PPC: an algorithm for accurate estimation of SNP allele frequencies in small equimolar pools of DNA using data from high density microarrays

Robust estimation of allele frequencies in pools of DNA has the potential to reduce genotyping costs and/or increase the number of individuals contributing to a study where hundreds of thousands of genetic markers need to be genotyped in very large populations sample sets, such as genome wide association studies. In order to make accurate allele frequency estimations from pooled samples a correction for unequal allele representation must be applied. We have developed the polynomial based probe specific correction (PPC) which is a novel correction algorithm for accurate estimation of allele frequencies in data from high-density microarrays. This algorithm was validated through comparison of allele frequencies from a set of 10 individually genotyped DNA's and frequencies estimated from pools of these 10 DNAs using GeneChip 10K Mapping Xba 131 arrays. Our results demonstrate that when using the PPC to correct for allelic biases the accuracy of the allele frequency estimates increases dramatically

Rates and Patterns of Mutation in Microsatellite DNA

Sequence comparisons of orthologous microsatellite loci in cattle and sheep revealed that the substitution rate in microsatellite flanking sequences does not differ from the rate in presumably neutrally evolving intron sequences. This suggests that microsatellites are generally located in regions that are not subjected to selection. Interestingly, a propensity for substitutions to occur in the border region between flanking and repeat sequence was found. Pedigree analysis of large numbers of barn swallows revealed extremely high mutation frequencies for the tetranucleotide HrU6 and pentanucleotide HrU10 repeat loci. A detailed analysis showed that both the rate and the pattern of mutation differed significantly between the two loci. Further analysis of HrU6 and HrU10 mutations, as well as mutation data for another hypermutable locus (HrU9) in barn swallows, revealed that mutations were more likely to arise in some families than others. This was partly, but probably not only, due to an effect of allele length on mutation rate. The mutation rate was found to vary between colonies of breeding birds, but, overall, not between two different populations. Single molecule genotyping of DNA prepared from human sperm cells was used to detect mutations at the tetranucleotide repeat D21S1245. A tenfold difference in mutation rate between alleles was found. Three phylogenetically distinct allele lineages could be defined, which differed significantly in mutation rate. Unexpectedly, the mutation rate was not found to increase with male age. Microsatellites are commonly applied in a wide range of genetic contexts including linkage mapping, forensic science and population genetics. Obtaining a detailed picture of the evolution of these tandem repeats is important in order to fully understand how to interpret microsatellite data. In addition, studies of the mechanisms underlying microsatellite mutation will provide insights in the processes that shape the eukaryotic genome. This thesis demonstrates that microsatellite evolution is a highly heterogeneous process that is dependent on more factors than was previously thought. As the rate and pattern may vary between loci, caution must therefore be taken when building models to handle microsatellite data

Identification of a prostate cancer susceptibility gene on chromosome 5p13q12 associated with risk of both familial and sporadic disease

Genetic heterogeneity is a difficulty frequently encountered in the search for genes conferring susceptibility to prostate cancer. To circumvent this issue, we selected a large prostate cancer pedigree for genome-wide linkage analysis from a population that is genetically homogeneous. Selected cases and first-degree relatives were genotyped with Affymetrix 10K SNP arrays, identifying a 14 Mb haplotype on chromosome 5 (5p13–q12) inherited identical-by-descent (IBD) by multiple cases. Microsatellite genotyping of additional deceased case samples confirmed that a total of eight cases inherited the common haplotype (P=0.0017). Re-sequencing of eight prioritised candidate genes in the region in six selected individuals identified 15 SNPs segregating with the IBD haplotype, located within the ITGA2 gene. Three of these polymorphisms were selected for genotyping in an independent Tasmanian data set comprising 127 cases with familial prostate cancer, 412 sporadic cases and 319 unaffected controls. Two were associated with prostate cancer risk: rs3212649 (OR=1.67 (1.07–2.6), P=0.0009) and rs1126643 (OR=1.52 (1.01–2.28), P=0.0088). Significant association was observed in both familial and sporadic prostate cancer. Although the functional SNP remains to be identified, considerable circumstantial evidence, provided by in vivo and in vitro studies, supports a role for ITGA2 in tumour development