Genetic mapping with SNP markers in Drosophila.

Map-based positional cloning of Drosophila melanogaster genes is hampered by both the time-consuming, error-prone nature of traditional methods for genetic mapping and the difficulties in aligning the genetic and cytological maps with the genome sequence. The identification of sequence polymorphisms in the Drosophila genome will make it possible to map mutations directly to the genome sequence with high accuracy and resolution. Here we report the identification of 7,223 single-nucleotide polymorphisms (SNPs) and 1,392 insertions/deletions (InDels) in common laboratory strains of Drosophila. These sequence polymorphisms define a map of 787 autosomal marker loci with a resolution of 114 kb. We have established PCR product-length polymorphism (PLP) or restriction fragment-length polymorphism (RFLP) assays for 215 of these markers. We demonstrate the use of this map by delimiting two mutations to intervals of 169 kb and 307 kb, respectively. Using a local high-density SNP map, we also mapped a third mutation to a resolution of approximately 2 kb, sufficient to localize the mutation within a single gene. These methods should accelerate the rate of positional cloning in Drosophila.     

Genome-wide analysis of single-nucleotide polymorphisms in human expressed sequences

Single-nucleotide polymorphisms (SNPs) have been explored as a high-resolution marker set for accelerating the mapping of disease genes. Here we report 48,196 candidate SNPs detected by statistical analysis of human expressed sequence tags (ESTs), associated primarily with coding regions of genes. We used Bayesian inference to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimaeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing, sequencing error rates, context-sensitivity and cDNA library origin. Three separate validations-comparison with 54 genes screened for SNPs independently, verification of HLA-A polymorphisms and restriction fragment length polymorphism (RFLP) testing-verified 70%, 89% and 71% of our predicted SNPs, respectively. Our method detects tenfold more true HLA-A SNPs than previous analyses of the EST data. We found SNPs in a large fraction of known disease genes, including some disease-causing mutations (for example, the HbS sickle-cell mutation). Our comprehensive analysis of human coding region polymorphism provides a public resource for mapping of disease genes (available at http://www.bioinformatics.ucla.edu/snp).     

High-resolution haplotype structure in the human genome

Linkage disequilibrium (LD) analysis is traditionally based on individual genetic markers and often yields an erratic, non-monotonic picture, because the power to detect allelic associations depends on specific properties of each marker, such as frequency and population history. Ideally, LD analysis should be based directly on the underlying haplotype structure of the human genome, but this structure has remained poorly understood. Here we report a high-resolution analysis of the haplotype structure across 500 kilobases on chromosome 5q31 using 103 single-nucleotide polymorphisms (SNPs) in a European-derived population. The results show a picture of discrete haplotype blocks (of tens to hundreds of kilobases), each with limited diversity punctuated by apparent sites of recombination. In addition, we develop an analytical model for LD mapping based on such haplotype blocks. If our observed structure is general (and published data suggest that it may be), it offers a coherent framework for creating a haplotype map of the human genome.     

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.     

Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.     

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.     

Dopa decarboxylase (Ddc) affects variation in Drosophila longevity

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.     

Haplotype tagging for the identification of common disease genes

Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.     

Exhaustive allelic transmission disequilibrium tests as a new approach to genome-wide association studies

Genome-wide disease-association mapping has been heralded as the study design of the next generation, but the lack of analytical methods to use genotype data fully is a large stumbling block. Here we describe an algorithm and statistical method that efficiently and exhaustively exploits haplotype information by subjecting alleles (a marker or contiguous sets of markers) from sliding windows of all sizes to transmission disequilibrium tests. By applying our method to simulated data and to Hirschsprung disease, we show that it can detect both common and rare disease variants of small effect. These results show that the theoretical benefits of genome-wide association studies are at last realizable.     

Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis

Juvenile polyposis (JP; OMIM 174900) is an autosomal dominant gastrointestinal hamartomatous polyposis syndrome in which patients are at risk for developing gastrointestinal cancers. Previous studies have demonstrated a locus for JP mapping to 18q21.1 (ref. 3) and germline mutations in the homolog of the gene for mothers against decapentaplegic, Drosophila, (MADH4, also known as SMAD4) in several JP families. However, mutations in MADH4 are only present in a subset of JP cases, and although mutations in the gene for phosphatase and tensin homolog (PTEN) have been described in a few families, undefined genetic heterogeneity remains. Using a genome-wide screen in four JP kindreds without germline mutations in MADH4 or PTEN, we identified linkage with markers from chromosome 10q22-23 (maximum lod score of 4.74, straight theta=0.00). We found no recombinants using markers developed from the vicinity of the gene for bone morphogenetic protein receptor 1A (BMPR1A), a serine-threonine kinase type I receptor involved in bone morphogenetic protein (BMP) signaling. Genomic sequencing of BMPR1A in each of these JP kindreds disclosed germline nonsense mutations in all affected kindred members but not in normal control individuals. These findings indicate involvement of an additional gene in the transforming growth factor-beta (TGF-beta) superfamily in the genesis of JP, and document an unanticipated function for BMP in colonic epithelial growth control.     

Bayesian inference of epistatic interactions in case-control studies

Epistatic interactions among multiple genetic variants in the human genome may be important in determining individual susceptibility to common diseases. Although some existing computational methods for identifying genetic interactions have been effective for small-scale studies, we here propose a method, denoted 'bayesian epistasis association mapping' (BEAM), for genome-wide case-control studies. BEAM treats the disease-associated markers and their interactions via a bayesian partitioning model and computes, via Markov chain Monte Carlo, the posterior probability that each marker set is associated with the disease. Testing this on an age-related macular degeneration genome-wide association data set, we demonstrate that the method is significantly more powerful than existing approaches and that genome-wide case-control epistasis mapping with many thousands of markers is both computationally and statistically feasible.     

Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.     

Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse

Single-nucleotide polymorphisms (SNPs) have been the focus of much attention in human genetics because they are extremely abundant and well-suited for automated large-scale genotyping. Human SNPs, however, are less informative than other types of genetic markers (such as simple-sequence length polymorphisms or microsatellites) and thus more loci are required for mapping traits. SNPs offer similar advantages for experimental genetic organisms such as the mouse, but they entail no loss of informativeness because bi-allelic markers are fully informative in analysing crosses between inbred strains. Here we report a large-scale analysis of SNPs in the mouse genome. We characterized the rate of nucleotide polymorphism in eight mouse strains and identified a collection of 2,848 SNPs located in 1,755 sequence-tagged sites (STSs) using high-density oligonucleotide arrays. Three-quarters of these SNPs have been mapped on the mouse genome, providing a first-generation SNP map of the mouse. We have also developed a multiplex genotyping procedure by which a genome scan can be performed with only six genotyping reactions per animal.     

Highly effective SNP-based association mapping and management of recessive defects in livestock

The widespread use of elite sires by means of artificial insemination in livestock breeding leads to the frequent emergence of recessive genetic defects, which cause significant economic and animal welfare concerns. Here we show that the availability of genome-wide, high-density SNP panels, combined with the typical structure of livestock populations, markedly accelerates the positional identification of genes and mutations that cause inherited defects. We report the fine-scale mapping of five recessive disorders in cattle and the molecular basis for three of these: congenital muscular dystony (CMD) types 1 and 2 in Belgian Blue cattle and ichthyosis fetalis in Italian Chianina cattle. Identification of these causative mutations has an immediate translation into breeding practice, allowing marker assisted selection against the defects through avoidance of at-risk matings.     

Dynein mutations impair autophagic clearance of aggregate-prone proteins

Mutations that affect the dynein motor machinery are sufficient to cause motor neuron disease. It is not known why there are aggregates or inclusions in affected tissues in mice with such mutations and in most forms of human motor neuron disease. Here we identify a new mechanism of inclusion formation by showing that decreased dynein function impairs autophagic clearance of aggregate-prone proteins. We show that mutations of the dynein machinery enhanced the toxicity of the mutation that causes Huntington disease in fly and mouse models. Furthermore, loss of dynein function resulted in premature aggregate formation by mutant huntingtin and increased levels of the autophagosome marker LC3-II in both cell culture and mouse models, compatible with impaired autophagosome-lysosome fusion.     

