Counselling under genetic heterogeneity: a practical approach

Hereditary diseases due to mutation at different gene loci may be indistinguishable phenotypically. In these situations genetic risk predictions using polymorphic markers may be hampered if an individual family is not sufficiently informative to permit it to be assigned to one or the other linkage group. To provide the most usefull estimates of risk, the probability of linkage to a particular chromosome region should be determined prior to calculating risk estimates using the marker system. The probability can be calculated directly using the lod score generated for the family. The individual carrier risk is then the average of the carrier risks determined for linkage to different genetic loci, weighted by the probability of linkage to each group. Several examples are provided.     

Feasibility of using genetic linkage analysis to identify the genes encoding T cell-defined minor histocompatibility antigens

We have evaluated the utility of genetic linkage analysis to identify genes that encode minor histocompatibility antigens using vaccinia virus vectors as a simple and convenient method for transient expression of class I MHC molecules in lymphoblastoid cell lines. As a test case, we used a CTL clone that recognizes HA-8, a minor histocompatibility antigen encoded by the KIAA0020 gene and presented by HLA-A*0201. EBV-transformed B cell lines from individuals in three large pedigrees from the CEPH reference family collection were infected with a recombinant vaccinia virus vector encoding an HLA-A*0201 transgene, which led to high level expression of the MHC restricting allele HLA-A*0201 on the cell surface. HA-8 expression in the vaccinia-infected target cells was then determined using standard in vitro cytotoxicity assays. Pairwise linkage analysis of the segregation of HA-8 expression in these pedigrees demonstrated that the HA-8 gene was tightly linked with a cluster of marker loci located on the distal portion of chromosome 9p. Analysis of 9p marker haplotypes for individuals in the three families identified several individuals with recombinant haplotypes, and these recombination events were used to refine the precision of the HA-8 gene localization further. The data collectively indicate that the HA-8 gene is localized to a 10.3 cM (corresponding to 3.9 Mb) interval of distal 9p that is thought to encode at least 11 genes, including KIAA0020. These results demonstrate that linkage analysis can be used to map minor histocompatibility genes with high precision and accuracy. Over the next years, refinement and annotation of the human genome sequence will undoubtedly increase the utility of linkage analysis as a tool for identifying minor histocompatibility antigen genes.     

Evidence for the multigenic inheritance of schizophrenia

Schizophrenia is assumed to have complex inheritance because of its high prevalence and sporadic familial transmission. Findings of linkage on different chromosomes in various studies corroborate this assumption. It is not known whether these findings represent heterogeneous inheritance, in which various ethnic groups inherit illness through different major gene effects, or multigenic inheritance, in which affected individuals inherit several common genetic abnormalities. This study therefore examined inheritance of schizophrenia at different genetic loci in a nationally collected European American and African American sample. Seventy‐seven families were previously genotyped at 458 markers for the NIMH Schizophrenia Genetics Initiative. Initial genetic analysis tested a dominant model, with schizophrenia and schizoaffective disorder, depressed type, as the affected phenotype. The families showed one genome‐wide significant linkage (Z = 3.97) at chromosome 15q14, which maps within 1 cM of a previous linkage at the α7‐nicotinic receptor gene. Chromosome 10p13 showed suggestive linkage (Z = 2.40). Six others (6q21, 9q32, 13q32, 15q24, 17p12, 20q13) were positive, with few differences between the two ethnic groups. The probability of each family transmitting schizophrenia through two genes is greater than expected from the combination of the independent segregation of each gene. Two trait‐locus linkage analysis supports a model in which genetic alleles associated with schizophrenia are relatively common in the general population and affected individuals inherit risk for illness through at least two different loci. © 2001 Wiley‐Liss, Inc.     

Major gene model for the inheritance of catechol-o-methyltransferase activity in five large families

Five large families including 1,189 individuals were each ascertained through one proband with essential hypertension. Four of the probands were white and one was black. Erythrocyte catechol-o-methyltransferase (COMT) activity was measured in 551 family members. Standard statistical methods were used to investigate sex, age, and family differences in COMT activity. Maximum-likelihood methods were used to fit mixtures of normal distributions to COMT activity. COMT activity is distinctly bimodal. Pedigree segregation analyses were performed on the untransformed COMT values, their square roots, and natural logarithms in each family. In no family and under none of the three transformations was it possible to reject the hypothesis of Mendelian transmission of a major gene with two alleles in Hardy-Weinberg equilibrium. In most cases a genetic hypothesis with complete dominance or recessiveness, or a hypothesis of equal transmission probabilities was rejected. While the different transformations had a large effect on the skewness and kurtosis of the overall distribution of the data, they had little effect on the outcome of these segregation analyses. Therefore, this study strongly supports the concept that variation in COMT activity is due in large part to the effects of a major gene.     

Physical and genetic mapping of human chromosome 3 loci containing microsatellite repeats

One hundred and six microsatellite repeat-containing loci, including 59 CA-containing repeats from the CEPH/Genethon collection, were regionally assigned on human chromosome 3 using a somatic cell hybrid mapping panel, dividing the chromosome into 14 intervals. The others were dinucleotide and tetranucleotide repeat-containing loci newly developed for human chromosome 3, of which 26 were also localized by means of genetic linkage analysis against selected CEPH microsatellites. The regional assignment of these two marker sets in a common mapping panel facilitates their integration. Incorporation of these highly polymorphic loci into the developing physical and genetic maps should provide useful information for studies of various diseases involving chromosome 3.     

Heterogeneity of insulin-dependent diabetes–new evidence

We have performed complex segregation and linkage analysis in 182 families with at least one insulin-dependent diabetic proband. All families were typed for B histocompatibility (HLA) antigens and 118 for DR. The recessive model fit the data best, with maximum likelihood estimates of recombination between HLA DR and the susceptibility factor of 0.019. Substantial heterogeneity was suggested, with smallest estimated recombination for pedigrees whose probands have two high-risk DR alleles. The results are compatible with a strong, tightly HLA-linked susceptibility factor and evidence for additional non-HLA linked genetic factor(s).     

Effectiveness of Shrinkage and Variable Selection Methods for the Prediction of Complex Human Traits using Data from Distantly Related Individuals

Genome‐wide association studies (GWAS) have detected large numbers of variants associated with complex human traits and diseases. However, the proportion of variance explained by GWAS‐significant single nucleotide polymorphisms has been usually small. This brought interest in the use of whole‐genome regression (WGR) methods. However, there has been limited research on the factors that affect prediction accuracy (PA) of WGRs when applied to human data of distantly related individuals. Here, we examine, using real human genotypes and simulated phenotypes, how trait complexity, marker‐quantitative trait loci (QTL) linkage disequilibrium (LD), and the model used affect the performance of WGRs. Our results indicated that the estimated rate of missing heritability is dependent on the extent of marker‐QTL LD. However, this parameter was not greatly affected by trait complexity. Regarding PA our results indicated that: (a) under perfect marker‐QTL LD WGR can achieve moderately high prediction accuracy, and with simple genetic architectures variable selection methods outperform shrinkage procedures and (b) under imperfect marker‐QTL LD, variable selection methods can achieved reasonably good PA with simple or moderately complex genetic architectures; however, the PA of these methods deteriorated as trait complexity increases and with highly complex traits variable selection and shrinkage methods both performed poorly. This was confirmed with an analysis of human height.     

Investigation of the Ability of Haplotype Association and Logistic Regression to Identify Associated Susceptibility Loci

