A pedigree-based linkage study of coeliac disease: failure to replicate previous positive findings

Coeliac Disease (CD) is a gluten sensitive enteropathy characterised by villous atrophy and crypt cell hyperplasia. There is a tight HLA association between CD and the HLA DQ alleles DQA1*0501, DQB1*0201 (DQ2), arranged in either cis- or trans- configuration, are found in 98.9% of cases in Northern European populations and 80% in Greeks and Ashkenazi Jews resident in Israel. We have previously shown that the HLA alleles and CD do not co-segregate in families multiply affected with CD, suggesting that the HLA association is entirely due to the necessity to have these normal DQ alleles for CD to manifest, and that the main genetic predisposition lies at a locus other than the MHC. It is therefore possible to conduct genetic linkage studies in order to isolate the non HLA genes which predispose to CD. Recently a group conducted a genome screen for the non HLA genes in an affected sib-pair analysis and identified four non HLA loci with positive lod scores. We examined these loci using a pedigree based linkage study. Our pedigree sample consisted of a cohort of 21 families with 60 affected individuals and 125 unaffected family members. We used 11 microsatellite markers at the loci implicated and analysed the genotype data using both MLINK and MFLINK to detect linkage. The MLINK and MFLINK analyses did not provide any evidence to support the earlier findings, although the difficulties involved in analysing complex diseases mean that one cannot be certain that these regions do not harbour susceptibility loci, at least in some families.     

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.     

Assignment of the linkage group EAM-TYRP2-TPP2 to chromosome 11 in pigs byin situ hybridization mapping of the TPP2 gene

Restriction fragment length polymorphisms are described for the genes coding for tripeptidyl peptidase II (TPP2) and tyrosinase related protein II (TYRP2) in pigs. A linkage group comprising these loci and the locus for blood group M (EAM) was established by two-point lod score analysis in a three-generation pedigree. Multipoint analysis indicated the linear order EAM-1.1-TYRP2-8.4-TPP2 (recombination distances are given as Kosambi cM). The linkage group was assigned to porcine chromosome 11—the first on this chromosome—throughin situ hybridization mapping of the TPP2 gene. TPP2 is the first gene localized on this chromosome usingin situ hybridization.     

Using risk calculation to implement an extended relative pair analysis

A new nonparametric method of linkage analysis is described based on identity by descent relationships between all pairs of affected relatives within a pedigree. This approach is an extension of ESPA, which only uses information from pairs of affected siblings. The new method, called ERPA, uses the risk calculation facilities of the LINKAGE programs to obtain the necessary information in a fashion which is simple to implement and which automatically generalizes to allow for marker loci which may be multiple, non-codominant and sex-linked. We have investigated the relative performance of ERPA, ESPA and the lod score method on simulated data. ERPA appears to be more sensitive than ESPA for detecting linkage in pedigrees with small sibships, though both nonparametric methods are inferior to the lod score method when the true mode of transmission can be specified.     

Linkage studies in spinocerebellar ataxia (SCA)

Data are now available on 9 pedigrees in detail and 4 pedigrees as lod scores only. Linkage to HLA is significant (? = 5.53 at recombination rates of 0.223 in males and 0.327 in females). Tight linkage is excluded. Nine pedigrees which appear to be typical olivopontocerebellar atrophy (OPCA I) have recombination rates of 0.150 in males and 0.300 in females. The remaining 4 pedigrees are clinically atypical or include discrepant data and give no evidence for linkage. The symbol SCA1 is proposed for a locus on chromosome 6 (loosely linked to HLA), at which at least one allele produces OPCA I (Menzel type). It is not yet clear whether other clinical types are determined by alleles at different loci, although this is suggested by several pedigrees, including a Danish pedigree of OPCA with dementia. Linkage evidence will be decisive in delineating the ataxias.     

