The roads from phenotypic variation to gene discovery: mutagenesis versus QTLs

In model organisms, chemical mutagenesis provides a powerful alternative to natural, polygenic variation (for example, quantitative trait loci (QTLs)) for identifying functional pathways and complex disease genes. Despite recent progress in QTLs, we expect that mutagenesis is will ultimately prove more effective because the prospects of gene identification are high and every gene affecting a trait is potentially a target.     

Analysing complex genetic traits with chromosome substitution strains

Many valuable animal models of human disease are known and new models are continually being generated in existing inbred strains,. Some disease models are simple mendelian traits, but most have a polygenic basis. The current approach to identifying quantitative trait loci (QTLs) that underlie such traits is to localize them in crosses, construct congenic strains carrying individual QTLs, and finally map and clone the genes. This process is time-consuming and expensive, requiring the genotyping of large crosses and many generations of breeding. Here we describe a different approach in which a panel of chromosome substitution strains (CSSs) is used for QTL mapping. Each of these strains has a single chromosome from the donor strain substituting for the corresponding chromosome in the host strain. We discuss the construction, applications and advantages of CSSs compared with conventional crosses for detecting and analysing QTLs, including those that have weak phenotypic effects.     

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.     

The genetically isolated populations of Finland and sardinia may not be a panacea for linkage disequilibrium mapping of common disease genes

The choice of which population to study in the mapping of common disease genes may be critical. Isolated founder populations, such as that found in Finland, have already proved extremely useful for mapping the genes for specific rare monogenic disorders and are being used in attempts to map the genes underlying common, complex diseases. But simulation results suggest that, under the common disease-common variant hypothesis, most isolated populations will prove no more useful for linkage disequilibrium (LD) mapping of common disease genes than large outbred populations. There is very little empirical data to either support or refute this conclusion at present. Therefore, we evaluated LD between 21 common microsatellite polymorphisms on chromosome 18q21 in 2 genetic isolates (Finland and Sardinia) and compared the results with those observed in two mixed populations (United Kingdom and United States of America). Mean levels of LD were similar across all four populations. Our results provide empirical support for the expectation that genetic isolates like Finland and Sardinia will not prove significantly more valuable than general populations for LD mapping of common variants underlying complex disease.     

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.     

Regulatory changes underlying expression differences within and between Drosophila species

Differences in gene expression are an important source of phenotypic variation, and can be caused by changes in cis and/or trans regulation. cis-regulatory variants alter allele-specific expression, whereas trans-regulatory variants influence expression of both alleles in a diploid cell. Because of this difference, we hypothesize that natural selection may favor one type of change over the other. Here, we investigate contributions of cis- and trans-regulatory changes to variable intra- and interspecific gene expression using four strains of Drosophila melanogaster, three strains of D. simulans and a total of 78 genes. We show that cis-regulatory changes account for a greater proportion of the expression differences observed between rather than within species. These data are inconsistent with a neutral model assuming equal probabilities of fixation for cis- and trans-regulatory polymorphisms, suggesting that natural selection influences the molecular mechanisms underlying divergent gene expression. Specifically, cis-regulatory changes seem to accumulate preferentially over time.     

A phenylalanine in DGAT is a key determinant of oil content and composition in maize

Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.     

Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease

Linkage disequilibrium (LD) mapping provides a powerful method for fine-structure localization of rare disease genes, but has not yet been widely applied to common disease. We sought to design a systematic approach for LD mapping and apply it to the localization of a gene (IBD5) conferring susceptibility to Crohn disease. The key issues are: (i) to detect a significant LD signal (ii) to rigorously bound the critical region and (iii) to identify the causal genetic variant within this region. We previously mapped the IBD5 locus to a large region spanning 18 cM of chromosome 5q31 (P<10(-4)). Using dense genetic maps of microsatellite markers and single-nucleotide polymorphisms (SNPs) across the entire region, we found strong evidence of LD. We bound the region to a common haplotype spanning 250 kb that shows strong association with the disease (P< 2 x 10(-7)) and contains the cytokine gene cluster. This finding provides overwhelming evidence that a specific common haplotype of the cytokine region in 5q31 confers susceptibility to Crohn disease. However, genetic evidence alone is not sufficient to identify the causal mutation within this region, as strong LD across the region results in multiple SNPs having equivalent genetic evidence-each consistent with the expected properties of the IBD5 locus. These results have important implications for Crohn disease in particular and LD mapping in general.     

Functional screening of an asthma QTL in YAC transgenic mice.

Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.     