While finely spaced markers are increasingly being used in case‐control association studies in attempts to identify susceptibility loci, not enough is yet known as to the optimal spacing of such markers, their likely power to detect association, the relative merits of single marker versus multimarker analysis, or which methods of analysis may be optimal. Some investigations of these issues have used markers simulated under different theoretical models of population evolution. However the HapMap project and other sources provide real datasets which can be used to obtain a more realistic view of the performance of these approaches. SNPs around APOE and from two HapMap regions were used to obtain information regarding linkage disequilibrium (LD) relationships between polymorphisms, and these real patterns of LD were used to simulate datasets such as would be obtained in case‐control studies were these SNPs to influence susceptibility to disease. The datasets obtained were analysed using tests for heterogeneity of estimated haplotype frequencies and using logistic regression analyses in which only main effects from each marker were considered. All markers surrounding the putative susceptibility locus were analysed, using sets of either 1, 2, 3 or 4 markers at a time. Some markers within 150 kb of the susceptibility locus were able to detect association. At distances less than 100 kb there was no correlation between the distance from the susceptibility locus and the strength of evidence for association. When the average inter‐locus spacing is 25 kb many loci would not be detected, while when the spacing is as low as 2 kb one can be fairly confident that at least one marker will be in strong enough LD with the susceptibility locus to enable association to be detected, if the susceptibility locus has a strong enough effect relative to the sample size. With an inter‐locus spacing of 4 kb some susceptibility loci did not have a marker locus in strong LD, potentially undermining the ability to detect association. There was little difference in the performance of haplotype‐based analysis compared with logistic regression considering effects of each marker as separate. Multimarker analysis on occasion produced results which were much more highly significant than single marker analysis, but only very rarely. Our results support the view that if markers are randomly selected then a spacing as low as 2 kb is desirable. Multimarker analysis can sometimes be more powerful than single marker analysis so both should be performed. However, because it is rare for multimarker analysis to be much more highly significant than single marker analysis one should strongly suspect that when such results occur they may be due to mistakes in genotyping or through some other artefact. Haplotype analysis may be more prone to such problems than logistic regression, suggesting that the latter method might be preferred.     

Using multivariate genetic modeling to detect pleiotropic quantitative trait loci

Large numbers of sibling pairs or other relatives are needed to detect linkage between a quantitative trait locus (QTL) and a marker, especially if the variance of the QTL is low relative to the total phenotypic variance of the trait. One strategy to increase the power to detect linkage is to reduce the environmental variance in the trait under analysis. This approach was explored by carrying out a series of simulation studies in which multivariate observations were used to estimate individual genotypic values at a QTL, that pleiotropically affected more than one trait. Simulations for different QTL allele frequencies with a completely informative marker showed that the power to detect the QTL increased substantially when estimates of individual genotypic values at the QTL were used in the linkage analysis instead of phenotypic observations. An advantage of this approach is that, rather than employing phenotypic selection, individuals with extreme genotypes may be selected when ascertaining a sample of extreme families.     

Attention-deficit/hyperactivity disorder (ADHD): feasibility of linkage analysis in a genetic isolate using extended and multigenerational pedigrees

Segregation analyses converge in explaining the predisposition to attention-deficit/hyperactivity disorder (ADHD) as the consequence of a major gene and exclude purely environmental or cultural transmission. As a result of the ADHD phenotype restrictions, collection of extended families or design of linkage studies using families has been extremely difficult and thus currently linkage studies have been performed using only concordant or discordant sib-pairs rather than large families. On the other hand, intergenerational studies are represented by the transmission disequilibrium test (TDT) using trios. We collected pedigree data on ADHD from the Paisa community from Antioquia, Colombia, a genetic isolate. The goal of this study was to genetically map a putative gene predisposing to ADHD in a set of 27 multigenerational Paisa families. Here we present the results of a power simulation using SIMLINK to detect linkage of ADHD. ADHD was assumed to be a dichotomous trait with incomplete penetrance and a phenocopy rate of 3% in males and 0.2% in females. We simulated cosegregation of the trait and a marker locus in our pedigrees. We assumed Hardy-Weinberg and linkage equilibrium, equally frequent marker alleles and evaluated power at several recombination fractions between the trait and marker loci. Also, the ADHD trait was assumed to be genetically heterogeneous and different functions of age-dependent penetrance were simulated. We found exceptionally good power to detect linkage (expected LOD > 14 if theta is 0.1 or less), and that the presence of heterogeneity up to 50% does not affect substantially the projected LOD scores even for a theta recombination value of 0.05 (eLOD > 5.87). Having now obtained blood samples and confirmatory interviews in five families (representing 20% of the projected number of families), we performed a new analysis. The expected mean LOD in these five families reached values close to 10 and remained invariant when heterogeneity and different penetrance models were considered. We discuss the relative benefits of using extended and multigenerational families for genetic mapping studies as opposed to using nuclear families, affected sib pairs or sporadic cases which require the collection of over 1000 analytical units to get the same power exhibited by the small number of pedigrees described here.     