Exact Trait-Model-Free Tests for Linkage Detection in Pedigrees

A number of trait-model-free tests have been proposed for linkage detection between a genomic region and a trait. These tests involve testing the dependence in segregation between a trait and marker alleles by assigning a score to every possible identity-by-descent configuration of the pedigree members without modeling the trait, and then averaging the scores over all such configurations compatible with the observed marker genotypes and genealogical relationship of the pedigree members. In this paper we propose a permutation test as an alternative to the existing exact trait-model-free tests for linkage detection. The proposed test is computationally efficient and is applicable on complex multigeneration pedigree structures. In this paper, we have compared the performance of the permutation test with two other exact trait-model-free tests for linkage detection on simulated datasets. We have demonstrated that the proposed permutation test is fully robust against mispecification of marker allele frequencies and has very good power for linkage detection. The permutation test is implemented in the program lm_ibdtests within the framework of MORGAN 2.8 ( http://www.stat.washington.edu/thompson/Genepi/MORGAN/Morgan.shtml ).     

Shared Genomic Segment Analysis: The Power to Find Rare Disease Variants

Shared genomic segment (SGS) analysis uses dense single nucleotide polymorphism genotyping in high‐risk (HR) pedigrees to identify regions of sharing between cases. Here, we illustrate the power of SGS to identify dominant rare risk variants. Using simulated pedigrees, we consider 12 disease models based on disease prevalence, minor allele frequency and penetrance to represent disease loci that explain 0.2–99.8% of total disease risk. Pedigrees were required to contain ≥15 meioses between all cases and to be HR based on significant excess of disease (P < 0.001 or P < 0.00001). Across these scenarios, the power for a single pedigree ranged widely. Nonetheless, fewer than 10 pedigrees were sufficient for excellent power in the majority of models. Power increased with the risk attributable to the disease locus, penetrance and the excess of disease in the pedigree. Sharing allowing for one sporadic case was uniformly more powerful than sharing using all cases. Furthermore, an SGS analysis using a large attenuated familial adenomatous polyposis pedigree identified a 1.96 Mb region containing the known causal APC gene with genome‐wide significance. SGS is a powerful method for detecting rare variants and a valuable complement to genome‐wide association studies and linkage analysis.     

一个遗传性脊髓小脑共济失调大家系的连锁分析

目的检测一遗传性脊髓小脑共济失调家系的遗传缺陷基础。方法收集湖南地区一遗传性脊髓小脑共济失调家系，对患者的临床表型进行评估。采集家系成员48人（包括患者8人）外周静脉血提取基因组DNA，对候选染色体区域选取合适的微卫星标记，采用多重PCR和电泳方法进行基因组扫描，数据经Linkage软件包进行连锁分析以对该家系进行基因定位，以确定疾病相关基因所在的染色体区域。结果两点间连锁分析结果显示，该家系在脊髓小脑共济失调遗带瘟童SCAl、SCA27SCA3、SCA57SCA67SCA77SCA87SCAl07SCAl，？SCAl27SCAl37SCAl47SCAl57SCAl67SCAl7、SCA20、SCA27、SCA28、SCA31、DRPLA处的最大LOD值仅为0．49，提示该家系致病基因与这些位点无连锁关系。结论排除该家系与目前已知的20个常染色体显性小脑共济失调相关染色体位点的连锁关系，提示可能存在其它未知的SCAs位点。     

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.     

Genetic linkage mapping for a susceptibility locus to bipolar illness: Chromosomes 2,3,4,7,9,10p,11p,22, and Xpter

We are conducting a genome search for a predisposing locus to bipolar (manicdepressive) illness by genotyping 21 moderate-sized pedigrees. We report linkage data derived from screening marker loci on chromosomes 2, 3, 4, 7, 9, 10p, 11p, 22, and the pseudoautosomal region at Xpter. To analyze for linkage, two-point marker to illness lod scores were calculated under a dominant model with either 85% or 50% maximum penetrance and a recessive model with 85% maximum penetrance, and two affection status models. Under the dominant high penetrance model the cumulative lod scores in the pedigree series were less than ?2 at Θ = 0.01 in 134 of 142 loci examined, indicating that if the disease is genetically homogeneous linkage could be excluded in these marker regions. Similar results were obtained using the other genetic models. Heterogeneity analysis was conducted when indicated, but no evidence for linkage was found. In the course of mapping we found a positive total lod score greater than +3 at the D7S78 locus at Θ = 0.01 under a dominant, 50% penetrance model. The lod scores for additional markers within the D7S78 region failed to support the initial finding, implying that this was a spurious positive. Analysis with affected pedigree member method for COL1A2 and D7S78 showed no significance for linkage but for PLANH1, at the weighting functions f(p) = 1 and f(p) = 1/sqrt(p) borderline P values of 0.036 and 0.047 were obtained. We also detected new polymorphisms at the mineralocorticoid receptor (MLR) and calmodulin II (CALMII) genes. These genes were genetically mapped and under affection status model 2 and a dominant, high penetrance mode of transmission the lod scores of < ?2 at Θ = 0.01 were found. © 1994 Wiley-Liss, Inc.     

