Henry A. Erlich

Dissecting the Genetic Complexity of the Association between Human Leukocyte Antigens and Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an inflammatory disease with a complex genetic component. An association between RA and the human leukocyte antigen (HLA) complex has long been observed in many different populations, and most studies have focused on a direct role for the HLA-DRB1 shared epitope in disease susceptibility. We have performed an extensive haplotype analysis, using 54 markers distributed across the entire HLA complex, in a set of 469 multicase families with RA. The results show that, in addition to associations with the DRB1 alleles, at least two additional genetic effects are present within the major histocompatibility complex. One of these lies within a 497-kb region in the central portion of the HLA complex, an interval that excludes DRB1. This genetic risk factor is present on a segment of a highly conserved ancestral A1-B8-DRB1*03 (8.1) haplotype. Additional risk genes may also be present in the HLA class I region in a subset of DRB1*0404 haplotypes. These data emphasize the importance of defining haplotypes when trying to understand the HLA associations with disease, and they clearly demonstrate that such associations with RA are complex and cannot be completely explained by the DRB1 locus.

HLA Class II Gene Polymorphism: DNA Typing, Evolution, and Relationship to Disease Susceptibility

The detection of HLA class II polymorphism is valuable in the areas of individual identification, tissue typing for transplantation, and genetic susceptibility to specific autoimmune diseases. Polymorphism in the HLA class II region (see Figure 1 for map) has been identified using serologic reagents (HLA-DR and -DQ specificities), by cellular techniques (Dw and DPw specificities) and, more recently, by restriction fragment length polymorphism (RFLP) analysis. For HLA class II typing, RFLP analysis is based on the presence or absence of polymorphic restriction sites located primarily in non-coding regions which are in linkage disequilibrium with allelic variation in coding sequences. Until recently, the direct analysis of coding sequence polymorphism has been difficult. However, the enzymatic amplification of specific DNA sequences using the PCR has provided a new approach to genetic typing.1-4 The capacity of the PCR to amplify a specific segment of genomic DNA has made it an invaluable tool in the study of polymorphism and evolution, as well as in the analysis of genetic susceptibility to disease. In all of these areas, a particular gene must be examined in a variety of individuals; either within a species, in different closely related species, or in patient and in healthy control populations. We have used PCR, initially with the Klenow fragment of E. colf DNA polymerase I and more recently with the thermostable Taq DNA polymerase, to determine the allelic sequence diversity of the HLA class II genes (HLA-DRβ, HLA-DQα, HLA-DQβ, and HLA-DPβ).

Newborn HLA-DR,DQ genotype screening: age- and ethnicity- specific type 1 diabetes risk estimates

Objective:u2002 Certain human leukocyte antigen (HLA)‐DR,DQ genotypes have been associated with type 1 diabetes mellitus (T1DM) risk, although it is unknown whether the association is due to alleles, haplotypes, genotypes, the formation of heterodimers, or all of the above. To characterize the role of the HLA‐DR,DQ genotype and ethnicity on the onset age of T1DM, we analyzed these factors in patients with T1DM and the general population.

Pathway-based analysis of genetic susceptibility to cervical cancer in situ: HLA-DPB1 affects risk in Swedish women.

We have conducted a pathway-based analysis of genome-wide single-nucleotide polymorphism (SNP) data in order to identify genetic susceptibility factors for cervical cancer in situ. Genotypes derived from Affymetrix 500k or 5.0 arrays for 1076 cases and 1426 controls were analyzed for association, and pathways with enriched signals were identified using the SNP ratio test. The most strongly associated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were Asthma (empirical P=0.03), Folate biosynthesis (empirical P=0.04) and Graft-versus-host disease (empirical P=0.05). Among the 11 top-ranking pathways were 6 related to the immune response with the common denominator being genes in the major histocompatibility complex (MHC) region on chromosome 6. Further investigation of the MHC revealed a clear effect of HLA-DPB1 polymorphism on disease susceptibility. At a functional level, DPB1 alleles associated with risk and protection differ in key amino-acid residues affecting peptide-binding motifs in the extracellular domains. The results illustrate the value of pathway-based analysis to mine genome-wide data, and point to the importance of the MHC region and specifically the HLA-DPB1 locus for susceptibility to cervical cancer.

[44] – Genetic Analysis Using Polymerase Chain Reaction-Amplified DNA and Immobilized Oligonucleotide Probes: Reverse Dot-Blot Typing

Publisher Summary nThe difficulty of analyzing single-copy genes in genomic DNA has been overcome by use of the polymerase chain reaction (PCR) to first amplify the target sequence to high abundance, followed by hybridization with the allele-specific oligonucleotide probe. This chapter discusses the analysis of PCR-amplified DNA with sequence-specific oligonucleotide hybridization probes. The conventional dot-blot method in conjunction with PCR amplification and oligonucleotide probes has greatly simplified the analysis of any DNA or RNA sequence, including those involved in genetic diseases, HLA polymorphisms, cancer, and so on. If the sample size is large, the PCR/dot-blot method is convenient to use, especially when the number of probes required is small. However, as the number of probes required for genetic typing increases, this method becomes cumbersome because the PCR product must be immobilized on a number of membranes, each of which is hybridized to a different labeled oligonucleotide probe. To alleviate this difficulty, a method known as the “reverse dot blot” is used. In this technique, the allele- or gene-specific oligonucleotide probes are bound to the filter instead of the PCR products, and the amplified DNA labeled during the PCR is used to hybridize to the immobilized array of probes.

Detection of new HLA-DPB1 alleles generated by interallelic gene conversion using PCR amplification of DPB1 second exon sequences from sperm

The rate at which allelic diversity at the HLA class I and class II loci evolves has been the subject of considerable controversy as have the mechanisms which generate new alleles. The patchwork pattern of polymorphism, particularly within the second exon of the HLA-DPB1 locus where the polymorphic sequence motifs are localized to 6 discrete regions, is consistent with the hypothesis that much of the allelic sequence variation may have been generated by segmental exchange (gene conversion). To measure the rate of new DPB1 variant generation, we have developed a strategy in which DPB1 second exon sequences are amplified from pools of FACS-sorted sperm (n=50) from a heterozygous sperm donor. Pools of sperm from these heterozygous individuals are amplified with an allele-specific primer for one allele and analyzed with sequence-specific oligonucleotide probes (SSOP) complementary to the other allele. This screening procedure, which is capable of detecting a single variant molecule in a pool of parental alleles, allows the identification of new variants that have been generated by recombination and/or gene conversion between the two parental alleles. To control for potential PCR artifacts, the same screening procedure was carried out with mixtures of sperm from DPB1 *0301/*0301 and DPB1 *0401/ 0401morexa0» individuals. Pools containing putative new variants DPB1 alleles were analyzed further by cloning into M13 and sequencing the M13 clones. Our current estimate is that about 1/10,000 sperm from these heterozygous individuals represents a new DPB1 allele generated by micro-gene conversion within the second exon.«xa0less