Linkage and risk assessment in fragile X families using new DNA probes at Xq27.

Until recently few polymorphic loci had been genetically mapped close to the fragile X syndrome locus [FRAXA]. Six polymorphic loci, DXS369, DXS297, DXS296, DXS304, IDS and DXS374, have now been mapped closer to the fragile X FRAXA than in the present study. We report the results of genetic linkage analysis of 32 fragile X [fra(X)] families using 12 polymorphic loci including these new markers. Cytogenetic and molecular data were combined in two-point linkage analysis for the estimation of lod scores and carrier probabilities in potential carriers. Combined with results from previous studies, recombination fractions (0) corresponding to the maximum lod scores (Z max) were obtained for each of the 12 loci versus FRAXA. Recombination fractions between marker loci in the families were also calculated. The data were evaluated to determine the efficacy of using the strategy suggested by Suthers et al. (1991a) for molecular studies in fra(X) families. The large proportion of females heterozygous for at least one locus (83%) and of females heterozygous for flanking loci (60%) indicate that this is a very useful diagnostic strategy. Use of these new marker loci substantially changed the carrier risk estimates for members of 7 of the 32 families from the risk estimates previously calculated on the basis of less closely linked probes available prior to 1989.     

Genome-wide linkage analysis of a composite index of neuroticism and mood-related scales in extreme selected sibships

There is considerable evidence to suggest that the genetic vulnerabilities to depression and anxiety substantially overlap and quantitatively act to alter risk to both disorders. Continuous scales can be used to index this shared liability and are a complementary approach to the use of clinical phenotypes in the genetic analysis of depression and anxiety. The aim of this study (Genetic and Environmental Nature of Emotional States in Siblings) was to identify genetic variants for the liability to depression and anxiety after the application of quantitative genetic methodology to a large community-based sample (n = 34,371), using four well-validated questionnaires of depression and anxiety. Genetic model fitting was performed on 2658 unselected sibships, which provided evidence for a single common familial factor that accounted for a substantial proportion of the genetic variances and covariances of the four scales. Using the parameter estimates from this model, a composite index of liability (G) was constructed. This index was then used to select a smaller--but statistically powerful--sample for DNA collection (757 individuals, 297 sibships). These individuals were genotyped with more than 400 microsatellite markers. After the data were checked and cleaned, linkage analysis was performed on G and the personality scale of neuroticism using the regression-based linkage program MERLIN-REGRESS. The results indicated two potential quantitative trait loci (QTL): one on chromosome 1p (LOD 2.2) around 64 cM (43-70 cM) near marker D1S2892 and another on chromosome 6p (LOD 2.7) around 47 cM (34-63 cM) near marker D6S1610. Further exploratory sex-specific analyses suggested that these QTLs might have sex-limited effects.     

不完全外显的遗传性痉挛性截瘫一家系临床与致病基因排除定位分析

目的 描述一个遗传了4代的不完全外显的遗传性痉挛性截瘫(hereditary spastic paraplegia,SPG)大家系的临床特征,并进行致病基因排除定位分析.方法 对SPG家系内11例患者的临床资料进行回顾性分析,并采用荧光多重PCR、毛细管凝胶电泳、Linkage软件包,选择对已定位常染色体显性遗传致病基因位点附近微卫星标记进行连锁分析.结果 该SPG家系的11例患者的发病年龄2～10岁,表现为缓慢进展的双下肢僵硬无力,四肢肌张力轻度增高,双上肢为主的腱反射亢进,剪刀步态和病理征阳性,无小便失禁或尿频、感觉障碍、眼震、痴呆等;遗传学分析该家系符合常染色体显性遗传,但外显不完全,连锁分析和突变分析发现该家系与已知的常染色体显性遗传SPG致病基因位点不连锁.结论 该SPG家系具有典型的"单纯型"痉挛性截瘫临床特点,发病年龄早,上肢体征较下肢明显,遗传学分析不支持该家系与已定位常染色体显性遗传位点相连锁,是一种新的SPG亚型.     

