Allele Frequency Distributions in Pooled DNA Samples: Applications to Mapping Complex Disease Genes
TL;DR: The studies show that accurate, quantitative data on allele frequencies, suitable for identifying markers for complex disorders, can be identified from pooled DNA samples, and this approach promises to drastically reduce the labor and cost of genotyping in the initial identification of disease loci.
Abstract: Genetic studies of complex hereditary disorders require for their mapping the determination of genotypes at several hundred polymorphic loci in several hundred families. Because only a minority of markers are expected to show linkage and association in family data, a simple screen of genetic markers to identify those showing linkage in pooled DNA samples can greatly facilitate gene identification. All studies involving pooled DNA samples require the comparison of allele frequencies in appropriate family samples and subsamples. We have tested the accuracy of allele frequency estimates, in various DNA samples, by pooling DNA from multiple individuals prior to PCR amplification. We have used the ABI 377 automated DNA sequencer and GENESCAN software for quantifying total amplification using a 58 fluorescently labeled forward PCR primer and relative peak heights to estimate allele frequencies in pooled DNA samples. In these studies, we have genotyped 11 microsatellite markers in two separate DNA pools, and an additional four markers in a third DNA pool, and compared the estimated allele frequencies with those determined by direct genotyping. In addition, we have evaluated whether pooled DNA samples can be used to accurately assess allele frequencies on transmitted and untransmitted chromosomes, in a collection of families for fine-structure gene mapping using allelic association. Our studies show that accurate, quantitative data on allele frequencies, suitable for identifying markers for complex disorders, can be identified from pooled DNA samples. This approach, being independent of the number of samples comprising a pool, promises to drastically reduce the labor and cost of genotyping in the initial identification of disease loci. Additional applications of DNA pooling are discussed. These developments suggest that new statistical methods for analyzing pooled DNA data are required.
Citations
More filters
29 Jan 2015
TL;DR: The current state of the genetic dissection of complex traits is summarized in this paper, which describes the methods, limitations, and recent applications to biological problems, including linkage analysis, allele-sharing methods, association studies, and polygenic analysis of experimental crosses.
Abstract: Medical genetics was revolutionized during the 1980s by the application of genetic mapping to locate the genes responsible for simple Mendelian diseases. Most diseases and traits, however, do not follow simple inheritance patterns. Geneticists have thus begun taking up the even greater challenge of the genetic dissection of complex traits. Four major approaches have been developed: linkage analysis, allele-sharing methods, association studies, and polygenic analysis of experimental crosses. This article synthesizes the current state of the genetic dissection of complex traits—describing the methods, limitations, and recent applications to biological problems.
1,805 citations
TL;DR: With the discovery of massive numbers of genetic markers and the development of better tools for genotyping, association studies will inevitably proliferate and now is the time to consider critically the design of such studies to avoid the mistakes of the past and to maximize their potential to identify new components of disease.
Abstract: Assessing the association between DNA variants and disease has been used widely to identify regions of the genome and candidate genes that contribute to disease. However, there are numerous examples of associations that cannot be replicated, which has led to skepticism about the utility of the approach for common conditions. With the discovery of massive numbers of genetic markers and the development of better tools for genotyping, association studies will inevitably proliferate. Now is the time to consider critically the design of such studies, to avoid the mistakes of the past and to maximize their potential to identify new components of disease.
1,499 citations
TL;DR: Recent developments in quantitative genotyping assays and in the design and analysis of pooling studies are discussed.
Abstract: DNA pooling is a practical way to reduce the cost of large-scale association studies to identify susceptibility loci for common diseases. Pooling allows allele frequencies in groups of individuals to be measured using far fewer PCR reactions and genotyping assays than are used when genotyping individuals. Here, we discuss recent developments in quantitative genotyping assays and in the design and analysis of pooling studies. Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and inter-loci interactions, and that allow the analysis of haplotypes.
582 citations
TL;DR: By providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.
Abstract: We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r2 = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.
439 citations
Cites methods from "Allele Frequency Distributions in P..."
...Pooling of DNA samples has been successfully used with both microsatellite markers and SNPs (Arnheim et al. 1985; Pacek et al. 1993; Syvänen et al. 1993; Kwok et al. 1994; Barcellos et al. 1997; Shaw et al. 1998)....
[...]
TL;DR: A highly parallel method for genotyping single nucleotide polymorphisms (SNPs) using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligon nucleotide tags, which can be used for allele-frequency estimation in pooled DNA samples.
Abstract: Large scale human genetic studies require technologies for generating millions of genotypes with relative ease but also at a reasonable cost and with high accuracy We describe a highly parallel method for genotyping single nucleotide polymorphisms (SNPs), using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligonucleotide tags First, marker-specific primers are used in PCR amplifications of genomic regions containing SNPs Second, the amplification products are used as templates in single base extension (SBE) reactions using chimeric primers with 3' complementarity to the specific SNP loci and 5' complementarity to specific probes, or tags, synthesized on the array The SBE primers, terminating one base before the polymorphic site, are extended in the presence of labeled dideoxy NTPs, using a different label for each of the two SNP alleles, and hybridized to the tag array Third, genotypes are deduced from the fluorescence intensity ratio of the two colors This approach takes advantage of multiplexed sample preparation, hybridization, and analysis at each stage We illustrate and test this method by genotyping 44 individuals for 142 human SNPs identified previously in 62 candidate hypertension genes Because the hybridization results are quantitative, this method can also be used for allele-frequency estimation in pooled DNA samples
400 citations
References
More filters
Book•
01 Feb 1987
TL;DR: Recent developments of statistical methods in molecular phylogenetics are reviewed and it is shown that the mathematical foundations of these methods are not well established, but computer simulations and empirical data indicate that currently used methods produce reasonably good phylogenetic trees when a sufficiently large number of nucleotides or amino acids are used.
