P080 HLA disease association assignment by multiplexed NGS assay

Abstract

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. 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). 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. 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.