Long-range multilocus haplotype phasing of the MHC

Abstract

Haplotypes are a powerful tool for identifying the genetic basis of common complex diseases. Disease-association mapping requires molecular methods for haplotyping biallelic SNP variation and highly complex polymorphisms. We developed a method for phasing HLA-A, HLA-B, and HLA-DRB1 alleles on chromosome 6 in unrelated individuals. This method uses the highly polymorphic HLA-B locus to discriminate the two HLA haplotypes in heterozygous individuals and its ideal location 1.4 Mbp telomeric to HLA-DRB1 and 1.2 Mbp centromeric to HLA-A to capture 2-Mbp-long genomic DNA. Genomic DNA representing a single HLA-B-captured haplotype is genotyped for HLA-A and HLA-DRB1 alleles and linkage to HLA-B is established. Proof of principle was established in a large blinded study of phase-known samples. Availability of an efficient method for MHC haplotype phase determination will facilitate the mapping of causative MHC-resident genes in many human diseases and has the potential to be broadened to other polymorphic gene complexes.

Fig. 1.

Procedure for haplotyping 2-Mbp-long human genomic DNA. (a) Genomic DNA was hybridized with two HLA-B oligonucleotide probes immobilized on glass surface. (b) Excess genomic DNA was removed from surface through stringent washes, retaining template DNA with an HLA-B sequence complementary to the probe. (c) Captured genomic DNA fragments were collected into separate tubes, each containing one haplotype. (d) HLA-A and HLA-DRB genes were PCR-amplified and typed by using oligonucleotide arrays. HLA-A and HLA-DRB1 alleles were assigned by evaluating the relative signal intensities of quadruplicate probe hybridizations derived from the quantarray software program. For illustration purposes, hybridization images and relative intensities for probes A1–A3 for HLA-A and 188A–188E for HLA-DRB1 are shown (complete probes are listed in Tables 4 and 5).