PubMed Health⌕ Search

Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 19 recordsLinked to original sources
PubMed1992

Oligonucleotide genotyping of HLA polymorphism on microtitre plates.

Molecular analysis of mutations and polymorphisms that are of medical importance requires both accuracy and simplicity. In organ transplantation there is a need for an HLA typing procedure that combines the remarkable accuracy of oligonucleotide genotyping with the simplicity of conventional serological typing. We describe a simple semiautomated method of HLA class II typing consisting of an oligonucleotide hybridisation assay done on microtitre plates followed by automatic colorimetric reading. Individual HLA-DR generic typing for 30 DR specificities, including subtypes of DR1, DR2, DR13, DR14, and DR52, is done on a single plate. The entire typing assay can be completed in less than 4 hours. The procedure has been validated on more than a thousand haplotypes in prospective DR typing of kidney transplant patients, leukaemic patients, and their potential donors. The simplicity of this assay makes it suitable for routine laboratory use. It can be applied to genetic testing in general, including the testing of patients with multiple mutations.

Base Sequence↗
PubMed1992

The evolutionary origin of the HLA-DR3 haplotype.

The human HLA-DR3 haplotype consists of two functional genes (DRB1*03 and DRB3*01) and one pseudogene (DRB2), arranged in the order DRB1...DRB2...DRB3 on the chromosome. To shed light on the origin of the haplotype, we sequenced 1480 nucleotides of the HLA-DRB2 gene and long stretches of two other genes, Gogo-DRB2 from a gorilla, "Sylvia" and Patr-DRB2 from a chimpanzee, "Hugo". All three sequences (HLA-DRB2, Gogo-DRB2, Patr-DRB2) are pseudogenes. The HLA-DRB2 and Gogo-DRB2 pseudogenes lack exon 2 and contain a twenty-nucleotide deletion in exon 3, which destroys the correct translational reading frame and obliterates the highly conserved cysteine residue at position 173. The Patr-DRB2 pseudogene lacks exons 1 and 2; it does not contain the twenty-nucleotide deletion, but does contain a characteristic duplication of that part of exon 6 which codes for the last four amino acid residues of the cytoplasmic region. When the nucleotide sequences of these three genes are compared to those of all other known DRB genes, the HLA-DRB2 is seen as most closely related to Gogo-DRB2, indicating orthologous relationship between the two sequences. The Patr-DRB2 gene is more distantly related to these two DRB2 genes and whether it is orthologous to them is uncertain. The three genes are in turn most closely related to HLA-DRBVI (the pseudogene of the DR2 haplotype) and Patr-DRB6 (another pseudogene of the Hugo haplotype), followed by HLA-DRB4 (the functional but nonpolymorphic gene of the DR4 haplotype). These relationships suggest that these six genes evolved from a common ancestor which existed before the separation of the human, gorilla, and chimpanzee lineages. The DRB2 and DRB6 have apparently been pseudogenes for at least six million years (myr). In the human and the gorilla haplotype, the DRB2 pseudogene is flanked on each side by what appear to be related genes. Apparently, the DR3 haplotype has existed in its present form for more than six myr.

Animals↗
PubMed1992

Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients.

To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.

DNA Probes, HLA↗
PubMed1992

The genes for MHC class II regulatory factors RFX1 and RFX2 are located on the short arm of chromosome 19.

RFX1 is a transacting DNA-binding regulatory factor involved in the control of MHC class II gene expression. RFX2 is a structurally very similar protein with identical DNA binding features. A member of the family of RFX factors is affected in an autosomal recessive disease, MHC class II deficient combined immunodeficiency (CID), caused by a defect in a trans-acting regulatory factor controlling MHC class II gene expression. In situ hybridization with 3H-labeled RFX1 cDNA has allowed us to identify two distinct targets on the short arm of chromosome 19 (19p13.1 and 19p13.2-p13.3). With the use of biotinylated genomic cosmid clones specific for RFX1 and RFX2, respectively, it was then possible to localize RFX1 at 19p13.1 and RFX2 at 19p13.2-p13.3. These two regulatory genes are thus assigned to a region of high gene density and RFX1 is close to another DNA-binding factor, LYL1.

Chromosome Mapping↗
PubMed1992

The DNA-binding defect observed in major histocompatibility complex class II regulatory mutants concerns only one member of a family of complexes binding to the X boxes of class II promoters.

The X box of major histocompatibility complex class II promoters is essential for proper expression of class II genes. Here we show that two distinct protein-DNA complexes (A and B), which exhibit similar binding characteristics and identical contact points on the X box, can be formed. This suggests the existence of a family of related X box-binding factors. Complex B (and not complex A) is specifically affected in primary combined immunodeficiency, a congenital defect in class II gene regulation. RFX1, the first X box-binding protein cloned, encodes a functionally relevant factor present in complex A and not in complex B as originally suspected. This report also illustrates the need for caution in correlating specific cloned proteins with nuclear factors identified by DNA-binding assays, particularly when dealing with families of related proteins.

Base Sequence↗
PubMed1992

HLA-DR polymorphism in a Senegalese Mandenka population: DNA oligotyping and population genetics of DRB1 specificities.

