Experimental generation of SNP haplotype signatures in patients with sickle cell anaemia.

backgroundSickle cell anemia is caused by a single type of mutation, a homozygous A→T substitution in the ß globin gene. Clinical severity is diverse, partially due to additional, disease-modifying genetic factors. We are studying one such modifier locus, HMIP (HBS1L-MYB intergenic polymorphism, chromosome 6q23.3). Working with a genetically admixed patient population, we have encountered the necessity to generate haplotype signatures of genetic markers to label genomic fragments with distinct genealogical origin at this locus. With the goal to generate haplotype signatures from patients experimentally, we have investigated the suitability of an existing nanofluidic assay platform to perform phase alignment with single-nucleotide polymorphism alleles. METHODOLOGY/PRINCIPAL

findingsPatient DNA samples were loaded onto Fluidigm Digital Arrays and individual DNA molecules were assayed with allele-specific probes for SNP markers. Here we present data showing the utility of the nanofluidic approach, yielding haplotype data identical to those obtained with a family-based method. We then determined haplotype composition in a group of patients with sickle cell disease, including in those where a mathematical inference approach gave ambiguous or misleading results. Experimental phasing of genotypes across 3.8 kb for rs9399137, rs9402685, and rs11759553 created unequivocal haplotype signatures for each of the patients. In 68 patients, we found 8 copies of a haplotype signature ('C-C-T'), which is known to be prevalent in Europeans but to be absent in West African populations. We have confirmed the identity of our phased allele pairs by single-molecule sequencing and have demonstrated, in principle, that three-allele phasing (using three colors) is a potential extension to this method. CONCLUSIONS/SIGNIFICANCE: Phased haplotypes yield more information than the individual marker genotypes. Procedures such as the one described here would therefore benefit genetic mapping and functional studies as well as diagnostic procedures where the identity or parental origin of short genetic fragments is of importance.