kb apart in EVC, whereas no LD was found between a pair of markers located about 5 kb apart in CRMP1. However, in general, LD correlated with the distance between loci. The CRMP1 and EVC genes are located near WFS1, the Wolfram syndrome type 1 gene, in which mutations also cause low frequency sensorineural hearing loss (LFSNHL). The haplotypes obtained from these polymorphisms will be useful to track the segregation of phenotypes in families with Ellis-van Creveld syndrome, Weyers acrodental dysostosis, LFSNHL and Wolfram syndrome type 1.     

Can long-range microsatellite data be used to predict short-range linkage disequilibrium?

The distribution of linkage disequilibrium (LD) across the genome is highly complex. Little is known about the relationship between long-range and short-range LD in a genomic region. We assessed whether a dense set of microsatellite data could be used to predict short-range LD in family samples. We analyzed intermarker LD in data derived from chromosomal regions 18q22 and 10q25-26, densely genotyped with microsatellite markers. The pattern of LD was highly heterogeneous within and between both chromosomal regions. On 10q25-26, very little LD was detected. On 18q22, where marker density was higher, many marker pairs were in LD. We modeled the decay of LD over distance in this region. A classical model accounted for most of the relationship between LD and distance (R (2)=63%). We used this model to predict the proportion of markers expected to show useful levels of LD at short distances. This prediction agreed with estimates based on single-nucleotide polymorphism (SNP) marker genotypes in the region. Both microsatellite and SNP data predict that about 80% of marker pairs would display levels of LD that are useful for association studies at distances of up to 15 kb in this region. These projections also agree with levels of LD directly measured in a 10 kb set of SNP genotypes generated in a nearby region of finished sequence. Our results suggest that existing sets of microsatellite data, if sufficiently dense, may be used to develop good initial estimates of the density of additional markers needed to screen a region for disease alleles by association analysis.