A rapid sample method for HLA haplotype typization. A preliminary study on celiac patients

The aim of the present work was to determine the human leukocyte (HLA) haplotype in 64 Sardinian patients affected with celiac disease, using a rapid and easy to apply sampling method that permits samples from blood drawing to be stored more easily. Numerous studies have demonstrated how the HLA system plays a very important role in immune system regulation, determining a link between this gene and a high number of pathologies including celiac disease. In fact a genetic susceptibility exists in celiac sprue, linked to HLA-DQB1*0201 and -DQB1*0302 genes which represent sierologic groups -DQ2 and -DQ8 whose early identification could be fundamental in obtaining a diagnosis of celiac disease.

To realize this study a collecting method of samples was developed through the brushing of oral mucosa, which is extremely less traumatic than the classic sampling method using blood drawing, and which also allows a long conservation period before sample analysis. Samples were later analyzed with Van Embden's DNA extraction method to extract the patient's DNA, on which we executed the Polymerase Chain Reaction (PCR). To obtain the HLA haplotype from each patient we used 8 specific primers that amplified the HLA-DQB1 allele in low-resolution.

Out of the 64 patients we found 26 HLA-DQB1*02 homozygotes, 28 HLA-DQB1*02 heterozygotes and 10 negative samples for the HLA-DQB1*02 allele, thus confirming what had emerged from previous blood draws.

These results show how the method we developed using oral brushing is a sure method to obtain samples for determining the HLA haplotype in extra-hospital areas. This could allow the use of this method to obtain early diagnosis for chronic pathologies linked to the HLA groups and for recognizing this genotype in extensive population studies.