HLA class II loci are useful markers in human population genetics, because they are extremely variable and because new molecular techniques allow large-scale analysis of DNA allele frequencies. Direct DNA typing by hybridization with sequence-specific oligonucleotide probes (HLA oligotyping) after enzymatic in vitro PCR amplification detects HLA allelic polymorphisms for all class II loci. A detailed HLA-DR oligotyping analysis of 191 individuals from a geographically, culturally, and genetically well-defined western African population, the Mandenkalu, reveals a high degree of polymorphism, with at least 24 alleles and a heterozygosity level of .884 for the DRB1 locus. The allele DRB1*1304, defined by DNA sequencing of the DRB1 first-domain exon, is the most frequent allele (27.1%). It accounts for an unusually high DR13 frequency, which is nevertheless within the neutral frequency range. The next most frequent specificities are DR11, DR3, and DR8. Among DRB3-encoded alleles, DR52b (DRB3*02) represents as much as 80.7% of all DR52 haplotypes. A survey of HLA-DR specificities in populations from different continents shows a significant positive correlation between genetic and geographic differentiation patterns. A homozygosity test for selective neutrality of DR specificities is not significant for the Mandenka population but is rejected for 20 of 24 populations. Observed high heterozygosity levels in tested populations are compatible with an overdominant model with a small selective advantage for heterozygotes.

Base Sequence↗
PubMed1991

Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization.

As the demand for donors for bone marrow transplantation increases, the use of HLA-matched, genetically unrelated donors represents a promising strategy. It is well documented that the clinical outcome of bone marrow transplantation is directly dependent on optimal matching for HLA class I and class II specificities. Molecular studies have revealed the existence of a much larger number of HLA class II alleles than was anticipated, many of which cannot be recognized by routine serological typing. Currently this "hidden" polymorphism represents a major limitation to the generalized use of unrelated donors for bone marrow transplantation. It has recently become possible, however, to identify HLA allelic polymorphism directly at the DNA level by hybridization with sequence-specific oligonucleotide probes ("HLA oligotyping") after amplification of DNA by polymerase chain reaction. In this study, we have investigated whether donor-recipient pairs that are fully matched for HLA by serology are truly HLA-DR, -DQ, and -DP identical and to what extent class II differences influence the primary mixed lymphocyte culture. We show that HLA oligotyping, performed on 50 pairs of HLA class I and II serologically matched individuals, can indeed reveal phenotypically relevant allelic differences at either DRB or DQB loci in 56% of these pairs and can therefore improve HLA class II typing and the choice of bone marrow donors quite significantly. Oligotyping for DRB/DQB/DPB polymorphism also allows prediction of a positive mixed lymphocyte culture, as established in 31 donor/recipient combinations, and even detection of polymorphic differences that were not revealed by this test. This approach is well suited for accurate HLA typing of large pools of bone marrow donors and was successfully applied to select fully matched donors for bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed1991

Regulatory factor-X binding to mutant HLA-DRA promoter sequences.

The class II genes of the major histocompatibility complex (MHC) encode a family of cell surface glycoproteins that present processed antigen to the T cell receptor. Class II genes are regulated coordinately, responding to both immunologic and developmental signals. Conserved sequence elements 5' to class II genes have been shown to be important in transcriptional control. One of these sequences, the X box, is a specific target for the binding of the factor RF-X. In the hereditary HLA class II deficiency, a form of primary immunodeficiency, a regulatory defect in expression of class II genes is associated with a defect in the binding of RF-X. To determine the basepairs that are important for this binding interaction, a series of single basepair substitutions spanning the X box motif of the DRA gene was constructed and tested for binding of RF-X by gel electrophoresis mobility shift assays (EMSAs). Several, but not all, of the mutants severely affected binding of RF-X. In addition, the binding of both the natural and the recombinant form of RF-X was affected with the same specificity. A comparison of X box basepair positions important for RF-X binding to DRA with sequences conserved between X boxes of other class II alpha chain genes suggests that high affinity RF-X binding is important for a high level of expression and may explain differences in the levels of class II expression of different class II alpha chains.

Animals↗
PubMed1991

Additional complexity within the HLA-D region: sequence analysis of two new DRw13-DQw7 haplotypes.

HLA-DRB1 is by far the most polymorphic locus within the HLA-D region with now well over 40 alleles. Nearly one fourth of these alleles are subtypes of DRw6, and these are in most cases undetectable by routine typing procedures. In this paper we present the molecular characterization of two new Caucasian DRw13-DQw7 haplotypes by DNA sequencing of the polymorphic first domain exons of DRB1 and DRB3 loci. The first haplotype, DRB1*1301-DRB3*0101-DQB1*0301, has arisen by a recombination between locus DRB1 from a DRw13-DQw6 haplotype and DQA1 from a DR4-DQw7 haplotype, as determined by DNA sequencing, DQ oligotyping, and restriction fragment length polymorphism typing. The second haplotype, DRB1*1305-DQB1*0301, is characterized by the novel DRB1*1305 allele differing from DRB1*1301 by three amino acids. It probably arose by a gene conversion event between a DRw13-DQw6 allele and DRB1*1101. This allele represents a DRw11/DRw13 hybrid DR molecule with a DRw13 serological epitope in the second hypervariable region and a Dw5 cellular epitope in the third hypervariable region. As determined by sequencing of locus DRB3, this allele is associated with DRw52b. Our molecular analysis of the complex HLA-DRw13 group now allows unambiguous DNA typing of all five DRw13 alleles with seven oligonucleotides, a significant improvement in the context of organ transplantation.

Amino Acid Sequence↗