Mapping Of Disease Loci Research Articles

SPINK5: both rare and common skin disease

Atopic dermatitis (AD or eczema) is an inflammatory skin disease with clinical manifestations presenting in early childhood, often with subsequent development of asthma. AD and asthma are examples of atopic diseases (caused by allergy) which are characterized by increased levels of IgE. During recent years, there has been a steady increase of atopic diseases in western society with prevalence of AD now at approximately 15%. Genetic mechanisms underlying complex gene traits are difficult to elucidate and the atopies are no exception. A complex interaction between various genes and environmental factors are thought to underlie AD and other atopic disease. Genetic linkage and association studies have been undertaken to try map disease loci for AD and, although no single gene has been identified unequivocally, linkage to a number of chromosomal regions has been demonstrated. For example, a major susceptibility locus on chromosome 3q21 has been identified spanning approximately 10 cM and including the genes encoding CD80 and CD86, two type-1 membrane proteins of the immunoglobulin superfamily.Clues as to a key genetic component of AD came as a result of the study of a rare recessive skin disease, Netherton's syndrome, in which atopy is characteristic. Netherton's syndrome was recently shown to be caused by mutations in the SPINK5 gene, which encodes LEKTI, a 15-domain serine protease inhibitor expressed in epithelial and mucosal surfaces and also in the thymus. Intriguingly, SPINK5 is contained within the distal end of the cytokine cluster on 5q31–33, a chromosomal region previously to have shown linkage to AD. Walley et al. [1xGene polymorphism in Netherton and common atopic disease. Walley et al. Nat. Genet. 2001; 29: 175–178Crossref | PubMed | Scopus (277)See all References[1] have recently reported an association (computed by transmission disequilibrium tests) between AD and a Glu420→Lys polymorphism in SPINK5. In addition, five more coding polymorphisms were found in this gene. Interestingly, a parent-of-origin effect was seen, with maternal Asn368 and Lys420 conveying a significantly increased risk of atopy to offspring when compared with paternally inherited alleles. A similar effect was seen with associations between AD and serum IgE concentration. One mechanistic hypothesis proposed is that the Glu420→Lys polymorphism in SPINK5 introduces an additional basic residue (optimal site for proteolysis) into the linker sequence between LEKTI proteinase inhibitor domains, which might lead to variable inhibition of various serine proteinase allergens.The role of SPINK5 in rare and common skin disease demonstrates the value of studying rare but less genetically complex diseases to gain insight into the mechanisms underlying the more common complex traits. From the identification of the genes involved in monogenic disorders and the cellular pathways involved in their clinical manifestation, parallels can be drawn to similar but more prevalent complex genetic diseases such as AD and atopy. The application of similar genetic approaches to that described by Walley and co-workers is likely to pave the way for new therapies and further understanding of AD, atopy and other multigenic disorders.

Molecular genetics and structural genomics of the human protein kinase C gene module.

Protein kinase C (PKC) has become a major focus among cell biologists interested in second-messenger signal transduction and much has been learned about differences in the cellular localization and function of its different isotypes. In this study we systematically address the genomic locations and gene structures of the human PKC gene module. We first carried out fine chromosomal mapping of all nine PKC genes by fluorescence in situ hybridization (FISH), using cosmid and BAC probes. The PKC genes are found to be dispersed throughout the genome, and in some positions distinct from those previously reported: PKCalpha is at 17q24, PKCbeta at 16p12, PKCgamma at 19q13.4, PKCdelta at 3p21.2, PKCepsilon at 2p21, PKCzeta at 1p36.3, PKCeta at 14q22-23, PKCtheta; at 10p15 and PKCiota at 3q26. For PKCiota, an additional FISH signal mapped on Xq21.3 revealed a pseudogene (derived by retrotransposition). PKCgamma, zeta, and theta; are found to map to the most distal positions on the chromosomes, potentially implicating telomere position effects in their expression. Using the complete human genome draft sequence and bioinformatics tools, we then carried out a systematic analysis of PKC gene structure, including determination of the occurrence of single-nucleotide polymorphisms corresponding to the PKC loci. This resource of genomic information now facilitates investigation of the PKC gene module in structural chromosomal abnormalities and human disease locus mapping studies.

The genetics of systemic lupus erythematosus.