Genome-wide genetic association of complex traits in heterogeneous stock mice

Difficulties in fine-mapping quantitative trait loci (QTLs) are a major impediment to progress in the molecular dissection of complex traits in mice. Here we show that genome-wide high-resolution mapping of multiple phenotypes can be achieved using a stock of genetically heterogeneous mice. We developed a conservative and robust bootstrap analysis to map 843 QTLs with an average 95% confidence interval of 2.8 Mb. The QTLs contribute to variation in 97 traits, including models of human disease (asthma, type 2 diabetes mellitus, obesity and anxiety) as well as immunological, biochemical and hematological phenotypes. The genetic architecture of almost all phenotypes was complex, with many loci each contributing a small proportion to the total variance. Our data set, freely available at http://gscan.well.ox.ac.uk, provides an entry point to the functional characterization of genes involved in many complex traits.     

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.     

The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana

Light has an important role in modulating seedling growth and flowering time. We show that allelic variation at the PHYTOCHROME C (PHYC) photoreceptor locus affects both traits in natural populations of A. thaliana. Two functionally distinct PHYC haplotype groups are distributed in a latitudinal cline dependent on FRIGIDA, a locus that together with FLOWERING LOCUS C explains a large portion of the variation in A. thaliana flowering time. In a genome-wide scan for association of 65 loci with latitude, there was an excess of significant P values, indicative of population structure. Nevertheless, PHYC was the most strongly associated locus across 163 strains, suggesting that PHYC alleles are under diversifying selection in A. thaliana. Our work, together with previous findings, suggests that photoreceptor genes are major agents of natural variation in plant flowering and growth response.     

Detection of regulatory variation in mouse genes

Functional polymorphism in genes can be classified as coding variation, altering the amino-acid sequence of the encoded protein, or regulatory variation, affecting the level or pattern of expression of the gene. Coding variation can be recognized directly from DNA sequence, and consequently its frequency and characteristics have been extensively described. By contrast, virtually nothing is known about the extent to which gene regulation varies in populations. Yet it is likely that regulatory variants are important in modulating gene function: alterations in gene regulation have been proposed to influence disease susceptibility and to have been the primary substrate for the evolution of species. Here, we report a systematic study to assess the extent of cis-acting regulatory variation in 69 genes across four inbred mouse strains. We find that at least four of these genes show allelic differences in expression level of 1.5-fold or greater, and that some of these differences are tissue specific. The results show that the impact of regulatory variants can be detected at a significant frequency in a genomic survey and suggest that such variation may have important consequences for organismal phenotype and evolution. The results indicate that larger-scale surveys in both mouse and human could identify a substantial number of genes with common regulatory variation.     

Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees. Australian Breast Cancer Family Study

Mutations in BRCA1 (ref. 1) confer an increased risk of female breast cancer. In a genome-wide scan of linkage disequilibrium (LD), a high level of LD was detected among microsatellite markers flanking BRCA1 (ref. 3), raising the prospect that positive natural selection may have acted on this gene. We have used the predictions of evolutionary genetic theory to investigate this further. Using phylogeny-based maximum likelihood analysis of the BRCA1 sequences from primates and other mammals, we found that the ratios of replacement to silent nucleotide substitutions on the human and chimpanzee lineages were not different from one another (P=0.8), were different from those of other primate lineages (P=0.004) and were greater than 1 (P=0.04). This is consistent with the historic occurrence of positive darwinian selection pressure on the BRCA1 protein in the human and chimpanzee lineages. Analysis of genetic variation in a sample of female Australians of Northern European origin showed evidence for Hardy-Weinberg (HW) disequilibrium at polymorphic sites in BRCA1, consistent with the possibility that natural selection is affecting genotype frequencies in modern Europeans. The clustering of between-species variation in the region of the gene encoding the RAD51-interaction domain of BRCA1 suggests the maintenance of genomic integrity as a possible target of selection.     

Susceptibility to testicular germ-cell tumours in a 129.MOLF-Chr 19 chromosome substitution strain.

The identification of genes that control susceptibility to testicular germ-cell tumours (TGCTs), the most common cancer affecting young men, has been difficult. In laboratory mice, TGCTs arise from primordial germ cells in only the 129 inbred strains, and susceptibility is under multigenic control. The spontaneously arising mutation Ter (ref. 5) on mouse chromosome 18 (Refs 6,7) increases TGCT frequency on a 129/Sv background. We originally used Ter in genetic crosses to identify loci that control tumorigenesis. A genome scan of tumour-bearing progeny from backcrosses between the 129/Sv-Ter/+ and MOLF/Ei strains provided modest evidence that MOLF-derived alleles on chromosome 19 enhance development of bilateral TGCTs (ref. 9). To obtain independent evidence for linkage to the MOLF chromosome, we made an autosomal chromosome substitution strain (CSS; or 'consomic strain') in which chromosome 19 of 129/Sv+/+ was replaced by its MOLF-derived homologue. The unusually high frequency of TGCTs in this CSS (even in the absence of the Ter mutation) provides evidence confirming the genome survey results, identifies linkage for a naturally occurring strain variant allele that confers susceptibility to TGCTs and illustrates the power of CSSs in complex trait analysis.