Stephen J. Scharf

HLA-DR, DO AND DP TYPING USING PCR AMPLIFICATION AND IMMOBILIZED PROBES

A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence‐based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non‐Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all ‘new’ alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing.

Rapid typing of DNA sequence polymorphism at the HLA-DRB1 locus using the polymerase chain reaction and nonradioactive oligonucleotide probes

An HLA-DR typing system that uses sequence-specific oligonucleotide (SSO) probes conjugated to horseradish peroxidase (HRP) for analyzing DRB alleles amplified by the polymerase chain reaction has been developed. Using 25 HRP-SSO probes and two primer pairs for generic and for DRB1 locus-specific amplification, we can distinguish 31 of 34 HLA-DRB1 alleles. This procedure is suitable for typing heterozygous samples from a variety of sources, including cDNA templates, and can detect in a simple and rapid dot-blot format allelic variants not distinguishable by serological methods. It should prove valuable for tissue typing, determining individual identity, and studies of disease susceptibility.

Sequence analysis of the HLA-DRß and HLA-DQß loci from three Pemphigus vulgaris patients

Pemphigus vulgaris (PV) is an autoimmune dermatologic disease that has been associated with the HLA serotypes DR4 and DRw6. In studying this association at the level of coding sequence polymorphism, we have determined the nucleotide sequences of the second variable exons from the HLA-DR beta and DQ beta loci from three PV patients with the HLA serotypes DR4/4, DR4/5, and DR4/5. These exons were enzymatically amplified by polymerase chain reaction (PCR) and cloned directly into an M13 vector for DNA sequencing. Analyses of amino acid sequences translated from the nucleotide sequence data show that all three patients contained a DR4 DR beta I sequence associated with the Dw10 DR4 subtype specificity, a relatively rare subtype among U.S. Caucasian DR4 haplotypes. The DQ beta sequence from three of the four DR4 haplotypes was identical to the sequence (DQB3.2) found on 60-80% of control DR4 haplotypes. These observations suggest that the amino acid residues at position 68, 69, and 72 of the DR beta I chain that distinguish Dw10 from the other DR4 subtypes may be involved in disease susceptibility.

A multilocus genotyping assay for cardiovascular disease.

Abstract In our efforts to develop diagnostic tests for complex multifactorial disorders, and to assist the research community in evaluating genetic markers for predisposition to cardiovascular disease, we have developed a prototype assay to genotype up to 35 variable sites among 15 genes. The candidate markers in this panel were selected from biological pathways likely to contribute to the development and progression of cardiovascular disease. Each sample is amplified in two multiplex polymerase chain reactions that are then hybridized to an array of immobilized oligonucleotide probes. The assay has been applied to a population-based cohort representing 238 families; allele frequencies observed among 455 unrelated parents from this cohort agree with available literature values. Data from a cohort of 142 lipid-clinic patients were used to explore locus associations with arterial occlusion, as measured by quantitative angiography. This prototype assay provides a research tool for studies to assess the association of multiple markers with disease, and for clinical studies to evaluate marker association with patient responsiveness to experimental therapies.

Sequencing of HLA-D in responders and nonresponders to short ragweed allergen, Amb a V

We investigated the molecular basis for the striking association between HLA-DR2,Dw2 and human immune responsiveness to the Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a V by sequencing the second exons of the DRB and DQBI genes of 17 selected ragweed-allergic Caucasoid subjects. We also studied the DQA1 allelic polymorphic regions (APRs) in these patients by dot-blotting using sequence-specific oligonucleotides (SSOs). The deduced amino acid sequences of the respective class II β and α polypeptides were compared, with particular emphasis on residues in the APRs that are implicated in antigen binding. No evidence for “new” HLA-DRB or DQB sequences unique to Amb a V responders were found on sequencing seven Dw2+ subjects. This suggests that the presence of a particular Dw2-associated class II molecule usually provides a necessary, but not always sufficient condition for responsiveness to Amb a V. The HLA phenotypes of three subjects suggest that they possess novel recombinant haplotypes containing either DRB1*1501 and DRB5*0101 (Dr2.2-associated) or DQB1*0602 (DQ1.2-associated) sequences. In these subjects, responsiveness to Amb a V was associated with the DR2.2 but not the DQ1.2 sequences, suggesting that DRαβI or DRαβV class II molecules are involved in antigen presentation. We investigated whether there may be shared HLA-D-encoded responder sequences present in all responders, including some exceptional DR2−Amb a V responders. The 13 subjects producing antibody (Ab) responses to Amb a V [either from natural exposure and/or after ragweed immunotherapy (Rx)] possessed DRB1*1501, 1601, 1602, 0103, 0402, 0404, 0801 or 1101 sequences, which share the majority of their aa residues in the APRs 2–4. Some of these shared residues might be important for the binding of a common Amb a V agretope prior to presentation of the class II Amb a V complex to the T-cell receptor (Tcr). An alternative postulate is that the recognition of two different Amb a V agretopes may be determined by the βI polypeptides of molecules having the DR2 and DQw3 specificities.

DNA-dependent DNA polymerases.

This unit presents characteristics and reaction conditions of the DNA‐dependent DNA polymerases, including E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, native and modified T7 DNA polymerase, and Taq DNA polymerase.

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.

HLA Class II Sequence Polymorphism and Autoimmunity

Specific alleles at the HLA-DR and -DQ loci have been associated with a variety of autoimmune diseases. The role of HLA-DP polymorphism in susceptibility has not been as fully explored as that of the other class II antigens due to the complexity of the primed lymphocyte typing (PLT) method for determining DPw specificities. These class II loci encode a heterodimer consisting of an alpha (ca 32 kDa) and a beta (ca 29 kDa) glycoprotein chain. These α-β heterodimers are highly polymorphic transmembrane proteins which bind peptides derived from the processing of foreign antigens (Babbitt et al., 1985; Guillet et al., 1987; Sette et al., 1987; Unanue et al., 1987; Watts et al., 1986). The activation of CD4+ T lymphocytes results from the recognition of this class II-peptide complex by the alpha-beta T cell receptor. In addition to their role in peptide binding on antigen-presenting cells, the polymorphic class II molecules present on thymic stromal cells influence the specificity of the mature T cell repertoire (Kappler et al., 1987; Kappler et al., 1988; MacDonald et al., 1988; Teh et al., 1988).