Evidence for the multigenic inheritance of schizophrenia.

Schizophrenia is assumed to have complex inheritance because of its high prevalence and sporadic familial transmission. Findings of linkage on different chromosomes in various studies corroborate this assumption. It is not known whether these findings represent heterogeneous inheritance, in which various ethnic groups inherit illness through different major gene effects, or multigenic inheritance, in which affected individuals inherit several common genetic abnormalities. This study therefore examined inheritance of schizophrenia at different genetic loci in a nationally collected European American and African American sample. Seventy-seven families were previously genotyped at 458 markers for the NIMH Schizophrenia Genetics Initiative. Initial genetic analysis tested a dominant model, with schizophrenia and schizoaffective disorder, depressed type, as the affected phenotype. The families showed one genome-wide significant linkage (Z = 3.97) at chromosome 15q14, which maps within 1 cM of a previous linkage at the alpha 7-nicotinic receptor gene. Chromosome 10p13 showed suggestive linkage (Z = 2.40). Six others (6q21, 9q32, 13q32, 15q24, 17p12, 20q13) were positive, with few differences between the two ethnic groups. The probability of each family transmitting schizophrenia through two genes is greater than expected from the combination of the independent segregation of each gene. Two trait-locus linkage analysis supports a model in which genetic alleles associated with schizophrenia are relatively common in the general population and affected individuals inherit risk for illness through at least two different loci.     

显性视网膜色素变性家系已知连锁位点的筛查

目的 用连锁分析法对1个中国人显性视网膜色素变性家系进行已知位点的筛查,寻找其致病基因.方法 随机选取已知致病基因上下约5cM(JB)范围内的27对微卫星标记,确立单倍型,用两点法计箅最大优势对数(Lod score)值.结果 所选微卫星标记与该家系表型间最大Lod值小于1.结论 基本排除由已知常染色体显性遗传视网膜色素变性的候选基因导致该家系的病变.     

Childhood manifestation of autosomal dominant polycystic kidney disease: no evidence for genetic heterogeneity

Autosomal dominant polycystic kidney disease (ADPKD) usually becomes symptomatic between the third and fifth decades. We studied ten families segregating for ADPKD in which children were observed with typical manifestations of the disease at birth or in early childhood. In these families, linkage analysis was carried out with a cloned DNA sequence from the alphaglobin locus known to be closely linked to the disease gene in adult onset ADPKD. In the families studied here, close linkage ( θ _max= 0.09 at Z_max= 2.32) was also observed between the marker and disease loci. These results provide no evidence for genetic heterogeneity of ADPKD in families with early and adult onset.     

Genetic linkage analysis of epidermolysis bullosa dystrophia, Cockayne-Touraine type

Genetic linkage relationships between 27 informative marker loci and the locus for epidermolysis bullosa dystrophica, Cockayne-Touraine type (EBD-CT), were examined in a single large kindred. Linkage could not be demonstrated to any of the marker loci, further adding to the exclusion map for EBD-CT. The dominant forms of EBD so far delineated by clinical criteria and electron microscopy remain genetically undefined in terms of loci and allelism. Further investigation will be undertaken using restriction fragment length polymorphisms mapped to regions outside the existing exclusion map.     