Genetic refinement of the chromosome 5q lattice corneal dystrophy type I locus to within a 2 cM interval.

Lattice corneal dystrophy type I (LCDI) is a relatively common corneal dystrophy which can cause severe visual impairment. Recent studies have suggested a genetic localisation for the disease to chromosome 5q. Independent genetic linkage analysis in a six generation LCDI pedigree confirmed linkage to the 5q region bounded by marker loci IL9 and D5S436 suggesting genetic homogeneity. A maximum two point lod score of 7.51 (theta = 0.03) was obtained with marker D5S393. Multipoint and haplotype data positioned the disease between loci D5S393 and D5S396 corresponding to a genetic distance of 2cM, thus refining linkage sufficiently to allow for physical mapping of this disorder.     

A computer-oriented linkage analysis scheme

A comprehensive, computer-oriented scheme for genetic linkage analysis is described. It computes the likelihood of a complex pedigree allowing for many non-standard features including multiple alleles (up to four per locus), multiple mates, more than one mating in the eldest generation, consanguinity, missing or partially tested persons, and different recombination fractions between males and females.
The program works by computing weighted likelihoods in a vertical, descending fashion. First, the set of possible genotypes for the members of the eldest generation is determined and prior probabilities assigned to these states of nature (based on gene frequency calculations). Phenotypic information is used to modify these probabilities in the standard manner. Then, for a particular set of genotypes, the conditional likelihood of the remainder of the pedigree is computed. This calculation may itself be many-branched and therefore a computer is necessary to keep track of the stages of the process. When the youngest generation has been taken into account, a final likelihood is determined, reflecting all information in the pedigree.
The current version of the program successfully calculates the exact likelihood of a complex pedigree which was designed to exploit the non-standard features of the program. It has also computed likelihoods for a published pedigree, and the results are essentially equal to those obtained by the other authors using a previously available computer scheme for linkage analysis.     

An approach to investigating linkage for bipolar disorder using large Costa Rican pedigrees

Despite the evidence that major gene effects exist for bipolar disorder (BP), efforts to map BP loci have so far been unsuccessful. A strategy for mapping BP loci is described, focused on investigation of large pedigrees from a genetically homogenous population, that of Costa Rica. This approach is based on the use of a conservative definition of the BP phenotype in preparation for whole genome screening with polymorphic markers. Linkage simulation analyses are utilized to indicate the probability of detecting evidence suggestive of linkage, using these pedigrees. These analyses are performed under a series of single locus models, ranging from recessive to nearly dominant, utilizing both lod score and affected pedigree member analyses. Additional calculations demonstrate that with any of the models employed, most of the information for linkage derives from affected rather than unaffected individuals. © 1996 Wiley-Liss, Inc.     

