Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis

Summary Two yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100kb centromeric to the HLA-A locus, or on the telomeric side.     

Allergic contact dermatitis to nickel is associated with a Taq I HLA-DQA allelic restriction fragment

RFLP analysis of the HLA class II genes DRA, DQA, and DQB was performed in 33 patients with allergic contact eczema to nickel. A significant association with a Taq I HLA-DQA allelic restriction fragment was found. Twenty-two of 33 patients compared to 31 of 100 healthy controls had a 4.5-kb DQA fragment (corrected P value <0.05, relative risk 4.5, and etiologic fraction 0.52). In order to study whether the magnitude of the response in a lymphocyte proliferative assay to nickel sulfate was controlled by HLA class II genes or not, the patients were divided into low, intermediate, and high responders. No significant differences were found in the distribution of Taq I HLA class II allelic patterns between the groups.     

DNA polymorphism related to the idiopathic hemochromatosis gene: evidence in a recombinant family

Summary The metabolic error involved in idiopathic hemochromatosis, as well as the underlying genetic defect remain unknown. It has, however, been recently shown that this genetic lesion occurs at a locus linked to the major histocompatibility complex, probably close to the HLA-A locus, and that the disease is recessively transmitted. Therefore, in a family where one subject has idiopathic hemochromatosis his HLA-identical siblings should also be affected. We present here the restriction polymorphism with two MHC class I probes and one DR probe in an exceptional family with three HLA-identical siblings: one (the proband) has a major form of idiopathic hemochromatosis, while the other two are free of any clinical or biochemical signs of the disease. The restriction patterns observed after DNA digestion by enzymes EcoRI, EcoRV, BglII, BamHI, PvuII, TaqI, HincII, and HindIII led to the conclusion that one of the proband's chromosome 6 had undergone two alterations: one, a deletion in the DR region, was revealed by missing fragments all correlated with DR5; the other was an unbalanced cross-over or a genetic conversion in the MHC class I region. This latter alteration was revealed by modifications in the patterns of high molecular weight HindIII bands which hybridize with probe pHLA2 and also by the absence of a HindIII fragment of 7.4 kb hybridized by another class I probe. This latter alteration most likely involved the hemochromatosis gene and could be the first step toward a molecular approach to this gene.     

DNA sequence of HLA-A11: Remarkable homology with HLA-A3 allows identification of residues involved in epitopes recognized by antibodies and T cells

The human class I alleles HLA-A11 and HLA-A3 have a well-documented history of serological cross-reactivity. This cross-reactivity suggests that they are closely related, a suggestion which is supported by the fact that the HLA-A11 and HLA-A3 genes are distinguished from all other A-locus genes by a restriction fragment length polymorphism observed in Bam HI digests. To examine the extent of sequence homology between HLA-A11 and HLA-A3, we have cloned the HLA-A11 gene and sequenced the coding regions (exons). The results reveal that HLA-A11 and HLA-A3 display the highest degree of homology reported for any pair of serologically defined class I alleles. Only nine base differences resulting in six amino acid differences were observed in exons 2–8. One of the amino acid substitutions is in the 1 domain and the other five are in the 2 domain. Comparison of this sequence with that of other human class I molecules implicates Gln₆₂ as a critical residue involved in HLA-A11 – HLA-A3 serological cross-reactivity. In addition, the amino acid sequence allowed us to successfully predict cross-reactive recognition of HLA-A11 by cytotoxic T lymphocytes specific for a rare subtype of HLA-A3, HLA-A3.2. This result provides further support for the importance of the 2 domain residues 152 and 156 in forming determinants on class I molecules that are recognized by cytotoxic T lymphocytes.Abbreviations used in this paper CTL cytotoxic T lymphocyte - PBL peripheral blood lymphocyte - PHA phytohemagglutinin     

TaqI polymorphism in the LDL receptor gene and a TaqI 1.5-kb band associated with familial hypercholesterolemia

Summary The low density lipoprotein (LDL) receptor gene was analyzed in 67 unrelated healthy Japanese and 38 members of six consecutive families with familial hypercholesterolemia (FH) by Southern blot hybridization with TaqI, an LDL receptor cDNA fragment containing exons 1 to 8 being used as a probe. A new TaqI RFLP at the LDL receptor locus was detected with allele frequencies of 0.67 and 0.33. The data obtained with smaller cDNA subfragment probes revealed that the TaqI RFLP site is located within 1.1 kb of the 5 side of the EcoRI site of exon 5. The TaqI RFLP was in linkage disequilibrium with the PstI RFLP but showed no significant linkage disequilibrium with the RFLPs for AvaII, ApaLI/I15, PvuII, NcoI, and ApaLI/3. Among the seven RFLPs at the LDL receptor locus, the TaqI RFLP was the only useful genetic marker in one of the six families with FH. Furthermore, the association of an additional TaqI 1.5-kb band with a mutant LDL receptor gene was observed in another family with FH in which the proband was homozygous for all of the seven RFLPs. The data obtained with various restriction enzymes and smaller cDNA subfragments probes suggested that a minor change in nucleotide sequences in the region including exons 5 to 8 is present in the mutant gene. These data suggest that the TaqI RFLP is a useful genetic marker at the LDL receptor locus and that TaqI serves for the analysis of some mutant LDL receptor genes, when used with small LDL receptor cDNA probes.     

The polymorphic Fcγ receptor II gene maps to human chromosome 1q

Human receptors for IgG (FcR) play important roles in the immune response. Expression of the human FcRII gene may be relevant in immune complex related disorders such as systemic lupus erythematosus and Sjogren's syndrome. We have used spot blot analysis of dual laser-sorted human chromosomes to localize the FcRII gene to human chromosome 1. Spot blot analysis of sorted derivative chromosomes sublocalized the gene to the chromosome 1 long arm (1q1225.1). This subchromosomal localization involved reassigning a reciprocal chromosome translocation breakpoint. We also identified Xmn I and Taq I FcRII polymorphic restriction sites that arose before the races diverged. These common Xmn I and Taq I polymorphisms are predicted to be informative for segregation analysis with human diseases in 85% of all matings.     

The chimpanzee major histocompatibility complex class II DR subregion contains an unexpectedly high number of beta-chain genes

The major histocompatibility complex (MHC) class II DR subregion of the chimpanzee was studied by restriction fragment length polymorphism (RFLP) analysis. Genomic DNA obtained from a panel of 94 chimpanzees was digested with the restriction enzyme Taq I and hybridized with an HLA-DR probe specific for the 3' untranslated (UT) region. Such a screening revealed the existence of 14 distinct DRB/Taq I gene-associated fragments allowing the definition of 11 haplotypes. Segregation studies proved that the number of chimpanzee class II DRB/Taq I fragments is not constant and varies from three to six depending on the haplotype. Comparison of these data with a human reference panel manifested that some MHC DRB/Taq I fragments are shared by man and chimpanzee. Moreover, the number of HLA-DRB/Taq I gene-associated fragments detected in a panel of homozygous typing cells varies from one to three and corresponds with the number of HLA-DRB genes present for most haplotypes. However, a discrepancy is observed for the HLA-DR4,-DR7, and -DR9 haplotypes since a fourth HLA-DRB pseudogene present within these haplotypes lacks its 3' UT region and thus is not detected with the probe used. These results suggest that chimpanzees have a higher maximum number of DRB genes per haplotype than man. As a consequence, some chimpanzee haplotypes must show a dissimilar organization of the MHC DR subregion compared to their human equivalents. The implications of these findings are discussed in the context of the trans-species theory of MHC polymorphism. Address correspondence and offprint requests to: R. E. Bontrop.     

Altered structure of HLA class I heavy chains associated with mouse beta-2 microglobulin

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2 microglobulin ( ₂m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse ₂-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human ₂m restores these reactivities. Since most of the amino acid differences between mouse and human ₂m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.     

Characterization of two novel polymorphisms at the human parathyroid hormone gene locus

Summary Two new polymorphisms within the human parathyroid hormone (PTH) gene are described. One corresponds to a CA transversion that destroys DraII and NlaIV restriction sites. The other is revealed by the enzyme XmnI, and its position has been mapped with respect to the PTH gene. We have also identified a sequence change that results in the TaqI restriction fragment length polymorphism (RFLP) described previously at this locus and have found that this sequence change also results in disruption of a BstBI site. Finally, we describe a polymerase chain reaction (PCR)-based method that permits a rapid evaluation of the DraII and BstBI (TaqI) polymorphisms. The introduction of these two additional RFLPs and this PCR-based assay should considerably extend the power of genetic analyses of the human PTH gene.     

