Haplotyping the human leukocyte antigen system from single chromosomes

Nicholas M Murphy,Don Cooper,Matthew Burton,Helen R Irving,David R Powell,Abha Chopra,David C Sayer,Fernando J Rossello,Lavinia Gordon,Ming Je Hsieh,Mark D Pertile,Colin W Pouton,Brian D Tait

doi:10.1038/srep30381

Abstract

We describe a method for determining the parental HLA haplotypes of a single individual without recourse to conventional segregation genetics. Blood samples were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 amplification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function.

Highlights

Genetic haplotyping and genome phasing are increasingly prominent components of both sequencing technologies and clinical genotyping/phenotyping
It is not feasible to establish which donors are haplotype-matched with a particular patient, current recruitment and human leukocyte antigen (HLA) typing of donors on to haematopoietic stem cell transplantation (HSCT) registries does not include definition of HLA haplotypes
We haplotyped the most relevant HLA alleles by single chromosome isolation using bivariate flow cytometry followed by whole chromosome amplification

Summary

Introduction

Genetic haplotyping and genome phasing are increasingly prominent components of both sequencing technologies and clinical genotyping/phenotyping. We sought to adapt published methods using flow cytometry to sort chromosomes in suspension This allowed us to acquire single chromosomes and to rapidly determine the haplotype phase of genes which are of critical importance to immune function in bone-marrow transplantation[1]. Located on the short arm of human chromosome 6, the major histocompatibility complex (MHC) has been extensively haplotype sequenced due to its relevance to transplant matching and disease diagnosis[2]. The highly polymorphic human leukocyte antigen (HLA) system genes are central to the recognition of self from non-self. For this reason, the matching of HLA loci sequences is a cornerstone of donor/recipient matching in organ and haematopoietic stem cell transplantation (HSCT)[3]. Due to the critical need of haplotyping in HSCT, we chose to exemplify the method for this manuscript using chromosome 6 (Fig. 1)

Methods

Results

Conclusion