Glenn Thomas Horn

Sequence analysis of HLA class II genes from insulin-dependent diabetic individuals

To examine the nature of HLA-linked genetic susceptibility to insulin-dependent diabetes mellitus (IDDM), we compared HLA class II gene sequences from IDDM patients and control individuals. Genomic libraries were constructed from two siblings with IDDM, typed serologically as DR3,w6 and DR3,4. These libraries represent the HLA haplotypes (DR3, DR4) most frequently associated with IDDM, as well as one haplotype found less often. Individual genomic clones were identified and assigned to specific loci and haplotypes. The nucleotide sequence was then determined from the variable second exon from the HLA-DQ alpha, DQ beta, and DR beta genes from all three haplotypes. Sequence variation within the DQ alpha genes could not be correlated with the disease. For all three haplotypes, the DQ alpha sequence from the IDDM patient was identical to the DR-matched control sequence. Similarly, for the DR3 haplotype, the DQ beta sequences matched all control DR3 alleles. The DQ beta sequence from the DR4 haplotype was identical to the predominant DR4 allele (DQ beta 3.2) but differed at four amino acid residues from the other major DR4 DQ beta sequence (DQ beta 3.1) found rarely among IDDM patients. Sequence analysis of the DQ beta gene from the DRw6 haplotype revealed a new allele that differed from the DQ beta allele from a control DR6 allele at two residues. The DR beta genes from these three haplotypes also did not show any sequence features uniquely associated with IDDM, although the frequency of certain allelic variants in all three of these haplotypes may be altered in the IDDM population. A particular group of amino acids was found to be shared between the DR beta-1 alleles from the DR4 and DRw6 haplotypes and may be involved in genetic susceptibility to IDDM.

Analysis of Isotypic and Allotypic Sequence Variation in the HLA-DRβ Region Using the In Vitro Enzymatic Amplification of Specific DNA Segments

DRβ sequence polymorphism has been analyzed using the polymerase chain reaction in vitro amplification method. The amplified DRβ segments have been cloned and sequenced to determine the extent of allotypic (allele) and isotypic (locus) variation. The sequences have been assigned to either the DRβ 1 or the second DRβ chain locus on specific haplotypes. This sequence information can serve as the basis for a rapid DNA typing method using a dot-blot format with sequence-specific oligonucleotide probes. The patchwork pattern of polymorphism revealed by this analysis may reflect gene conversion-like mechanisms between different alleles and different loci. A previously unreported sequence, termed DRβX, is described and helps define one of the three major evolutionary groups of DR haplotypes.

Allelic Sequence Variation in the HLA-DQ Region: Implications for Localization of Serological Specificities and for Susceptibility to Autoimmune Disease

The pattern of nucleotide sequence variation in the HLA-DQ genes has been analyzed with respect to the known serologically defined HLA types and to susceptibility to insulin-dependent diabetes (IDDM). Thus far, 8 allelic variants have been found for the DQα gene, and 12 alleles have been seen for DQβ. The DQw1 specificity correlates best with a related set of DQα variants, whereas the DQw2, DQw3, and DQ(blank) specificities correlate with alleles of DQβ. A pattern of amino acid substitutions at position 57 in the DQβ protein appears to reflect the degree of susceptibility to IDDM for the different HLA haplotypes. This portion of DQβ also shows significant homology with several human viruses, suggesting that it may be a preferred site for molecular mimicry.