TaqI HLA-B and -DRB RFLP analysis can predict disease in siblings of affected children with 21-hydroxylase deficiency

Summary The possibility of using TaqI restriction fragment length polymorphism (RFLP) analysis of the HLA-B locus and the HLA-DR-DQ subregions, flanking the 21-hydroxylase genes, for predicting disease in siblings of children with 21-hydroxylase deficiency was analyzed in 12 nuclear families with at least one affected child and a total of 18 at-risk off-spring. As part of the study allelic TaqI HLA-B RFLP patterns were determined in homozygous cell lines and families. The frequencies of individuals homozygous for TaqI allelic patterns of the different investigated HLA loci, each locus alone and in various combinations, were determined in 100 random controls. In all 12 families it was possible to make correct genetic diagnosis by the use of only one restriction enzyme, TaqI, and two locus-specific HLA cDNA probes, HLA-B and -DRB. In all families four haplotypes were obtained. Thus, affected siblings as well as carriers could be identified. Seven of the eight sibling pairs concordant for 21-hydroxylase deficiency had pairwise identical TaqI HLA-B-DRB-DQA-DQB haplotypes. The last disease-concordant sibling pair had inherited different haplotypes from their mother, who had nonclassical 21-hydroxylase deficiency. None of the ten healthy children shared both haplotypes with their affected sibling(s). Early prenatal suppression of the fetal adrenal cortex with fluorinated corticosteroids can prevent virilization of female fetuses with 21-hydroxylase deficiency. In most cases RFLP analysis of the 21-hydroxylase genes is not informative enough for prenatal diagnosis. Our results from the present retrospective family study indicate that TaqI HLA-B and -DRB RFP analysis will be a valuable tool for first trimester assessment of 21-hydroxylase deficiency. TagI HLA-B and -DRB RFLP analysis can be performed on DNA from chorionic villi biopsies obtained in the 8th week of pregnancy. Supplemented with sex determination, early withdrawal of prophylactic steroid therapy will thus be feasible when the mother carries a male or an unaffected female fetus.     

A novel NcoI polymorphism creates a fifth haplotype in the 3′ untranslated region of CKM

A novel NcoI polymorphism has been detected in the 3untranslated region of the creatine kinase (CKM) gene. The additional NcoI restriction site creates a fifth haplotype for the NcoI and TaqI restriction fragments length polymorphisms at this locus, and segregates with the myotonic dystrophy gene in 3 generations of an affected family.     

Molecular analysis of the human MHC class I region in hereditary haemochromatosis

Summary The unknown allele that predisposes to the development of haemochromatosis in man has been localized to the HLA class I region on the short arm of chromosome 6. We have utilized pulsed-field gel electrophoresis in conjunction with probes that map within, or in the vicinity of, this region to search for structural lesions that may further define the disease locus. Using the enzyme Mlu I, fragments that associated specifically with the HLA-A23, A31 and B8 alleles were identified. However, in members of three pedigrees affected by haemochromatosis, and in six unrelated patients with the disorder, no disease-specific differences were detected in the DNA fragments with four restriction enzymes and eight probes when compared with healthy individuals. These data suggest that the lesion responsible for hereditary haemochromatosis lies beyond the resolution of this technique and does not involve large structural deletions or extensive re-arrangements in this highly polymorphic region of the genome.     

The chromosome region containing the highly polymorphic HLA class I genes displays limited large scale variability in the human population

The large-scale organization and polymorphism of the HLA class I region was investigated by pulsed field gel (PFG) fractionation of DNA from various HLA-typed cell lines cleaved by different 'rare cutter' restriction enzymes, followed by hybridization with 'general' and locus-specific HLA probes. Results indicate that (i) most HLA class I sequences are contained in a 340 kb MluI DNA fragment which also carries the HLA-A gene; (ii) HLA-A, -B and -C genes are present on different fragments bounded by 'HTF islands' (CpG-rich, unmethylated DNA regions containing multiple sites for 'rare cutter' enzymes) which generally coincide with the 5' regions of expressed genes; and (iii) very little fragment size polymorphism is seen, implying that expansion/contraction events in the HLA class I region due to unequal crossing over (as documented in the mouse class I system) are infrequently found in the human population.     