Pairwise analysis of radiation hybrid mapping data

Two point lod scores are widely used in pedigree analysis as they provide a fast and efficient method of establishing linkage. Groups of markers that lie in close proximity to one another can be formed by admitting any locus that is linked to at least one existing member of the group with lod score greater than some predetermined value. It seems natural to extend this technique to Radiation Hybrid Mapping both for constructing groups of tightly linked loci that may then be analysed using more powerful statistics and as a method of ordering in its own right.
A general extension of two point analysis is derived and the problems associated with radiation hybrid data are discussed. In particular, the additional parameters representing the probabilities of different fragments being retained (which have no parallel in classical linkage analysis) lead to a range of estimators of the breakage probability, θ, which have equal and maximal likelihood. Ways of circumventing this problem are discussed along with the potential errors they introduce.
Importantly the ambiguity in estimation of θ is not carried through to the lod score as this depends only on the maximum value of the likelihood and not on the particular value of θ at which it occurs. Thus even though two point analysis fails to provide robust estimates of either breakage probabilities or the distance between loci, it represents a simple and effective method of constructing linkage groups that may be analysed with more powerful statistical methods. This is particularly important given the large number of microsatellites, ESTs and candidate genes currently being typed on radiation hybrids.     

A novel locus for parietal foramina maps to chromosome 4q21-q23

Parietal foramina [PFM], inherited usually in an autosomal dominant mode, is an extremely rare developmental defect characterized by a symmetrical, oval hole in the parietal bone. It can be present as either an isolated or a syndromic feature. PFM types 1 and 2 (PFM1 and PFM2) have been found to be caused by mutations in the MSX2 and ALX4 genes, located to chromosomes 5 and 11, respectively. After exclusion of both the above loci in a large Chinese pedigree with autosomal dominant PFM, a genome-wide search revealed a linkage of the PFM to markers at the 4q21-q23 region. The maximum LOD score from two-point linkage analysis is 3.87 for marker D4S2961. Analysis of co-segregated haplotype localized the region to a 20-cM interval that flanks D4S392 and D4S2945. Therefore, we concluded that the PFM in the family is a new PFM locus. Although three genes, BMPR1B, PP1 and IBSP, are located to 4q21-q25 and their functions are related to bone morphogenesis, no mutations were identified by sequencing analysis of their exons.     

Identification of a sixth locus for autosomal dominant retinitis pigmentosa on chromosome 19

We report the mapping of a sixth locus for autosomal dominantretinitis pigmentosa (adRP) to 19q13.4. After a total genomelinkage search using over 300 markers in a single large pedigree,marker loci on the long arm of chromosome 19 showed significantlinkage with the disease locus. Since the mapping informationfor the marker loci used in this study was derived from twodifferent genome maps, we established genetic distances betweenrelevant marker loci so that linkage information could be combinedfrom both maps. A conventional three point analysis betweenthe adRP phenotype and markers D19S180 and D19S214 gave a maximumlod score of 4.87. Combining data from these and other markers,we used the recently described multiple two point programmeFASTMAP to simulate a multipoint analysis of the full data set.This gave a lod score of 5.34 in the Interval between markersD19S180 and D19S214. Recently this laboratory has also reportedthe linkage of another form of retinal degeneration known ascone-rod dystrophy (CRD) to a genetically different set of markersfrom 19q. Linkage data presented here clearly supports the existenceof two separate retinal genes in this part of the genome.     

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.     

Somatic mutations at CA-repeat loci.

We found somatic mutations, detected as novel PCR bands, at three separate polymorphic CA-repeat loci. At one of these loci, analyzed in a three-generation pedigree, a new band generated from the same paternal allele was observed in four of six offspring. The other two children inherited the alternative paternal allele unchanged. Somatic mutations at two additional loci were identified upon subsequent comparison of banding patterns among 25 cancers and their corresponding normal tissues at 15 CA-repeat loci. Since somatic mutations of CA-repeats seem to be quite frequent, individuals who are mosaic for CA-repeat alleles at a particular locus probably are not unusual. Hence, the possibility of somatic mutation generating new length alleles at CA-repeat loci should be considered when one compares DNA samples, whether in forensic and paternity testing, loss of heterozygosity studies, or linkage analyses.     

The analysis of close linkage in large families

In large families, if two closely linked loci both have rare alleles and several distant members are ascertained through one having rare alleles at both loci, simple estimates of their recombination fraction are possible. This information is free from errors due to both reduced penetrance and erratic paternity. Simple estimates based on counting will often have high efficiency and limited bias. Some problems of linkage analysis between loci with codominant expression in complete three generation families are also considered. The omission of individuals of uncertain genotype at the test locus will be more efficient than their inclusion.