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 natural history of an HLA haplotype and its recombinants

 The presence of haplotype-specific recombination sites can be determined by analyzing the conservation of extended haplotypes in the population. This approach considers all meioses in the history of the population and requires the presence of characteristic markers that easily allow the identification of the haplotype or of its recombined segments. The recombination breakpoint can then be mapped by looking for shared alleles between haplotypes selected through the specific marker/s. We identified a rare perfect tandem duplication of a 145 base pair segment in the LTA promoter, which tags a B60 (B60D) haplotype. The duplication was detected in 16/90 B60 ⁺ Europeans, while absent in 101 B60 ⁺ Orientals. The conservation of the class I end and the extreme variability of the class II end suggested that the present-day B60D haplotypes originated from an ancestral haplotype by recombination events centromeric to the duplicated sequence. Through a fine mapping using markers of the HLA central region a preferential recombination site was localized in the 60 kilobase interval between TNFd,e, and D6S273/K11 Amicrosatellite loci (i.e., between LST1 and BAT3 genes). This site behaves as a potent recombination enhancer leading to fragmentation in most of the extant B60D haplotypes and can be considered responsible for their “instability”. In the relatively recently founded Finnish population, where the LST1/BAT3 interval recombination has probably not yet had the chance to occur, a founder effect can explain the presence of a rare DP (DPB1 ^* 1601) allele in most B60D haplotypes in this population. Received: 15 October 1997 / Revised: 23 December 1997     

Immunofixation for C2 typing: C2 allotypes in Spaniards in relation to HLA,Bf and C4

Summary C2 typing is performed by immunofixation with anti-C2 antiserum instead of by a hemolytic overlay. This method gives sharp band definition, is less cumbersome than the hemolytic overlay, gel files are easily made, and it also enables one to describe putative new nonhemolytic variants. C2 allele frequencies were studied in a sample of the normal Spanish population and were found to be similar to other Caucasoids. HLA-Bw62,-Cw3, and-DR4 were significantly associated with C2 B. Concordantly, the only C2^*B extended HLA haplotype found in family material was Bw62-Cw3-Bw6-(DR4)-Bf^*S-C2^*B-C4A^*3 B^*2-(GLO^*1). C4A^*4 B^*2 and C4A^*4 B^*4 are not found within the same haplotype together with C2^*B and Bw62 or Bw22 respectively, nor do other C2^*B haplotypes occur with common HLA-B alleles. These results may favour the hypothesis that the Bw62-C2^*B haplotype is produced by one mutation arising in the Bw62-C2^*C haplotype and that subsequent crossovers can explain other C2^*B haplotypes (including Bw22-C2^*B).     

Human BF*F-subtypes: segregation analysis with inclusion of MHC haplotypes

Summary The segregation of factor B(BF)F subtypes was analyzed in conjunction with other MHC markers in 15 families with 89 offspring. Informative data for BF F subtypes were obtained from 11 families, 6 of them with known recombinant individuals for the HLA-B/DR/GLO region. The subtypes did not contribute further to the localization of the cross-overs, but followed the known segregation of conventional BF allotypes. In 2 families of one kinship, the recognition of heterozygous BF^*FAFB individuals could be established following the inclusion of three generations. The rarer of the two BF F subtype alleles, BF^*FA, is positively associated with the HLA haplotypes BW62, CW3, C4A^*3 and A29, CWX, B44, C4A^*3, B^*1, DR7. BF F subtypes are regarded as a very useful additional tool for studies of MHC organization and disease association.     

