Comparison of HLA DQA1 and DQB1 Genes by Multiplex Ligation-Dependent Probe Amplification and Next Generation Sequencing Technologies for Celiac Disease Risk Determination

Abstract

Introduction: Celiac disease is a systemic autoimmune disease with chronic gastrointestinal disorder caused by intolerance to gluten proteins. The most dominant genetic risk factors are the specific genotypes encoding the Human Leukocyte Antigen (HLA) class II molecule genes, DQA1 and DQB1. The DQ2.5 variant (DQA1*05/DQB1*02 alleles) is present in 90-95% of patients with celiac disease. The DQ8 variant (DQA1*03/DQB1*0302) and DQ2.2 variant (DQA1*02/DQB1*02) are found in most of the remaining patients. A positive test is indicative of genetic susceptibility but does not necessarily mean disease development. A negative test has a more significant value because gluten intolerance rarely occurs in the absence of specific HLA predisposing alleles. In the current study we compared Multiplex Ligation-dependent Probe Amplification (MLPA) and Next Generation Sequencing (NGS) technologies for identifying Celiac disease risk HLA alleles. Methods: DNA was extracted from either saliva or whole-blood archival samples obtained from commercial vendors using Qiagen DNA mini extraction kits. MLPA assay probe mix designed to detect the HLA alleles implicated in Celiac disease consists of 29 specific fluorescent labeled amplification products. The PCR products are separated by capillary gel electrophoresis on the ABI 3500 Genetic Analyzer yielding a specific electropherogram. Based on the presence and relative height of individual probe, as compared to the relative probe peak height in various reference samples, the HLA allele in the patient sample was determined. For Next Generation sequencing assay, long range PCR products of HLA DQA1 and DQB1 genes were pooled to prepare bar coded NGS libraries. Equimolar pools of the barcoded libraries were sequenced on the Illumina MiSeq platform by paired end 151 bp sequencing. Demultiplexed FASTQ files were aligned to HLA variant database and genotypes were identified. Results: A total of 26 samples with different HLA variants were analyzed by both MLPA and NGS assay panel. The samples included DQ2.5, DQ8, DQ2.2, both heterozygous and homozygous variants, negatives and positive control cell line DNA samples. Data is summarized in Table 1. There was 100% concordance between the two technologies in determining the Celiac disease risk alleles. DNA dilution studies showed that both technologies were optimal with a minimum of 40 ng of DNA input. The workflow for MLPA was simple with sample to data generation completed within 24 hours. NGS assay was laborious with highly technical library preparation, sequencing and data analysis with sample to data in 48 hours. However, NGS technology results in a high resolution genotyping whereas MLPA assay is limited to 5 alleles of DQA1 and DQB1 genes.Table: Table. HLA DQA1 and DQB1 genotyping data summary- MLPA and NGS dataConclusion: Both methods are reliable and efficient to provide necessary information for celiac disease risk evaluation. In the future if high resolution HLA genotyping is needed, NGS may be more applicable.