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

Abstract

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.

New insights in HLA‐E polymorphism by refined analysis of the full‐length gene

“…The PI of the total coding and non‐coding region was comparable (0.027 vs 0.024). Compared with the coding region of classical HLA class I genes, with a PI of 0.65 for HLA‐A, 0.62 for HLA‐B and 0.57 for HLA‐C, the HLA‐E polymorphism is still limited.…”

A new HLA‐C allele with an alternative splice site in exon 3: HLA‐C*03:23N

“…To sequence C*03:23N specifically, PCR amplification targeting the HLA‐C locus was performed. Each resultant PCR product was cloned, and then each nucleotide sequence was determined by Sanger sequencing . The resulting electropherogram of C*03:23N showed the expected alternative acceptor splice site and the resultant deletion of 64 nucleotides in exon 3 as compared with the sequence of C*03:04:01:02 (Figure ).…”

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

“…One transcript is longer and leads to the inclusion of an additional 52 amino acids, the other transcript is shorter and introduces a premature stop codon, both transcripts are not able to form a functional HLA class II molecule and thus causes a null expression . RNA sequencing will not only be able to identify full‐length polymorphism, but also has the ability to detect alternatively spliced mRNA variants as has been shown in our RNA‐SBT HLA class I method . Furthermore, we used our RNA‐SBT method with especially designed HLA‐DRB4‐specific amplification primers to identify the presence of alternatively spliced products in an individual positive for DRB4*01:03:01:02N (data not shown), thus confirming the suitability of this method for detection of aberrantly spliced mRNA products.…”

“…combined with the DRB-locus-specific primers in the 5 ′ and 3 ′ UTR in the same way as the group-specific primers described in Table 3, resulting in the full-length allele-specific nucleotide sequence of the HLA-DRB1*03:07 allele. PCR amplification was performed according to previously described SBT protocols (10,15). In detail, amplification was carried out in a volume of 30 μl, consisting of 67 mM Tris-HCl (pH 8.8) (Merck, Darmstadt, Germany), 16.6 mM ammonium sulfate (Merck), 0.01% Tween 20 (Merck), 1.5 mM MgCl 2 (Life Technologies), 0.2 mM of each dNTP (GE Healthcare, Diegem, Belgium), 0.1 μg/μl cresol red (Sigma-Aldrich, St. Louis, MO), 5% glycerol (Alfa Aesar, Karlsruhe, Germany), 15 pmol of each primer (Sigma-Aldrich), 1.4U expand high fidelity enzyme mix (Roche, Basel, Switzerland) and 3 μl of sample cDNA.…”