Exclusion of Linkage Between Bipolar Affective Disorder and Chromosome 16 in 12 Australian Pedigrees

Several recent reports of possible susceptibility loci for bipolar affective disorder (BAD) have identified sites on a number of chromosomes. Specifically, two Danish studies have suggested the presence of a susceptibility locus for BAD on chromosome 16p13. As the first step of a whole genome scan, we screened 12 Australian families with markers at 16p13 and also a number of markers spanning the entirety of chromosome 16. Linkage analysis was undertaken using both the parametric lod score method (two- and multipoint) with different models and diagnostic thresholds, and the nonparametric affected pedigree member (APM) method. Results of lod score analysis convincingly excluded the 16p13 region from linkage to BAD in these families, while APM provided no support for linkage. Furthermore, using the broad definition of BAD, with individuals affected by bipolar I and II and recurrent unipolar disorders included, the entire chromosome was excluded from linkage to BAD with autosomal-dominant transmission at a maximum age-specific penetrance of 60%, and with autosomal-dominant and recessive modes of transmission at a maximum age-specific penetrance level of 90%. Diagnostic thresholds which did not include unipolar affected individuals were somewhat less informative. However, a majority (between 63–96%, depending upon the model) of the chromosome was clearly excluded using narrow diagnostic thresholds. Moreover, no positive lod scores were obtained at θ = 0.00 for any tested model or diagnostic threshold. Our results indicate that no linkage exists between BAD and chromosome 16 markers in this group of Australian families. Am. J. Med. Genet. 74:304–310, 1997. © 1997 Wiley-Liss, Inc.     

A follow-up linkage study of Finnish pre-eclampsia families identifies a new fetal susceptibility locus on chromosome 18

Pre-eclampsia is a common vascular disorder of pregnancy. It originates in the placenta and targets the maternal endothelium. According to epidemiological research, >50% of the liability to this disorder can be accounted for by genetic factors. Both maternal and fetal genes contribute to the risk, but especially the fetal genetic risk profile is still poorly understood. We have previously detected linkage signals in multiplex Finnish families on chromosomes 2p25, 4q32, and 9p13 using maternal phenotypes. We performed a linkage analysis using updated maternal phenotypes and an unprecedented linkage analysis using fetal phenotypes. Markers genotyped were available from 237 individuals in 15 Finnish families, including 72 affected mothers and 49 affected fetuses. The MERLIN software was used for sample and marker quality control and linkage analysis. The results were compared against the original ones obtained by using the GENEHUNTER 2.1 software. The previous identification of the maternal susceptibility locus to a genetic location at 21.70 cM near marker D2S168 on chromosome 2 was confirmed by using both maternal and fetal phenotypes (maternal non-parametric linkage (NPL) score 3.79, P=0.00008, LOD (logarithm (base 10) of odds)=2.20 and fetal NPL score 2.95, P=0.002, LOD=1.71). As a novel finding, we present a suggestive linkage to chromosome 18 at 86.80 cM near marker D18S64 (NPL score 2.51, P=0.006, LOD=1.20) using the fetal phenotype. We propose that chromosome 18 may harbor a new fetal susceptibility locus for pre-eclampsia.     

A Novel Genetic Study of Chinese Families with Autosomal Recessive Retinitis Pigmentosa

Autosomal recessive retinitis pigmentosa (arRP) is the commonest form of RP worldwide. To date 22 loci have been implicated in the pathogenesis of this disease; however none of these loci independently account for a significant proportion of recessive RP. Linkage studies of arRP in consanguineous families have been mainly based on homozygosity mapping, but this strategy cannot be applied in the case of non-consanguineous families. Therefore, we implemented a systematic approach for identifying the disease locus in three non-consanguineous Chinese families with arRP. Initially, linkage analysis using SNPs/microsatellite markers or mutation screening of known arRP genes excluded all loci/genes except RP25 on chromosome 6. Subsequently a whole genome scan for the three families using the 10K GeneChip Mapping Array was performed, in order to identify the possible disease locus. To the best of our knowledge this is the first report on the utilisation of the 10K GeneChip to study linkage in non-consanguineous Chinese arRP. This analysis indicates that the studied families are probably linked to the RP25 locus, a well defined arRP locus in other populations. The identification of another ethnic group linked to RP25 is highly suggestive that this represents a major locus for arRP.