Haplotypes of the BoLA‐A,Bf, CYP21 and DQB loci in Angus cattle

Abstract

The genetic diversity of bovine major histocompatibility complex (BoLA) haplotypes within the Angus breed was studied. Four BoLA loci were chosen for our study; BoLA‐A, complement factor B (Bf), cytochrome P450 steroid 21‐hydroxylase (CYP21) and BoLA‐DQB. Polymorphism of BoLA‐A products was determined by serology. Alleles of the Bf, CYP21 and BoLA‐DQB loci were distinguished by restriction fragment length polymorphism (RFLP) analysis. Thirteen different haplotypes were identified, eleven of which were confirmed by segregation analysis in a paternal half‐sibling family of Angus cattle (n=9 offspring). These thirteen haplotypes were comprised of 9 BoLA‐A alleles and a “blank”, two Bf alleles detected with TaqI, two CYP21 alleles detected with PstI, and eight DQB alleles detected with Taql and PvuII. Two haplotypes containing the supertypic BoLA‐URAA specificity were clearly differentiated by Bf and DQB typing. Two haplotypes distinguished by the BoLA‐A alleles w2 and w3, shared the same DQB?2 allele. Nine and four haplotypes carried the Bf?1 and Bf?2 alleles, respectively. Three haplotypes carried CYP21?A and ten contained CYP21?B. Linkage between BoLA‐A and the CYP21 locus, both previously mapped to chromosome 23 by synteny mapping, was confirmed by segregation analysis. These results demonstrate that extended BoLA haplotypes are useful for studying genetic diversity within a breed.     

TNFB polymorphisms characterize three lineages of TNF region microsatellite haplotypes

 The human major histocompatibility complex (MHC) contains a variety of genes, many of which are highly polymorphic and of immunological importance. A database of MHC extended haplotypes was used to integrate experimental, cell line, and population data. Three alleles of the human TNF-beta (lymphotoxin-alpha) gene were identified, named TNFB ^*1SL, TNFB ^*2LL, and TNFB ^*1LS, each representing a different lineage in the evolution of TNF region haplotypes. Lower variability in the length of the associated microsatellite alleles indicates that ^*1SL characterizes the youngest of the three haplotype lineages. Microsatellite haplotypes in the two older lineages show evidence for a coevolution of alleles through concerted expansions. Genetic predispositions to high and low TNF-alpha (cachectin) responses seem to have evolved independently in more than one lineage. The literature data suggest different, or even opposite, associations concerning the regulation of TNF-alpha in macrophages and lymphoid cells. Microsatellite ud may be the most informative marker for studies of the associations of individual TNF region markers with secretion levels, immunity, and disease. Received: 10 December 1996 / Revised: 21 May 1997     

Population-specific recombination sites within the human MHC region

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.     

Duplication of the MHC-linked Xenopus complement factor B gene

We have previously reported the molecular cloning of the mammalian major histocompatibility complex (MHC) class III gene, complement factor B (Bf) from Xenopus laevis, and linkage of the gene to the frog MHC. Here, we estimated the copy number of the Xenopus Bf gene by genomic Southern blotting analysis and demonstrated that Xenopus laevis has two copies of the Bf gene. Both genes co-segregated with the MHC-linked HSP70 genes among 19 offspring of an f/r × f/r cross, indicating a close linkage of the two Bf genes to the frog MHC. Both genes are transcribed and contain open reading frames. When compared with the previously determined cDNA sequence (Xenopus Bf A), the predicted amino acid sequence of the second cDNA species (Xenopus Bf B) shows 82% overall identity. Polymerase chain reaction analysis indicated that all of the partially inbred frogs with the f, r, g, and j MHC haplotypes, as well as 12 outbred frogs tested have both Bf genes, suggesting that the duplicated Bf genes are stable genetic traits in Xenopus laevis.     

Sequence-based association analysis of HLA class I and II alleles in Japanese supports conservation of common haplotypes

 Alleles of HLA-A, B, C, DRB1, DQB1, and DPB1 loci were fully determined in 117 healthy Japanese. A ^* 2402, A ^* 3303, A ^* 1101, A ^* 0201, B ^* 4403, B ^* 5201, Cw ^* 0102, Cw ^* 1403, Cw ^* 0304, Cw ^* 0702, Cw ^* 0801, and Cw ^* 1202 showed frequencies of over 10%. Multi-locus haplotype frequencies were estimated by the maximum likelihood method. Strength of association between C and B loci was comparable with that between DRB1 and DQB1 loci. Alleles unidentified by a serological method and having very similar nucleotide sequences (A2: A ^* 0201, A ^* 0206, A ^* 0207, B61: B ^* 4002, B ^* 4006) were carried by different haplotypes. Several frequent five-locus haplotypes were identified including A ^* 3303-Cw ^* 1403-B ^* 4403-DRB1 ^* 1302-DQB1 ^* 0604, and A ^* 2402-Cw ^* 1202-B ^* 5201-DRB1 ^* 1502-DQB1 ^* 0601. These sequence-based haplotypes corresponded to serology-based common haplotypes which have already been described in Japanese. These findings indicate that common HLA haplotypes consist of particular sets of HLA alleles and that these haplotypes have been conserved through recent human evolution. Received: 25 November 1996 / Revised: 20 January 1997     

Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family

Summary A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F ^* 3 C4F ^*2.2 is composed of two C4F alleles and the other C4S ^* 5.1 C4S ^*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB ^*35, Bf ^* F, and HLAD/DR ^*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(a+) variant.This work was supported by grant No 12-1727 from the Danish Medical Research Council     

HLA Haplotypes with C4B5; evidence for further allelic heterogeneity

Twenty-three individuals from various disease groups and normal controls were identified by immunofixation with anti-C4, C4-dependent lysis, determination of Rg (Rodgers) and Ch (Chido) phenotypes, and immunoblotting with C4-specific mouse monoclonal antibody. We found that one haplotype predominates with the C4B ^* 5 allele, HLA-A11, B22(55), Cw3, Bf ^* S, C4A ^* 4B ^* 5, which also carries the Ch ^{1,–2, 3} haplotype. The B5 allotype was also found with HLA-1360, HLA-1335 in Caucasoids, and HLA-B18 in non-Caucasoids; these carried the Ch ^{–1, –2, –3} haplotype. Our results are in accord with an earlier report of two B5 subtypes, B5Rg⁺ and B5Rg^– (Roos et al. 1984). The specificity of the mouse monoclonal antibodies IC4 and 21312 had been previously related to C4A and C4B, respectively, but our results suggest that they relate more closely to Rg and Ch determinants.     

Diversity of Extended HLA-DRB1 Haplotypes in the Finnish Population

The Major Histocompatibility Complex (MHC, 6p21) codes for traditional HLA and other host response related genes. The polymorphic HLA-DRB1 gene in MHC Class II has been associated with several complex diseases. In this study we focus on MHC haplotype structures in the Finnish population. We explore the variability of extended HLA-DRB1 haplotypes in relation to the other traditional HLA genes and a selected group of MHC class III genes. A total of 150 healthy Finnish individuals were included in the study. Subjects were genotyped for HLA alleles (HLA-A, -B, -DRB1, -DQB1, and -DPB1). The polymorphism of TNF, LTA, C4, BTNL2 and HLA-DRA genes was studied with 74 SNPs (single nucleotide polymorphism). The C4A and C4B gene copy numbers and a 2-bp silencing insertion at exon 29 in C4A gene were analysed with quantitative genomic realtime-PCR. The allele frequencies for each locus were calculated and haplotypes were constructed using both the traditional HLA alleles and SNP blocks. The most frequent Finnish A∼B∼DR -haplotype, uncommon in elsewhere in Europe, was A*03∼B*35∼DRB1*01∶01. The second most common haplotype was a common European ancestral haplotype AH 8.1 (A*01∼B*08∼DRB1*03∶01). Extended haplotypes containing HLA-B, TNF block, C4 and HLA-DPB1 strongly increased the number of HLA-DRB1 haplotypes showing variability in the extended HLA-DRB1 haplotype structures. On the contrary, BTNL2 block and HLA-DQB1 were more conserved showing linkage with the HLA-DRB1 alleles. We show that the use of HLA-DRB1 haplotypes rather than single HLA-DRB1 alleles is advantageous when studying the polymorphisms and LD patters of the MHC region. For disease association studies the HLA-DRB1 haplotypes with various MHC markers allows us to cluster haplotypes with functionally important gene variants such as C4 deficiency and cytokines TNF and LTA, and provides hypotheses for further assessment. Our study corroborates the importance of studying population-specific MHC haplotypes.     

Parakeratosis Mibelli and skin carcinoma

Summary Bf allele frequencies in a material of 172 unrelated Norwegians are given. Bf/HLA linkage relations in 49 informative matings with 178 children, and Bf/HLA association data of a material of 212 Bf-HLA haplotypes are presented. Of 171 informative meioses, there were no Bf-HLA-B recombinations, while 3 out of 158 Bf-HLA-A informative meioses showed recombination. There is significant association between the Bf ^F and the HLA-BW 35 allele.It is concluded that the Bf locus is situated on the HLA-B side of HLA-A within the HLA region, in very close proximity to HLA-B.     

Gene order in the major histocompatibility complex of the rat

The loci in the major histocompatibility complex (MHC) of the rat which code for class I and class II antigens—RT1.A and RT1.B, respectively — have previously been separated by laboratory-derived recombinants and by observations in inbred and wild rats. Closely linked to the MHC is the growth and reproduction complex (Grc) which contains genes influencing body size (dw-3) and fertility (ft). These phenotypic markers were used in this study to orient the A and B loci of the MHC. Two recombinants were used for mapping. The BIL(R1) animal is a recombinant between the MHC and Grc, and it carries the haplotype RT1.A ^lB^lGrc⁺. The r10 animal is an intra-MHC recombinant, and it has the haplotype RT1.A ⁿB¹ Grc. These recombinants were characterized serologically, by mixed lymphocyte reactivity, by immune responsiveness to poly (Glu⁵²Lys³³Tyr¹⁵) and by the presence of the dw-3 gene. The data demonstrate that the gene order of the loci is: dw-3-RT1.B-RT1.A.     

High KIR diversity in Amerindians is maintained using few gene-content haplotypes

Interaction between killer cell immunoglobulin-like receptors (KIR) and cognate HLA class I ligands influences the innate and adaptive immune response to infection. The KIR family varies in gene content and allelic polymorphism, thereby, distinguishing individuals and populations. KIR gene content was determined for 230 individuals from three Amerindian tribes from Venezuela: the Yucpa, Bari and Warao. Gene-content haplotypes could be assigned to 212 individuals (92%) because only five different haplotypes were present—group A and four group B. Six different haplotype combinations accounted for >80% of individuals. Each tribe has distinctive genotype frequencies. Despite few haplotypes, all 14 KIR genes are at high frequency in the three tribes, with the exception of 2DS3. Each population has an even frequency of group A and B haplotypes. Allele-level analysis of 3DL1/S1 distinguished five group A haplotypes and six group B haplotypes. The high frequency and divergence of the KIR haplotypes in the Amerindian tribes provide greater KIR diversity than is present in many larger populations. An extreme case being the Yucpa, for whom two gene-content haplotypes account for >90% of the population. These comprise the group A haplotype and a group B haplotype containing all the KIR genes, except 2DS3, that typify the group B haplotypes. Here is clear evidence for balancing selection on the KIR system and the biological importance of both A and B haplotypes for the survival of human populations.Electronic Supplementary Material Supplementary material is available for this article at     

Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes

After separating the *F and *S alleles by electrophoresis the allele-specific hemolytic activity was detected by agarose overlay method using the programmable densitometer for scanning. The hemolytic activity of BF allotypes was analyzed from 81 individuals. In thirteen FS heterozygous serum samples BF F had lower hemolysis than BF S. Four FF homozygous samples also exhibited lower hemolysis than a homozygous control sample. The low hemolytic activity of F in FS heterozygotes was not due to decreased protein concentrations relative to S. On the contrary, BF F was associated with higher protein concentration than BF S. The relative quantitation of the allele specific BF protein was done by crossed immunoelectrophoresis. BF F with low hemolytic activity but with high protein concentration associated strongly with HLA B35 phenotype and the family material confirmed the association with the haplotypes A3, Cw4, B35, DR1, BFFB, C4A3BQO (or A2BQO, A3,2BQO). The results suggest that particular MHC haplotypes contain a factor B allele with encoding for poor hemolytic activity or that MHC haplotype specific regulatory elements affect pre- or post-translational activity levels.     

