Abstract
When susceptibility to diseases is caused by cis-effects of multiple alleles at adjacent polymorphic sites, it may be difficult to assess with confidence the genetic phase and identify individuals carrying the risk haplotype. Experimental assessment of genetic phase is still challenging and most population studies use statistical approaches to infer haplotypes given the observed genotypes. While these statistical approaches are powerful and have been proven very useful in large scale genetic population studies, they may be prone to errors in studies with small sample size, especially in the presence of compound heterozygotes. Here, we describe a simple and novel approach using the popular PCR–RFLP based strategy to assess the genetic phase in compound heterozygotes. We apply this method to two extensively studied SNPs in two clustered immune-related genes: The −308 (G > A) and the +252 (A > G) SNPs of the tumor necrosis factor (TNF) alpha and the lymphotoxin alpha (LTA) genes, respectively. Using this method, we successfully determined the genetic phase of these two SNPs in known compound heterozygous individuals and in every sample tested. We show that the A allele of TNF −308 is carried on the same chromosome as the LTA +252(G) allele.
Highlights
SNPs are useful in mapping disease susceptibility genes
The SNPs identified from typical genetic mapping studies often are not the causal SNPs but may tag the true causal mutation if they are in linkage disequilibrium with each other (Ardlie et al, 2002; Gabriel et al, 2002; Ke et al, 2004)
We present here a simple application of the popular PCR–RFLP based strategy to determine the genetic phase at two adjacent SNPs in compound heterozygous individuals
Summary
SNPs are useful in mapping disease susceptibility genes. the SNPs identified from typical genetic mapping studies often are not the causal SNPs but may tag the true causal mutation if they are in linkage disequilibrium with each other (Ardlie et al, 2002; Gabriel et al, 2002; Ke et al, 2004). Several experimental methods for haplotype and genetic phase determination have been reported, including cloning (Burgtorf et al, 2003; Kitzman et al, 2011), hybridization (Yan et al, 2000; Douglas et al, 2001; Boldt and Petzl-Erler, 2002), polony technology (Mitra et al, 2003), MALDI-TOF mass spectrometry (Tost et al, 2002), sequencing (Levy et al, 2007), and chromosome micro-dissection (Ma et al, 2010) These methods are labor-intensive and/or require specialized and expensive equipment. All of the above methods may not be applicable in settings where cost is a premium and specialized equipment is not available or personnel have little or no training in labor-intensive methodologies
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