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Aim Strong association of HLA molecules with autoimmune and inflammatory diseases have been identified, strengthening the value of HLA genetic screening for diagnostic purposes. There’s a strong association between Ankylosing Spondylitis (AS) and HLA-B27. In ∼90% of European patients, B27 alleles strongly predispose for AS. In ∼90% of patients with Coeliac Disease, the HLA-DQ2.5 phenotype is expressed, encoding HLA-DQA1∗05:01 and DQB1∗02:01 alleles, with the remaining 10% mostly expressing the HLA-DQ8 molecule, encoding the DQA1∗03 variant and DQB1∗03:02 alleles. Also in the field of pharmacogenetics there’s a growing interest in the role of HLA. HLA-B∗57:01 screening to prevent Abacavir hypersensitivity syndrome is now a routine clinical use in the developed world. We developed a novel NGS-based genetic screening test that enables 2nd field resolution typings of HLA-B, DQA1, and DQB1 in a single tube. Methods A multiplexed amplification assay was established that encompass HLA-B, HLA-DQA1 and HLA-DQB1 locus-specific amplification of exon 2 and 3 in a single tube. The assay was compatible with the multiplex PCR kit (Qiagen) and required a PCR enhancer (GenDx). Analytical performance studies were assessed including screening of a large gDNA reference panel (n = 96 samples) to verify robustness. Amplicons were pooled and processed into the NGSgo workflow for Illumina (GenDx) and analyzed by NGSengine software (GenDx). Results For all samples tested (n = 96) strong amplicons of the expected sizes were generated in a multiplexed PCR. NGS data showed high locus mappability (>94%), and full coverage of the amplicon with even distribution of reads, especially for HLA-B, and HLA-DQA1, having average read depths >1500. For all samples, HLA-B, HLA-DQB1 and HLA-DQA1 typings were obtained that are in concordance with the pre-types. The multiplexed assay showed to be robust and capable of detecting both alleles when present in a balanced manner. Conclusions We developed a novel NGS-based genetic screening test that enables HLA-B, DQA1, and DQB1 typings in a single tube. The genetic screening test proved to be robust and useful for pharmacogenetic screening of HLA-B alleles and identification of autoimmune disease associated genetic susceptibilities like celiac disease. N. Westerink: Employee; Company/Organization; GenDx.I. van Rooy: Employee; Company/Organization; Gendx. M. Van Heck: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. |
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