Linkage disequilibrium of HLA-SB1 with the HLA-A1, B8, DR3, SCO1 and of HLA-SB4 with the HLA-A26, Bw38, Dw10, DR4, SC21 extended haplotypes

Homozygous typing cells from 13 normal HLA-A1, B8, Dw3, DR3 and five normal HLA-A26, Bw38, Dw10, DR4 individuals were typed for the following markers: HLA-SB, MB, MT; complement proteins BF, C2, C4A, C4B; and GLO. Ninety-one percent of A1, B8, Dw3, DR3 homozygous individuals (HI) tested were homozygous for BF ^* S, C2 ^* C, C4A ^* QO, and C4B ^*1 (SCO1 complotype), which indicates that the SCO1 complotype is in linkage disequilibrium with the A1, B8, DR3 haplotype in randomly selected normal populations. Sixty-seven percent of HLA-A1, B8, Dw3, DR3, SCO1 positive HI also expressed SB1; since the frequency of SB 1 in random Caucasian populations is 11.2%, this finding indicates that SB1 is in linkage disequilibrium with the A1, B8, DR3, SCO1 extended haplotype. All HI with the A26, Bw38, Dw10, DR4 haplotype were homozygous for both SC21 and SB4, suggesting that SC21 and SB4 should be included in the A26, Bw38, Dw10, DR4 extended haplotype. On the other hand, neither of the GLO markers were found in association with either haplotype. The results of this study indicate that HLA-SB is included in some extended haplotypes and may be important in these markers for diseases such as insulin-dependent diabetes mellitus. This study also demonstrated an apparent influence of HLA-SB on primary mixed lymphocyte culture (MLC) responses. The mean relative response of primary MLCs between individuals matched for HLA-A, B, D, DR, MB and MT but not SB was 40% of that for the MLCs with mismatched HLA-D, significantly higher than the MLCs matched for all HLA and complotypes.     

MHC-linked class III genes. Analysis of C4 gene frequencies,complotypes and associations with distinct HLA haplotypes in German Caucasians

The class III complement components, C4, C2 and factor B (BF), are encoded in the human major histocompatibility complex (MHC). The two genes determining C4 (C4A and C4B) display considerable polymorphism and, thus, are important markers for HLA. In combination with alleles of C2 and BF they can be grouped into unique complotypes. We have analyzed the C4 alleles in a panel of 204 unrelated German Caucasians and studied their segregation with HLA haplotypes in 24 normal families. Inclusion of the class III markers with the class I and 11 alleles provides a more refined picture of the genetic structure of the MHC in these families. When charted according to the HLA-B locus specificities the MHCs can be clustered into groups showing distinctly homogenous or heterogenous complotypes. The identification of such groups is valuable for the selection of genetic material to analyze the molecular genetics of the human MHC.Abbreviations BF factor B - C2 second component of complement - C4 fourth component of complement - EDTA ethylenediamine tetraacetate - GLO glyoxalase-I - MHC major histocompatibility complex     

HLA genotypes and HLA-linked genetic markers in Italian patients with classical 21-hydroxylase deficiency

Summary HLA genotype and HLA-linked marker data for 40 unrelated patients from central Italy and 2 unrelated patients from Sardinia with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) were analyzed. The results confirm that the HLA-linked 21-OH-def gene is associated with several different HLA determinants and complete HLA haplotypes, although the only determinant with significantly increased frequency was the complement C2 allele C2B. The HLA antigens B8 and DR3 were found in significantly decreased frequencies. The haplotype A3, Cw6, Bw47, BfF, DR7, which is exceptionally rare in the general population but which has been found in many other 21-OH-def patients from diverse geographical origins, was also found in one of the Italian patients. This and other HLA haplotype associations found among the Italian patients may represent mutations that have occurred on HLA haplotypes with genetic linkage disequilibrium or, alternatively, may represent mutations that have not yet had time to become randomly associated with different HLA complex determinants. The marked negative associations with B8 and DR3 could, however, result from an interaction between the gene products of the HLA complex and the 21-OH-def phenotype.