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Using sequence-based typing, we have identified a novel human leukocyte antigen (HLA)-A*30 allele, HLA-A*3014L, with a low expression pattern. The sequence of HLA-A*3014L is identical to that of HLA-A*3001 except for a G to C substitution in exon 3 at nucleotide position 563, resulting in an amino acid difference at position 164 (Cys to Ser). Due to the cysteine substitution, a disulfide bridge in the alpha2 domain of the HLA class I heavy chain cannot be formed. By using the standard microlymphocytotoxicity test, the HLA-A30 antigen cannot be detected. By flow cytometric analysis of the cell-surface expression at either 37 degrees C or 30 degrees C, a temperature-sensitive expression pattern of the HLA-A*3014L antigen was observed. Only by incubating the cells at 30 degrees C, which increases the stability of HLA class I heavy chains, was a weak but clearly detectable HLA-A*3014L expression found. The mRNA expression level of the HLA-A*3014L allele was not affected by the nucleotide substitution. The intrachain disulfide bond formation in the alpha2 domain is essential for the normal expression of the HLA molecules. Reduced protein expression is probably caused by incorrect HLA class I heavy chain folding and HLA class I complex assembly.

Due to the extensive polymorphism of the major histocompatibility complex (MHC), only limited progress has been made in high-resolution MHC sequencing, one of the initial requirements to elucidate the molecular immunopathology of several hundred MHC-associated diseases. We present an approach for sequencing the entire MHC with ≈ 1200 specific capture primers. Using target sites known to be highly conserved, Region-Specific Extraction (RSE, Generation Biotech) was used to target MHC segments of up to 40 kb length and enrich the contiguous 4.4 Mb MHC for sequencing. RSE uses a single primer extension-step that attaches a specific “handle” to genomic DNA target segments and firmly locks the selected DNA onto magnetic beads for capture. The captured segments extend far into both directions from any single capture point. This makes an RSE capture primer set essentially immune to changes in variable sections and avoids difficulties caused by genomic duplications, pseudo-genes, gene homologues and new sequence insertions. After RSE, unique oligonucleotide indexes were ligated to each DNA sample for parallel, paired-end 2 × 250 bp sequencing on a HiSeq2500 (Illumina). Read alignment (hg19 reference genome), local re-alignment, variant calling and annotation were performed by an in-house data analysis pipeline consisting of the CLC Genomics Workbench (Qiagen) and custom developed scripts. In our experiments, an up to 80-fold target enrichment was achieved, which provides an average coverage of about 200 across the 4.4 Mb target region. In samples with high coverage, > 90% of the target region can uniformly be sequenced with > 20× coverage. We expect that optimized target enrichment enables to sequence at least 16 samples per flow-cell with appropriate coverage for > 90% of the MHC region while maintaining the advantages of RSE in avoiding variation-dependent capture and allelic dropout. The combination of the RSE enrichment and massively parallel sequencing on HiSeq is a feasible approach for whole MHC sequencing. Further improvements in target enrichment efficiency and long-read/high-accuracy sequencing can turn it into a cost-effective and user-friendly tool in many clinical and research settings such as transplantation medicine, inflammatory or autoimmune disorders, pharmacogenomics, and many more. H. Rabenstein: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. B. Bangol: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. S. Linke: Employee; Company/Organization; Generation Biotech. R. Brumm: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. S. Poggel: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. S. Eck: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. K. Ott: Employee; Company/Organization; Generation Biotech. J. Dapprich: Employee; Company/Organization; Generation Biotech. K. Hirv: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics.