The study of a French family with two duplicated C4A haplotypes

Summary The finding of two duplicated C4A haplotypes in a normal French family led to a detailed study of their C4 polymorphism. The father had an extremely rare A^*6A^*11, B^* QO haplotype inherited by all of his children and the mother had the more common A^*3A^*2, B^*QO haplotype. Two HLA identical daughters only have four C4A alleles. The father's A11 allotype expresses Ch: 1 (Chido) rather than Rg:1 (Rodgers) and represents a new Ch phenotype Ch: 1,-2,-3,-4,-5,-6. In order to clarify the genetic background in this unusual family, DNA studies of restriction fragment length polymorphisms (RFLPs) were undertake. The father's rare haplotype, which expresses two C4A allotypes, results from a long and a short C4 gene normally associated with the A^*6, B^*1 that also exhibits the BglII RFLP. As it travels in an extended MHC haplotype HLA A2, B57 (17), C2^*C, BF^*S, DR7 that is most frequently associated with A^*6, B^*1, we postulate that the short C4B has been converted in the chain region to a C4A gene which produces a C4A protein. This report of a short C4A gene is the first example in the complex polymorphism of C4.     

The genomic structure of two ancestral haplotypes carrying C4A duplications

Two major histocompatibility complex (MHC) ancestral haplotypes (AH) HLA A24, Bw52, C2C, BfS, C4A3 + 2, C4BQO, DRw15, DQw6 (52.1) and HLA A24, Cw7, B7, C2C, BfS, C4A3 + 3, C4B1, DR1, DQw5 (7.2), which occur with the haplotype frequencies of approximately 10% and 4% respectively in the Japanese population, carry duplicated C4A alleles by C4 allotyping. Southern blot analysis with Taq I indicated that the 52.1 AH has two C4 genes defined by 7.0 kilobase (kb) and 6.0 kb C4 hybridizing fragments but both encode C4A allotypes, being C4A3 and C4A2 respectively. The 7.2 AH carries two C4A3 and one C4B1 alleles and restriction lenght polymorphism (RFLP) analysis with Taq I showed that 6.0 kb and 7.0 kb fragments are in the proportion of 2:1. By pulsed field gel electrophoresis (PFGE) analysis, the lengths of the Pvul fragments carrying C4 and Cyp21 genes were approximately 390 kb for 52.1 and 440 kb to 7.2. The results indicate that the RFLP markers do not correlate with C4 isotype (A or B) or allotype and that the C4 gene copy number is a function of the number of genomic blocks containing C4 and Cyp21.     

The susceptibility to insulin-dependent diabetes mellitus bis associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3, 4 heterozygotes

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P=6 x 10^–6): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P=0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P=0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes. Since recent works have shown that the structural gene for the monokine tumor necrosis factor alpha (TNF-) is located between the HLA-B and C4 loci and that TNF- might be of importance in IDDM pathogenesis, the hypothesis is put forward that the C4-associated IDDM susceptibility reflects linkage dis-equilibrium between the C4 gene and a gene controlling TNF- production. The high relative risk for IDDM in HLA-DR3,4 heterozygotes might be explained by the combined action of IDDM-specific susceptibility genes on DR4 haplotypes and DR3-linked susceptibility genes associated with predisposition to autoimmunity.     

DNA polymorphism of the C2 locus

The extent of the C2 locus in the HLA class III region has been determined by Southern blotting techniques and by DNA sequence analysis. The gene is 18 kb in length and therefore provides a marked contrast to the adjacent factor B gene of 6 kb. A novel restriction fragment length polymorphism (RFLP) has been identified using the endonuclease Sst I and a genomic probe derived from the 5 region of the C2 gene. Four variants have been detected in a sample of unrelated individuals with haplotypes carrying the C2C allele. Further analysis using C2 and factor B cDNA probes has determined the relationship between this and the other RFLPs previously identified in this region of the genome. Together, the three polymorphisms identified so far make the subdivision of previously indistinguishable haplotypes possible. They therefore constitute a series of markers which increase the resolution of genetic variation in the C2 locus and they may be important in studies of diseases associated with this region of the major histocompatibility complex.     

DNA polymorphism of human HLA-linked complement C4 allotypes, including C4 null alleles, in the Finnish population

Human HLA-linked complement C4 gene products, C4A and C4B, show extensive genetic polymorphism. In both loci, an allele without a gene product, C4 null, is also observed. We have performed a restriction enzyme analysis of genomic DNA samples from individuals having all common (frequency over 1%) C4 protein allotypes observed in the Finnish population. Only one allotype-specific RFLP marker was observed. With some enzymes a DNA polymorphism was observed, which was not detectable by C4 protein typing. Analysis of 10 different C4B null haplotypes and 4 C4A null haplotypes suggested that only one haplotype, HLA-B8 C4A0 B1, carried a C4A gene deletion. This was observed in all 4 unrelated individuals homozygous for this haplotype.     

L3T4 but not LFA-1 participates in antigen presentation by Ak-positive L-Cell transformants

Genomic DNA was isolated from 29 t strains and 4 congenic lines of mice, digested with restriction endonucleases, and hybridized with a probe representing the complement component 4 (C4) gene. All but one of the enzymes revealed restriction fragment length polymorphism in this sample of C4-related genes. Double digestion analysis suggested the presence of three C4 gene copies in some of the t chromosomes and two copies in others. The enzymes distinguished 16 different haplotypes among the 33 strains tested. Based on their restriction fragment length patterns, the t strains could be divided into four groups with strains in each group more closely related to each other with respect to their C4-region genes than strains belonging to different groups. At least three of these four groups represent different branches of the evolutionary tree constructed for the t chromosomes. The C4-related genes of the chromosomes are in strong linkage disequilibrium with the class II genes of the H-2 complex. Typing for the Ss and Slp allotypes of C4 has revealed the presence of the Ss¹ phenotype in two t strains and of the Slp^a phenotype in one strain.     

Molecular genotyping of human T-cell antigen receptor variable gene segments

The gene complex encoding the chain of the T-cell antigen receptor (Tcr) in man was previously reported to contain a restriction fragment length polymorphism (RFLP) involving a single Bgl II site adjacent to the second constant region gene. This RFLP allowed assignment of Tcr genotypes in certain human families. In the present study, two different RFLP in a V gene family were detected using the murine probe V8.1 in genomic DNA samples digested with the restriction endonucleases Hind III and Bam HI. Use of these RFLP to mark the V gene complex allowed complete haplotype assignment in four of seven families studied and provided support for linkage of the V gene complex to the constant region genes. Different combinations of the C and two V region markers can result in eight possible distinct haplotypes. The observation of all but one of the eight possible haplotypes in parents of the families studied suggests that recombination events occur between the C and V region and among members of the V region subfamily marked by the V8.1 probe. These markers can be used for mapping studies of the V gene complex in man and will allow an appraisal of possible associations between Tcr genes and disease susceptibility.Abbreviations used in this paper: Tcr T-cell antigen receptor - RFLP restriction fragment length polymorphism - C2 second Tcr constant region gene - V Variable - C constant - J joining - D diversity     

DNA polymorphism of MHC III genes in inbred and wild mouse strains

Genes encoding the second component (C2), factor B, and complement protein C4 and Slp (sex-limited protein) are members of the major histocompatibility complex class III gene cluster. In this report we describe isolation of a mouse C2 cDNA clone and its use together with factor B and C4 cDNA clones to examine the S region in a panel of 42 haplotypes in laboratory and wild mice representing 5 species and subspecies of Mus. Conservation of the C2 factor B gene duplex was evidenced by relatively limited polymorphism associated with speciation and nucleotide sequence homology between mouse and human C2 and factor B The C4-Slp gene duplex, on the other hand, showed extensive polymorphism by DNA blot analysis. This polymorphism correlated poorly with the C2/factor B restriction fragment length polymorphism, suggesting independent evolution of these two segments of the S region. Taken together, these data will be of particular importance in studies of mouse strains with abnormal regulation of immune effector systems since the class III gene products are essential for activation of the complement cascade.     

Localization of C4 genes within the HLA complex by molecular genotyping

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.Abbreviations RFLP restriction fragment length polymorphisms - LCL lymphoblastoid cell lines     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

Strong associations between RFLP and protein polymorphisms for CD46

Human CD46 (membrane cofactor protein) is a cell surface glycoprotein with cofactor activity for the factor I mediated cleavage of components C3b and C4b. Using a CD46 cDNA clone, three restriction enzymes give simple two allele restriction fragment length polymorphisms (RFLPs) in samples of over 300 Caucasians. For Pvu II, P1 with a 16.5 kilobase (kb) fragment and P2 with 14.8 kb + 1.9 kb fragments have frequencies of .40 and .60. For Hin dIII, H1 with a 4.3 kb fragment and H2 with a 2.3 kb fragment have similar frequencies. For Bgl. II, B1 with a 10 kb fragment and B2 with 8.3 kb + 1.8 kb fragments have frequencies of 0.08 and 0.92. There is strong linkage disequilibrium between these polymorphic sites. Designating haplotypes by Hin dIII, Pvu II, Bgl II alleles, there are two common haplotypes P2, H2, B2 and P1, H1, B2, expected at frequencies of .6 and .32, one less common haplotype P1, H1, B1 expected at a frequency .08. The two major protein isoforms of CD46, as detected on peripheral blood lymphocytes by western blot, of M _r 66 000 () and 56 000 () are determined by differential splicing in production of the mRNA. A strong association between protein isoform and RFLP haplotypes in 30 unrelated subjects suggests that the splicing preference site is in linkage disequilibrium with the RFLPs. The results are consistent with haplotypes P2, H2, B2 and P1, H1, B1 producing predominantly ; P1, H1, B2 producing predominantly in about 72% of cases and in 28% of cases. Address correspondence and offprint requests to: A. Wilton, at the present address.     

Restriction fragment length polymorphism of the human T cell receptor alpha gene

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3 joining (J) exon and the most 5 constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3 untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.Abbreviations used in this paper RFLP restriction fragment length polymorphism - Ti-alpha alpha gene of the T-cell receptor for antigen     

Genetics of human C4 polymorphism: Detection and segregation of rare and duplicated haplotypes

Applying a combined technology for the detection of allotypec variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B -chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 -chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p. i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p. i. and the C4A1 and C4B51 allotypes as Ch p. i. were genetically inherited. The C4A1 allotype has a unique Rg^- Ch⁺ C4A -chain. Duplicated C4A loci, A ^*3, A ^*2, and A ^*5, A ^*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.Abbreviations used in this paper BF Factor B polymorphism of the alternative pathway of complement activation - C2 second component of complement - C4 fourth component of complement - C4D C4-deficient (C4^*QO/QO) - Ch Chido determinant on C4B^* products - EDTA ethylendiaminetetraacetic acid - GLO I glyoxalase I - HLA human leucocyte antigens, A, B, C and DR (D =related) loci - PAGE polyacrylamide gel electrophoresis - PGM3 phosphoglucomutase, third locus - p. i. partial inhibitor = serological inhibition of some, but not all anti-Ch and anti-Rg sera at selected dilutions - SDS sodium dodecyl sulphate; 94k/96k, 94 000 and 96 000 dalton molecular weight Presented in part at the 1V International Workshop on the Genetics of Complement, July 13–15, 1982, Boston, MA, and the Xth International Complement Workshop, May 25–27,1983 in Mainz, Federal Republic of Germany.     

Analysis of the C4 genes in baleen whales using a human cDNA probe

We have used a human C4 cDNA probe to investigate the complement component C4 gene in four members of the family Balaenopteridae: fin whale (Balaenoptera physalus), sei whale (B. borealis), minke whale (B. acutorostrata), and bryde's whale (B. edeni). Restriction mapping of genomic DNA from the first three species suggests the presence of only one locus in these species, and also shows that the C4 genes in the three species are very similar. We have used 14 restriction endonucleases to investigate the restriction fragment length polymorphism (RFLP) of fin whales, 13 enzymes for sei whales, and 8 enzymes for the minke whale. No polymorphism was seen in DNA from the five minke whale samples, but Rsa I and Taq I restriction enzymes gave polymorphism in fin and sei whales whereas Hind III and Msp I restriction enzymes showed polymorphism in sei whales only. Only one bryde's whale sample was available for investigation. The study of DNA available from mother-fetus pairs from the two polymorphic species demonstrated a simple, two-allele transmission of RFLP alleles.     