There is a genetic predisposition to human systemic lupus erythematosus (SLE). The genes that contribute to susceptibility are, for the most part, unknown. The introduction of new gene mapping techniques has opened the way to explore lupus genetics on a genome-wide basis. Microsatellites are simple sequence repeats widely distributed throughout eukaryotic genomes. They exhibit length variation. This polymorphism can be exploited to provide a panoply of genome-wide markers. Thereby, loci linked with lupus have been mapped in lupus-prone mouse strains and in recently published studies in multi-case human families. More than 20 non-MHC (major histocompatibility complex) loci have now been linked with murine lupus. Nine non-MHC loci have been corroborated in human SLE. Some of the mouse intervals are syntenic with human loci raising the tantalizing possibility of common susceptibility genes. Although we await the results of formal gene identification, functional studies in back-cross and congenic analyses indicate that, in the mouse at least, disease genes act at multiple levels in disease development. A large number of genes are involved in the pathogenesis of SLE. The data also suggest that even the MHC contribution is multiple. Having mapped disease loci, geneticists now face the task of closing down on the actual aetiological alleles and demonstrating how they might operate. This undertaking will add significantly to our understanding of disease development.

Fine-scale mapping of disease loci

We review the issues involved in the fine-scale mapping of disease loci via allelic association. We argue that it is vital to properly account for uncertainty about the genealogy of the disease locus and founding haplotypes at the surrounding markers: we sketch how this might be done in a Bayesian framework using Markov chain Monte Carlo tecniques, and show that this works well for the much analysed data on the location of the Δ508 mutation for cystic fibrosis.

Bayesian Fine-Scale Mapping of Disease Loci, by Hidden Markov Models

We present a new multilocus method for the fine-scale mapping of genes contributing to human diseases. The method is designed for use with multiple biallelic markers-in particular, single-nucleotide polymorphisms for which high-density genetic maps will soon be available. We model disease-marker association in a candidate region via a hidden Markov process and allow for correlation between linked marker loci. Using Markov-chain-Monte Carlo simulation methods, we obtain posterior distributions of model parameter estimates including disease-gene location and the age of the disease-predisposing mutation. In addition, we allow for heterogeneity in recombination rates, across the candidate region, to account for recombination hot and cold spots. We also obtain, for the ancestral marker haplotype, a posterior distribution that is unique to our method and that, unlike maximum-likelihood estimation, can properly account for uncertainty. We apply the method to data for cystic fibrosis and Huntington disease, for which mutations in disease genes have already been identified. The new method performs well compared with existing multi-locus mapping methods.

Fine scale association mapping of disease loci using simplex families

We present a new method for the fine scale mapping of disease loci based on samples of simplex families, each containing an affected child. The method is based on a generalisation of a single locus allele transmission model to multiple marker loci. The model is developed under the assumption of a single ancestral mutation and allows for the calculation of posterior probabilities that each allele at a particular marker was present on the founder chromosome. We illustrate the method using simulated family data for cystic fibrosis and Huntingtons disease, for which the locations of mutations in the disease genes are now known. For both diseases, our new method provides good estimates of the location of the mutations.

Adaptation of the extended transmission/disequilibrium test to distinguish disease associations of multiple loci: the Conditional Extended Transmission/Disequilibrium Test

Linkage and association studies in complex diseases are used to identify and fine map disease loci. The process of identifying the aetiological polymorphism, the molecular variant responsible for the linkage and association of the chromosome region with disease, is complicated by the low penetrance of the disease variant, the linkage disequilibrium between physically‐linked polymorphic markers flanking the disease variant, and the possibility that more than one polymorphism in the most associated region is aetiological. It is important to be able to detect additional disease determinants in a region containing a cluster of genes, such as the major histocompatibility complex (MHC) region on chromosome 6p21. Some methods have been developed for detection of additional variants, such as the Haplotype Method, Marker Association Segregation Chi‐squares (MASC) Method, and the Homozygous Parent Test. Here, the Extended Transmission/Disequilibrium Test is adapted to test for association conditional on a previously associated locus. This test is referred to as the Conditional Extended TDT (CETDT). We discuss the advantages of the CETDT compared to existing methods and, using simulated data, investigate the effect of polymorphism, inheritance, and linkage disequilibrium on the CETDT.

Adaptation of the extended transmission/disequilibrium test to distinguish disease associations of multiple loci: the Conditional Extended Transmission/Disequilibrium Test.

Linkage and association studies in complex diseases are used to identify and fine map disease loci. The process of identifying the aetiological polymorphism, the molecular variant responsible for the linkage and association of the chromosome region with disease, is complicated by the low penetrance of the disease variant, the linkage disequilibrium between physically-linked polymorphic markers flanking the disease variant, and the possibility that more than one polymorphism in the most associated region is aetiological. It is important to be able to detect additional disease determinants in a region containing a cluster of genes, such as the major histocompatibility complex (MHC) region on chromosome 6p21. Some methods have been developed for detection of additional variants, such as the Haplotype Method, Marker Association Segregation Chi-squares (MASC) Method, and the Homozygous Parent Test. Here, the Extended Transmission/Disequilibrium Test is adapted to test for association conditional on a previously associated locus. This test is referred to as the Conditional Extended TDT (CETDT). We discuss the advantages of the CETDT compared to existing methods and, using simulated data, investigate the effect of polymorphism, inheritance, and linkage disequilibrium on the CETDT.

Sampling among haplotype resolutions in a coalescent-based genealogy sampler.

Analysis of the coalescent structure of a population may provide information useful in mapping disease loci. Current coalescent-based genealogy samplers require haplotyped data, but haplotypes are not always available, and it is not practical to sum over all haplotype assignments for large data sets. We describe a method of adding haplotype re-evaluation to the sampler, so that it samples not only among genealogies explaining a given haplotype configuration, but also among different haplotype configurations. Several different haplotype-rearrangement strategies are considered, but the simplest-inverting the phase of a single site in a single individual-appears to be the most successful. The straightforward haplotype sampler does not mix well; heating approaches can greatly improve its performance.

A Monte Carlo procedure for two-stage tests with correlated data.

One strategy for mapping disease loci using marker-disease associations is to test for association with case-control samples and follow up a positive result with a family-based test. Using a family-based test in the second stage can help provide protection against false-positive results that can result from use of inappropriate controls and provides assurance that association identified in the first stage is occurring between linked loci. It is crucial for this two-stage strategy that the first stage be as powerful as possible to detect association since only positive results are tested in the second stage. In certain situations, the power of the first-stage test can be increased by combining the case-control and family data. However, this introduces correlation between the first- and second-stage tests, and treating them as independent tests causes a bias. Here we propose a Monte Carlo method that accounts for the correlation and provides the correct significance level for the second-stage test. We also discuss the use of a two-stage procedure when doing a genome scan for the data presented in the Genetic Analysis Workshop 9 study.

Linkage Disequilibrium and Physical Mapping of Pas1 in Mice

By using linkage disequilibrium (LD) analysis in 21 strains of known susceptibility to lung cancer and by assembling a YAC contig, we mapped to a approximately 1.5-Mb region on distal mouse chromosome 6 the Pas1 locus, the major determinant of lung cancer predisposition in mice. Our results, on the basis of haplotype and phenetic analysis, suggest that the Pas1(s) susceptibility allele is shared by several mouse-inbred strains of independent origin, which show either high or intermediate predisposition to lung tumorigenesis. Therefore, the Pas1(s) allele is probably derived from an ancestral mouse rather than from independent mutations of the same gene. We showed the feasibility of LD in common inbred strains for the fine mapping of disease loci, and provided the biological basis and the reagents for the cloning of the Pas1 gene.

A multi‐marker approach to fine scale association mapping of disease loci

A multi‐marker approach to fine scale association mapping of disease loci

Generation and chromosome mapping of expressed sequence tags (ESTs) from a human infant thymus.

In an effort to identify novel genes that are expressed differentially in an infant thymus, we constructed an oligo-d(T) primed cDNA library from a human infant thymus followed by single-run partial sequencing to generate expressed sequence tags (ESTs). Characterization of more than 1400 sequences enabled us to convert human thymus transcripts into 1223 useful ESTs. These ESTs consisted of 613 (50.1%) showing homology to known human genes, 51 (4.2%) matching to genes from other species, 289 (23.6%) matching ESTs of unknown functions, and 182 (14.9%) being novel transcripts. The expression profile of an infant thymus features a high number of genes related to cell division-DNA synthesis and gene-protein expression, indicating the active growth stage of an infant thymus. To identify the chromosomal localization of 43 thymus ESTs, PCR-based mapping was performed using a human-rodent somatic cell hybrid or radiation hybrid mapping panel. The results indicated that several novel genes were determined to be located in the vicinity of previously mapped disease loci; histidinemia loci, plasminogen Tochigi disease loci, Ehlers-Danlos syndrome, hypertriglyceridemia, thyroid resistance locus, ocular albinism, galactosemia, porphyria variegata, Charcot-Marie-tooth disease, FEOM (fibrosis of extraocular muscles), Prader-Willi syndrome.

GLC1F, a new primary open-angle glaucoma locus, maps to 7q35-q36.