Genome-wide variation and identification of vaccine targets in the Plasmodium falciparum genome

One goal in sequencing the Plasmodium falciparum genome, the agent of the most lethal form of malaria, is to discover vaccine and drug targets. However, identifying those targets in a genome in which approximately 60% of genes have unknown functions is an enormous challenge. Because the majority of known malaria antigens and drug-resistant genes are highly polymorphic and under various selective pressures, genome-wide analysis for signatures of selection may lead to discovery of new vaccine and drug candidates. Here we surveyed 3,539 P. falciparum genes ( approximately 65% of the predicted genes) for polymorphisms and identified various highly polymorphic loci and genes, some of which encode new antigens that we confirmed using human immune sera. Our collections of genome-wide SNPs ( approximately 65% nonsynonymous) and polymorphic microsatellites and indels provide a high-resolution map (one marker per approximately 4 kb) for mapping parasite traits and studying parasite populations. In addition, we report new antigens, providing urgently needed vaccine candidates for disease control.     

A high-resolution survey of deletion polymorphism in the human genome

Recent work has shown that copy number polymorphism is an important class of genetic variation in human genomes. Here we report a new method that uses SNP genotype data from parent-offspring trios to identify polymorphic deletions. We applied this method to data from the International HapMap Project to produce the first high-resolution population surveys of deletion polymorphism. Approximately 100 of these deletions have been experimentally validated using comparative genome hybridization on tiling-resolution oligonucleotide microarrays. Our analysis identifies a total of 586 distinct regions that harbor deletion polymorphisms in one or more of the families. Notably, we estimate that typical individuals are hemizygous for roughly 30-50 deletions larger than 5 kb, totaling around 550-750 kb of euchromatic sequence across their genomes. The detected deletions span a total of 267 known and predicted genes. Overall, however, the deleted regions are relatively gene-poor, consistent with the action of purifying selection against deletions. Deletion polymorphisms may well have an important role in the genetics of complex traits; however, they are not directly observed in most current gene mapping studies. Our new method will permit the identification of deletion polymorphisms in high-density SNP surveys of trio or other family data.     

Genetic basis of individual differences in the response to small-molecule drugs in yeast

Individual response to small-molecule drugs is variable; a drug that provides a cure for some may confer no therapeutic benefit or trigger an adverse reaction in others. To begin to understand such differences systematically, we treated 104 genotyped segregants from a cross between two yeast strains with a collection of 100 diverse small molecules. We used linkage analysis to identify 124 distinct linkages between genetic markers and response to 83 compounds. The linked markers clustered at eight genomic locations, or quantitative-trait locus 'hotspots', that contain one or more polymorphisms that affect response to multiple small molecules. We also experimentally verified that a deficiency in leucine biosynthesis caused by a deletion of LEU2 underlies sensitivity to niguldipine, which is structurally related to therapeutic calcium channel blockers, and that a natural coding-region polymorphism in the inorganic phosphate transporter PHO84 underlies sensitivity to two polychlorinated phenols that uncouple oxidative phosphorylation. Our results provide a step toward a systematic understanding of small-molecule drug action in genetically distinct individuals.     

Recombination and linkage disequilibrium in Arabidopsis thaliana

Linkage disequilibrium (LD) is a major aspect of the organization of genetic variation in natural populations. Here we describe the genome-wide pattern of LD in a sample of 19 Arabidopsis thaliana accessions using 341,602 non-singleton SNPs. LD decays within 10 kb on average, considerably faster than previously estimated. Tag SNP selection algorithms and 'hide-the-SNP' simulations suggest that genome-wide association mapping will require only 40%-50% of the observed SNPs, a reduction similar to estimates in a sample of African Americans. An Affymetrix genotyping array containing 250,000 SNPs has been designed based on these results; we demonstrate that it should have more than adequate coverage for genome-wide association mapping. The extent of LD is highly variable, and we find clear evidence of recombination hotspots, which seem to occur preferentially in intergenic regions. LD also reflects the action of selection, and it is more extensive between nonsynonymous polymorphisms than between synonymous polymorphisms.