Alterations of class I HLA genes in human colon cancers

Summary Alterations of HLA class I genes were found in 3 of 12 human colon cancers. Rearrangements in HLA class I genes were observed in 2 cancers and amplification of HLA-coding genes was observed in 1 cancer. All 3 cancers were at an advanced stage. No examples of amplification or rearrangement in the HLA genes were found in 10 other tumours of diverse types. No alterations in the ₂-microgubulin gene were observed in 22 human solid tumours included in this study. The association between alterations in HLA genes and proto-oncogenes in these tumours is discussed.     

The human complement component C8B gene: structure and phylogenetic relationship

The eighth component of human complement (C8) is a serum protein that consists of three chains (, and ), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the -subunit is non-covalently bound to the disulfide-linked - subunit. Using a full-length C8 cDNA probe, we isolated several clones from human genomic DNA libraries. Four clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were shotgun subcloned into M13. C8-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. By using polymerase chain reaction (PCR) primers located in the adjacent intron sequences, all 12 exons of the C8B gene could be amplified from genomic DNA. All fragments showed the expected sizes. The sizes of eight introns could be determined by using primer pairs that amplified two exons and the enclosed intron, and by restriction mapping. These analyses and the insert sizes of the genomic clones indicate that the C8B gene has a total size of approximately 40 kb. The polymorphic TaqI site of the C8B gene localized in intron 11 could be demonstrated by direct restriction fragment analysis of a PCR fragment containing exons 11 and 12, and the enclosed intron 11. Homology comparison of the C8B gene with C8A and C9 on the basis of the exon structure confirmed the ancestral relationship known from the protein level.     

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

DNA polymorphism of the human complement component C7 gene in familial deficiencies

Summary A C7 cDNA probe detecting a TaqI restriction fragment length polymorphism has been used to examine the segregation of the silent allele (C7^*Q0) in two familial deficiencies. Carrier diagnosis in healthy children is possible when both parents are heterozygotes. Only one of these two families was informative. The silent allele is linked to different TaqI alleles in both families. This suggests that at least two different C7^*Q0 alleles are present in our population. This paper gives a protocol for genetic studies of hereditary traits in which the C7 gene and other genes tightly linked to it are involved.     

The molecular genetics of haemophilia A: screening for point mutations in the factor VIII gene using the restriction enzyme TaqI

Summary A combination of Southern blotting and the analysis of polymerase chain reaction (PCR) amplified DNA fragments was used to screen the factor VIII genes of 527 haemophilia A patients for point mutations within TaqI restriction sites. Since this directed search strategy yielded only four gene lesions, it was concluded that its efficacy is less than that originally predicted. One novel point mutation was however found in a moderately severe haemophiliac; a CGA (Arg) to CTA (Leu) transversion at codon 2209, an evolutionarily conserved residue in the C2 domain of the factor VIII protein. The remaining three detected lesions, CGA (Arg)TGA (Term) transitions at codons 2116, 2147 and 2307, respectively, have been reported before and are consistent with recurrent mutation at these hypermutable sites. A number of TaqI restriction site polymorphisms/rare variants were also noted. These variants appear to be population-specific but are nevertheless potentially useful in individual cases as intragenic markers for carrier detection and antenatal diagnosis.     

DNA polymorphism in the 5′ flanking region of the human carbonic anhydrase II gene on chromosome 8

Summary A restriction-fragment-length polymorphism (RFLP) is described which is associated with the human carbonic anhydrase II gene (CA2) that codes for one of the three genetically distinct carbonic anhydrase isozymes, CA I, CA II, and CA III. The isolated DNA was cleaved with several restriction enzymes and subjected to Southern blot hybridization analysis using a DNA probe containing the 5 end of the human CA II gene. A two allele RFLP which was detected with the restriction endonuclease, Taq I, is expressed phenotypically on Southern blots as either a 5.4 kilobase (kb) fragment or as 4.0 and 1.4 kb fragments. These fragments result from the presence or absence of a Taq I recognition site in the 5 flanking region approximately 1.0kb from the initiation codon of the CA II gene. Segregation analysis showed that the alleles are inherited in a Mendelian fashion, with a frequency of 50%.