Molecular characterization of three Mhc class II B haplotypes in the ring-necked pheasant

We investigated the class II B genes in free-ranging population of the ring-necked pheasant Phasianus colchicus by a combination of restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), and DNA sequencing. Special attention was paid to the variation in the second exon, which encodes the peptide-binding ₁-domain. The population was introduced, but it still exhibited major histocompatibility complex polymorphism with at least three segregating class II B haplotypes and consequently six genotypes. We found two class II B genes associated with each haplotype. The class II B genes of birds had until then only been molecularly characterized in the domestic chicken. the pheasant genes were highly variable, although one of the amplified sequences was found in two different haplotypes. Taken together, the most polymorphic positions (residues 37 and 38) were not identical in any of the predicted protein sequences, but all except one of the motifs had already been foud in the domestic chicken. Structurally important features in mammalian class II B genes were generally conserved also in the pheasant sequences, but the loss of a potential salt bridge constituent (Arg₇₂) in several sequences may suggest a slightly different structure of the adjacent parts of the peptide-binding groove. The pheasant genes are most closely related to the so called B-LBII family in the chicken, indicating that this represents a major line of development among avian class II B genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X75403-X75407. Correspondence to: H. Wittzell, Department of Theoretical Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden.     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

DNA polymorphisms and linkage relationship of the human complement componentC6,C7, andC9 genes

In this report we describe the linkage between genes encoding human complement componentsC6,C7, andC9. Polymorphisms have been described at the DNA level for theC7 andC9 genes. We have studied 20 individuals by Southern blot analysis with fourC6 cDNA subclones to detect restriction fragment length polymorphisms (RFLPs). We have found a Taq I polymorphism defined by two alleles of 8.0 (C6 H) and 6.0 (C6 L) kilobases (kb). RFLP segregation for theC6, C7, andC9 loci in informative families allowed us to estimate the maximum Lod scores at a recombination fraction of =0.0 (C6–C7), =0.0 (C7–C9), and =0.0 (C6–C9). Significant linkage disequilibrium was found betweenC6 andC7 and betweenC7 andC9 loci in directly determined haplotypes of unrelated parents. Data from this study show that the genes encoding the human terminal complement componentsC6, C7, andC9 define a cluster in the short arm of chromosome 5. We propose that the clusters involving theC8A andC8B and theC6, C7, andC9 genes be referred to as MACI and MACH, respectively.     

Association of membranous nephropathy with T-cell receptor constant β chain and immunoglobulin heavy chain switch region polymorphisms

We have investigated T-cell antigen receptor constant chain genes (Tcr C ) and immunoglobulin (Ig) heavy chain switch region genes of HLA-DR-typed patients with membranous nephropathy (MN) employing DNA restriction fragment length polymorphism (RFLP) analysis. When a Tcr C probe in conjunction with the restriction endonuclease BgI II was used, a significant increase in the frequency of a 10.0;9.2 kb heterozygous RFLP phenotype was found in MN (75.0 % versus 42.1 in controls; P=0.002). When Sst I-restricted DNA from MN patients was hybridized with a DNA probe homologous to the switch region flanking the Ig C _µ heavy chain gene (S _µ), there was a significant decrease in the frequency of the 2.1; 2.6 kb heterozygous RFLP phenotype in MN (24.0% versus 54.6% in controls; P=0.004). These results suggest that Tcr beta and Ig heavy chain loci, as well as HLA antigens, may be important in the pathogenesis of MN.     

A Taq1 γ-globin DNA polymorphism: an African-specific marker

Summary The allele frequency of a Taq 1 -globin gene restriction fragment length polymorphism (RFLP) is reported in ten population groups. In four African populations the 3.0 kb RFLP is common (50/132 {ie90-1} chromosomes), whereas it is completely absent in six European/Asian populations (0/277 {ie90-2} chromosomes). This Taq 1 RFLP is thus a specific African population marker.     

New chicken Rfp-Y haplotypes on the basis of MHC class II RFLP and MLC analyses

New chicken Rfp-Y haplotypes were determined by the use of restriction fragment length polymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B ¹⁵ , B ¹⁹ , B ²¹ , B ²⁰¹ . The RFLP polymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLP polymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information of the B1 domain of the proposed Rfp-Y haplotypes was obtained which supported the functional data.