Abstract: Recent developments of statistical methods in molecular phylogenetics are reviewed. It is shown that the mathematical foundations of these methods are not well established, but computer simulations and empirical data indicate that currently used methods such as neighbor joining, minimum evolution, likelihood, and parsimony methods produce reasonably good phylogenetic trees when a sufficiently large number of nucleotides or amino acids are used. However, when the rate of evolution varies exlensively from branch to branch, many methods may fail to recover the true topology. Solid statistical tests for examining'the accuracy of trees obtained by neighborjoining, minimum evolution, and least-squares method are available, but the methods for likelihood and parsimony trees are yet to be refined. Parsimony, likelihood, and distance methods can all be used for inferring amino acid sequences of the proteins of ancestral organisms that have become extinct.
15,840 citations
TL;DR: The identification of the genetic basis of complex human diseases such as schizophrenia and diabetes has proven difficult as mentioned in this paper, and Risch and Merikangas proposed that they can best accomplish this goal by combining the power of the human genome project with association studies.
Abstract: The identification of the genetic basis of complex human diseases such as schizophrenia and diabetes has proven difficult. In their Perspective, Risch and Merikangas propose that we can best accomplish this goal by combining the power of the human genome project with association studies, a method for determining the basis of a genetic disease.
5,143 citations
TL;DR: Bulk segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome and will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.
Abstract: We developed bulked segregant analysis as a method for rapidly identifying markers linked to any specific gene or genomic region. Two bulked DNA samples are generated from a segregating population from a single cross. Each pool, or bulk, contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. The two bulks can be made for any genomic region and from any segregating population. The bulks are screened for differences using restriction fragment length polymorphism probes or random amplified polymorphic DNA primers. We have used bulked segregant analysis to identify three random amplified polymorphic DNA markers in lettuce linked to a gene for resistance to downy mildew. We showed that markers can be reliably identified in a 25-centimorgan window on either side of the targeted locus. Bulked segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome. Genetic walking will be possible by multiple rounds of bulked segregation analysis; each new pair of bulks will differ at a locus identified in the previous round of analysis. This approach will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.
4,492 citations
Journal Article•
TL;DR: The statistical basis for this "transmission test for linkage disequilibrium" (transmission/disequilibrium test] is described and the relationship of this test to tests of cosegregation that are based on the proportion of haplotypes or genes identical by descent in affected sibs is shown.
Abstract: A population association has consistently been observed between insulin-dependent diabetes mellitus (IDDM) and the "class 1" alleles of the region of tandem-repeat DNA (5' flanking polymorphism [5'FP]) adjacent to the insulin gene on chromosome 11p. This finding suggests that the insulin gene region contains a gene or genes contributing to IDDM susceptibility. However, several studies that have sought to show linkage with IDDM by testing for cosegregation in affected sib pairs have failed to find evidence for linkage. As means for identifying genes for complex diseases, both the association and the affected-sib-pairs approaches have limitations. It is well known that population association between a disease and a genetic marker can arise as an artifact of population structure, even in the absence of linkage. On the other hand, linkage studies with modest numbers of affected sib pairs may fail to detect linkage, especially if there is linkage heterogeneity. We consider an alternative method to test for linkage with a genetic marker when population association has been found. Using data from families with at least one affected child, we evaluate the transmission of the associated marker allele from a heterozygous parent to an affected offspring. This approach has been used by several investigators, but the statistical properties of the method as a test for linkage have not been investigated. In the present paper we describe the statistical basis for this "transmission test for linkage disequilibrium" (transmission/disequilibrium test [TDT]). We then show the relationship of this test to tests of cosegregation that are based on the proportion of haplotypes or genes identical by descent in affected sibs. The TDT provides strong evidence for linkage between the 5'FP and susceptibility to IDDM. The conclusions from this analysis apply in general to the study of disease associations, where genetic markers are usually closely linked to candidate genes. When a disease is found to be associated with such a marker, the TDT may detect linkage even when haplotype-sharing tests do not.
3,791 citations
TL;DR: This article synthesizes the current state of the genetic dissection of complex traits--describing the methods, limitations, and recent applications to biological problems.
Abstract: Medical genetics was revolutionized during the 1980s by the application of genetic mapping to locate the genes responsible for simple Mendelian diseases. Most diseases and traits, however, do not follow simple inheritance patterns. Genetics have thus begun taking up the even greater challenge of the genetic dissection of complex traits. Four major approaches have been developed: linkage analysis, allele-sharing methods, association studies, and polygenic analysis of experimental crosses. This article synthesizes the current state of the genetic dissection of complex traits--describing the methods, limitations, and recent applications to biological problems.
3,216 citations