HLA-G whole gene amplification reveals linkage disequilibrium between the HLA-G 3'UTR and coding sequence

Abstract

Polymorphic sites in the HLA-G gene may influence expression and function of the protein. Knowledge of the association between high-resolution HLA-G alleles and 3-prime untranslated (3'UTR) haplotypes is useful for studies on the role of HLA-G in transplantation, pregnancy, and cancer. We developed a next generation sequencing (NGS)-based typing assay enabling full phasing over the whole HLA-G gene sequence with inclusion of the 3'UTR region. DNA from 171 mother-child pairs (342 samples) was studied for: (a) HLA-G allele information by the NGSgo-AmpX HLA-G assay, (b) 3'UTR haplotype information by an in-house developed sequence-based typing method of a 699/713 base pair region in the 3'UTR, and (c) the full phase HLA-G gene sequence, by combining primers from both assays. The mother to child inheritance allowed internal verification of newly identified alleles and of association between coding and UTR regions. The NGSgo workflow compatible with Illumina platforms was employed. Data was interpreted using NGSengine software. In 99.4% of all alleles analyzed, the extended typing was consistent with the separate allele and 3'UTR typing methods. After repeated analysis of four samples that showed discrepancy, consistency reached 100%. A high-linkage disequilibrium between IPD-IMGT/HLA Database-defined HLA-G alleles and the extended 3'UTR region was identified (D' = 0.994, P < .0001). Strong associations were found particularly between HLA-G*01:04 and UTR-3, between HLA-G*01:01:03 and UTR-7, and between HLA-G*01:03:01 and UTR-5 (for all: r = 1). Six novel HLA-G alleles and three novel 3'UTR haplotype variants were identified, of which three and one, respectively, were verified in the offspring.

Keywords: 3′-UTR; HLA-G; allele; haplotype; next generation sequencing.

FIGURE 1

Full phasing over the whole HLA‐G gene sequence by extended next generation sequencing. HLA‐G allele typing was acquired by an NGSgo‐AmpX assay on an amplicon that starts at −57 and ends at +2727. A separate, in‐house developed sequencing assay targeted the 3′UTR region and generated an amplicon between +2940 and +3559. For a combined strategy of simultaneous assessment of the HLA‐G allele and 3′UTR haplotype, the forward primer (starting at −57) from the NGSgo‐AmpX assay was combined with the reverse primer (at 3559) from the in‐house developed sequencing assay