A large family with adult-onset primary open-angle glaucoma (POAG) was identified. To initiate a genome-wide scan to map the POAG locus in this family. Blood samples or buccal swabs were obtained from 25 members of a large family with POAG after informed consent was obtained. Members and their spouses were evaluated clinically for POAG on the basis of intraocular pressures, cupping of discs, and visual fields. DNA samples were used for a genome-wide screen using microsatellite markers. Ten affected family members in 4 generations showed evidence of POAG including intraocular pressures of 22 mm Hg or more, and/or optic cup-disc ratios of 0.6 or more, and/or visual field defects consistent with glaucomatous damage. Primary open-angle glaucoma segregated as an autosomal dominant trait, with the disease locus mapping to 7q35-q36 between markers D7S2442 and D7S483 with a multipoint lod score of 4.06. A sixth gene for POAG (GLC1F) has been mapped to 7q35-q36 in a family with at least 4 generations affected. The mapping of this locus further confirms that primary open-angle glaucoma is a heterogeneous group of diseases with at least 6 different loci resulting in a similar phenotype. The eventual ability to classify which major POAG gene an affected person carries could have ramifications for selecting the most effective treatment regimen for that person.

Toward a framework linkage map of the canine genome.

Selective breeding to maintain specific physical and behavioral traits has made the modern dog one of the most physically diverse species on earth. One unfortunate consequence of the common breeding practices used to develop lines of dogs with the desired traits is amplification and propagation of genetic diseases within distinct breeds. To map disease loci we have constructed a first-generation framework map of the canine genome. We developed large numbers of highly polymorphic markers, constructed a panel of canine-rodent hybrid cell lines, and assigned those markers to chromosome groups using the hybrid cell lines. Finally, we determined the order and spacing of markers on individual canine chromosomes by linkage analysis using a reference panel of 17 outbred pedigrees. This article describes approaches and strategies to accomplish these goals.

Complex Segregation Analyses: Uses and Limitations

Complex segregation analysis (CSA) is a general method for evaluating the transmission of a trait within pedigrees. It proceeds by testing models of varying degrees of generality, both to determine whether a Mendelian locus is likely to exert a large effect on the phenotype of interest and to estimate the magnitude of genetic sources of variation in the trait. This information is valuable both as a prelude to linkage analysis, which generally assumes Mendelian transmission, and in providing a model on which to base parametric linkage methods.

Age-related macular degeneration. Clinical features in a large family and linkage to chromosome 1q.

To identify the chromosomal location of a disease-causing gene and to describe the clinical characteristics of a large family with age-related macular degeneration (ARMD). An ARMD pedigree was identified, and the disease state of family members was documented by stereoscopic fundus photography and was classified using a modified version of the Wisconsin Age-Related Maculopathy Grading System. A genome-wide screen at approximately 6-centimorgan spacing using a DNA-pooling strategy combined with shared-segment analysis was used to identify likely chromosomal regions. The entire family was then screened at each likely locus, and 1 positive locus was refined by screening with markers at an average density of 0.5 centimorgan and subjected to parametric linkage analysis. In the 10 affected family members, ARMD was manifest by the presence of large, soft, confluent drusen accompanied by varying degrees of retinal pigment epithelial degeneration and/or geographic atrophy. Age-related macular degeneration segregated as an autosomal-dominant trait, with the disease locus mapping to chromosome 1q25-q31 between markers D1S466 and D1S413, with a multipoint lod score of 3.00. Age-related macular degeneration localized to chromosome 1q25-q31 (gene symbol, ARMD1) as a dominant trait in a large family with a predominantly dry phenotype. Identification of ARMD genes will facilitate early diagnosis and aid in understanding the molecular pathophysiological mechanisms of ARMD. This knowledge will contribute to the development of preventive and improved treatment strategies.

Association Mapping of Disease Loci, by Use of a Pooled DNA Genomic Screen

Genomic screening to map disease loci by association requires automation, pooling of DNA samples, and 3,000-6,000 highly polymorphic, evenly spaced microsatellite markers. Case-control samples can be used in an initial screen, followed by family-based data to confirm marker associations. Association mapping is relevant to genetic studies of complex diseases in which linkage analysis may be less effective and to cases in which multigenerational data are difficult to obtain, including rare or late-onset conditions and infectious diseases. The method can also be used effectively to follow up and confirm regions identified in linkage studies or to investigate candidate disease loci. Study designs can incorporate disease heterogeneity and interaction effects by appropriate subdivision of samples before screening. Here we report use of pooled DNA amplifications-the accurate determination of marker-disease associations for both case-control and nuclear family-based data-including application of correction methods for stutter artifact and preferential amplification. These issues, combined with a discussion of both statistical power and experimental design to define the necessary requirements for detecting of disease loci while virtually eliminating false positives, suggest the feasibility and efficiency of association mapping using pooled DNA screening.

Human Glutamate Pyruvate Transaminase (GPT): Localization to 8q24.3, cDNA and Genomic Sequences, and Polymorphic Sites

Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescencein situhybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the ratgptcDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a “half-YAC” from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of anNlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this worldwide polymorphism.

5.2 - Mapping disease loci within the MHC by typing microsatellite loci

5.2 - Mapping disease loci within the MHC by typing microsatellite loci