Replication of genome-wide association studies (GWAS) loci for fasting plasma glucose in African-Americans.

AIMS/HYPOTHESIS: Chronically elevated blood glucose (hyperglycaemia) is the primary indicator of type 2 diabetes, which has a prevalence that varies considerably by ethnicity in the USA, with African-Americans disproportionately affected. Genome-wide association studies (GWASs) have significantly enhanced our understanding of the genetic basis of diabetes and related traits, including fasting plasma glucose (FPG). However, the majority of GWASs have been conducted in populations of European ancestry. Thus, it is important to conduct replication analyses in populations with non-European ancestry to identify shared loci associated with FPG across populations. METHODS: We used data collected from non-diabetic unrelated African-American individuals (n = 927) who participated in the Howard University Family Study to attempt to replicate previously published GWASs of FPG. Of the 29 single nucleotide polymorphisms (SNPs) previously reported, we directly tested 20 in this study. In addition to the direct test, we queried a 500 kb window centred on all 29 reported SNPs for local replication of additional markers in linkage disequilibrium (LD). RESULTS: Using direct SNP and LD-based comparisons, we replicated multiple SNPs previously associated with FPG and strongly associated with type 2 diabetes in populations with European ancestry. The replicated SNPs included those in or near TCF7L2, SLC30A8, G6PC2, MTNR1B, DGKB-TMEM195 and GCKR. We also replicated additional variants in LD with the reported SNPs in ZMAT4 and adjacent to IRS1. CONCLUSIONS/INTERPRETATION: We identified multiple GWAS variants for FPG in our cohort of African-Americans. Using an LD-based strategy we also identified SNPs not previously reported, demonstrating the utility of using diverse populations for replication analysis.

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2.

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.

Universal DNA microarray method for multiplex detection of low abundance point mutations.

Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.