The role of gene polymorphism in HLA class I splicing.

Among the large number of human leucocyte antigen (HLA) alleles, only a few have been identified with a nucleotide polymorphism impairing correct splicing. Those alleles show aberrant expression levels, due to either a direct effect of the polymorphism on the normal splice site or to the creation of an alternative splice site. Furthermore, in several studies, the presence of alternatively spliced HLA transcripts co-expressed with the mature spliced transcripts was reported. We evaluated the splice site sequences of all known HLA class I alleles and found that, beside the consensus GT and AG sequences at the intron borders, there were some other highly conserved nucleotides for the different class I genes. In this review, we summarize the splicing mechanism and evaluate what is known today about alternative splicing of HLA class I genes.

Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

RNA and protein expression of HLA-A(∗)23:19Q.

The assignment of null alleles is clinically relevant in stem cell transplantation, in particular for donor selection. It is unclear how questionable (Q) alleles, having an unknown expression profile, should be considered in matching criteria. In this study we analyzed the RNA and protein expression profile of a questionable allele encountered in a sample of the Guadeloupe population: GD23Q, HLA-A(∗)23:19Q, 29:02:01. Full-length DNA sequencing of HLA-A(∗)23:19Q revealed a single polymorphism at position 619 (G>A) compared to HLA-A(∗)23:01:01. Serological typing showed only the presence of HLA-A29; HLA-A(∗)23:19Q was not detected on the cell surface. The absence of HLA-A(∗)23:19Q surface expression was shown by flow cytometry using a directly labeled monoclonal antibody and a panel of five indirectly labeled polyclonal antibodies all directed against HLA-A23 (HLA-A9) molecules. Allele specific amplification revealed the absence of intact full-length mRNA, but the presence of two major alternatively spliced mRNAs: sequencing identified that in one variant exon 3 is missing and in the other variant introns 2 and 3 are retained. Based upon the lack of HLA-A(∗)23:19Q surface expression and the presence of aberrant mRNA transcripts only, this study shows that HLA-A(∗)23:19Q is non-expressed.

An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles.