| Description |
Celiac disease (CD) is an immunologically mediated inflamatory disorder of the small intestine. CD is induced by dietary exposure to the gluten proteins found in wheat, rye and barley. This disorder affects as many as 1 in 300 people of European descent, in whom it is associated with, the HLA-DQA1*0501 - DQB1*0201 alleles. Although the biochemical mechanism of the HLA contribution to the pathogenesis of CD is not fully understood, the linkage of this locus to CD is established. This thesis describes my research of two hypotheses: 1) HLA typing on an automated DNA sequencing machine can provide typing that is as reliable as typing performed by traditional methods, and 2) genes other than HLA exist that contribute to predisposition to CD. To test hypothesis 1, I developed a computerized HLA typing strategy that is a modification of the sequence specific primer method. Primers were designed to preferentially amplify DNA fragments of the generic allelic groups of the DQAl and DQBl loci. Only three PCR reactions are required for low resolution typing of DQAl and DQBl. Use of different labeled primers enabled genotyping for both loci in a single gel lane or capillary tube. Automated allele assignments were determined using computer software, based on DNA migration distance through a polyacrylamide gel using a standard genotype allele-calling program. Accuracy of this method was greater than 96% for both loci. To test hypothesis 2,1 performed a comprehensive four-stage linkage study on 113 celiac famiUes. The analyses were of a candidate gene search, a genome wide search, a follow-up genotyping, and stratification of families by HLA using computer analysis software LINKAGE and GENEHUNTER. Multipoint evidence for linkage was minimal in this study. However several genetic markers show above nominal evidence for linkage to CD using 2-point analysis. When stratified by HLA, HLOD scores increased for several of these markers, two of which provide suggestive evidence for linkage. The work presented here represents the integration of molecular genetics and medical informatics to HLA type CD patients. Furthermore, we have preliminary evidence that non-HLA genes may be involved in the pathogenesis of celiac disease. |
