A population-wide applicable HLA-DQ2 and DQ8 genotyping using DNA from dried blood spots and duplex allele-specific qPCR amplification

• Published in: Scandinavian journal of clinical and laboratory investigation 2016-10, Vol.76 (7), p.581-587
• Main Authors: Aguayo-Patrón, Sandra, Beltrán-Sauceda, Lizbeth, Calderón de la Barca, Ana María
• Format: Article
• Language: English
• Subjects: Alleles; Celiac disease; Celiac Disease - diagnosis; Celiac Disease - genetics; Celiac Disease - immunology; Child; Child, Preschool; Diabetes Mellitus, Type 1 - diagnosis; Diabetes Mellitus, Type 1 - genetics; Diabetes Mellitus, Type 1 - immunology; DNA - genetics; DNA Primers - chemistry; Dried Blood Spot Testing - methods; dried blood spots; duplex reactions; Female; Genetic Testing; Genotyping Techniques - methods; Haplotypes; HLA-DQ Antigens - classification; HLA-DQ Antigens - genetics; HLA-DQ2/DQ8; Humans; Male; Multiplex Polymerase Chain Reaction - methods; Organic Chemicals - chemistry; qPCR; Real-Time Polymerase Chain Reaction - methods; SYBR Green; type 1 diabetes
• ISSN: 0036-5513; 1502-7686
• DOI: 10.1080/00365513.2016.1230773

Genotyping of HLA-DQ2 and DQ8 haplotypes is important for diagnosis or for screening of early risk detection of celiac disease or type 1 diabetes. Usually, venous blood DNA extraction and expensive and time consuming amplification are used, that hinder population-wide studies. We assayed a friendly HLA-DQ2 and DQ8 genotyping procedure using a combination of DNA from dried blood spot (DBS) and duplex allele-specific qPCR amplification using SYBR Green. DNA was extracted using home-made buffers and compared to an extraction commercial kit. Duplex reactions by qPCR were designed using each Tm allele amplicon for reference samples (positive HLA-DQ2 or DQ8) with allele-specific primers. DBS samples from 558 children (7.99 ± 2.47 y) were collected. The DNA final yield obtained by the home-made extractive procedure was higher than from the commercial kit (1.11 ± 0.56 vs 0.23 ± 0.14 μg), while the quality was similar for both DNA samples. There was concordance in the amplification profiles for DNA samples obtained with both methods. All of four alleles from DQ2 and DQ8 haplotypes were accurately identified in duplex reactions. By using DBS samples and DNA extraction home-made procedure, the costs were reduced by 60%. The whole procedure is cost-effective for HLA-DQ2 and DQ8 genotyping.