HLA-Cw alleles associated with HLA extended haplotypes and C2 deficiency

Abstract: There are four MHC-linked complement genes, BF, C2, C4A and C4B, that are inherited as single DNA units, known as complotypes. Extended haplotypes were initially defined by studying the distribution of complotypes in relation to HLA-B and HLA-DR loci in Caucasian families. In order to analyze the distribution of HLA-Cw alleles in relation to extended haplotypes, we studied a large panel of MHC homozygous and heterozygous cell lines representing previously described Caucasian-derived extended haplotypes and 14 patients with complete C2 deficiency. HLA alleles were assigned using sequence-specific oligonucleotide probe hybridization (SSOP). Family analysis served to assign haplotypes for heterozygous samples. We found distinctive HLA-Cw alleles for each independent extended haplotype. Their association in each instance was statistically significant All patients with C2 deficiency carrying the haplotype [HLA-B18, S042, DR2] were associated with HLA-Cw*1203. These conserved allelic combinations may become an important tool for the study of human evolution and may contribute to the expeditious selection of prospective donors in clinical transplantation.     

Pathogenesis of autoimmune diseases associated with 8.1 ancestral haplotype: effect of multiple gene interactions

Genetic studies have shown that individuals with certain HLA alleles have a higher risk of specific autoimmune disease than those without these alleles. Particularly, the association in all Caucasian populations of an impressive number of autoimmune diseases with genes from the HLA-B8,DR3 haplotype that is part of the ancestral haplotype (AH) 8.1 HLA-A1, Cw7, B8, TNFAB*a2b3, TNFN*S, C2*C, Bf*s, C4A*Q0, C4B*1, DRB1*0301, DRB3*0101, DQA1*0501, DQB1*0201 has been reported by different research groups. This haplotype, the more common one in northern Europe, is also associated in healthy subjects with a number of immune system dysfunctions. It has been proposed that a small number of genes within the 8.1 AH modify immune responsiveness and hence affect multiple immunopathological diseases. In this paper, the characteristic features of this haplotype that might give rise to these diverse conditions are reviewed, focusing on the role of multiple gene interactions in disease susceptibility of 8.1 AH.     

DNA polymorphism of major histocompatibility complex class II and class III genes in systemic lupus erythematosus

We investigated the Taq I digested DNA restriction fragment length polymorphism (RFLP) of the Major Histocompatibility Complex (MHC) class II genes: HLA-DRB, -DQA, and the class III genes: C4 and 21-hydroxylase(CYP21) in 56 caucasoid patients with systemic lupus erythematosus (SLE) and 62 control subjects in order to define the molecular variation of these genes and their association with SLE. The results showed that the gene frequencies of both HLA-DR2 and -DR3 were significantly increased in the SLE population compared to normal subjects (DR2: 21.4% vs 10.7% chi 2 = 4.5. DR3: 29.6% vs 13.3%; chi 2 = 8.3). A high frequency of C4A and CYP21A gene deletions was also found in SLE patients (SLE 52%, normals 24%). All of 22 SLE patients, and 12 of 15 normal subjects who had C4A and CYP21A gene deletions had a 10.0kb Taq 1 DRB RFLP attributable to the presence of HLA-DR3. Family studies showed linkage of C4A/CYP21A deletions with HLA-B8 and -DR3, and confirmed the previously demonstrated association of the HLA-B8, DR3, C4A*Q0, C4*B1, Bf*S, C2*C haplotype with SLE. Deletions affecting the C4A and CYP21A genes were the commonest cause of C4A null alleles in SLE. No strong association between C4 null phenotype or C4 gene deletion, as determined by RFLP, was observed in patients who possessed DR2.     

HLA associations with inclusion body myositis

Inclusion body myositis (IBM) is defined clinically by a characteristic pattern of progressive proximal and distal limb muscle weakness and resistance to steroid therapy, and histologically by the presence of distinctive rimmed vacuoles and filamentous inclusions as well as a mononuclear infiltrate in which CD8⁺ T cells are predominant. Muscle damage is believed to be mediated by autoimmune mechanisms, but very little information is available on the immunogenic features of IBM. MHC class I and DR antigens were typed on 13 caucasoid patients with IBM using standard serological techniques or by allele-specific oligonucleotide typing. Complement components C4 and properdin factor B (Bf) were typed by immunofixation after electrophoresis. Restriction fragment length polymorphisms (RFLP) in the class ITT region were analysed using cDNA probes for C4 and 21-hydroxylase (CYP21) after Taq 1 digestion. IBM was associated with DR3 (92%), DR52 (100%) and HLA B8 (75%). The phenotype data were examined for likely haplotypes by considering together the alleles at the class T, DR and complement loci along with the C4 and CYP21 RFLP. Ten of the DR3⁺ subjects had a 6 4-kb C4-hybridizing fragment characteristic of a deletion of C4A and CYP21-A. These patients probably carried all, or at least the class II and III regions, of the extended haplotype marked by B8/C4A*Q0/C4Bl/BfS/DR3/DR52, which has been associated with several autoimmune diseases and is present in 11% of the healthy Caucasoid population. Of the remaining subjects, two had evidence of the extended haplotype marked by B]8/C4A3/C4BQ*0/BfFl/DR3, which is present in less than 5% of the healthy population and has been associated with insulin-dependent diabetes mellitus. These data provide support for an autoimmune etiology for, and genetic predisposition to, IBM.     

Complete inherited deficiency of the fourth complement component in a child with systemic lupus erythematosus and his disease-free brother in a North African family

Although null alleles of complement C4 genes (C4A ^*Q0 andC4B ^*Q0) are frequent in the normal population, the occurrence of two null alleles on the same chromosome is very rare and therefore complete C4 deficiency is exceptional. We describe a 16-year-old North African boy who presented with systemic lupus erythematosus with renal involvement and persistent undetectable classical pathway activity and C4 protein and hemolytic activity in plasma, with normal C3 levels. Similar complement abnormalities were observed in his healthy 12-year-old brother. Complete C4 deficiency was documented in the two brothers by investigation of the family and the lack of C4A and C4B bands upon phenotyping of C4. Southern blot analysis of the C4/CYP21 gene organization in the family indicated that the deficiency resulted from a deletion of the C4B/CYP21A genes associated with nonexpression of a C4A gene. The double-null haplotype was found to be associated with homozygous A2 B17 C2C BFF C4 AQO BQO DR7 HLA haplotype. Thus, similar C4 deficiencies with HLA identity may lead to different clinical presentations.     

The HLA-A3, Cw6, B47, DR7 extended haplotypes in salt losing 21-hydroxylase deficiency and in the Old Order Amish: identical class I antigens and class II alleles with at least two crossover sites in the class III region

Abstract: The HLA-B47, DR7 haplotype in congenital adrenal hyperplasia (CAH) due to 21–hydroxylase deficiency contains a deletion of most of the active CYP21 gene and the entire adjacent C4B gene. The C4A gene produces a protein which is electrophoretically C4A but anti-genically C4B. In the Old Order Amish, the HLA-B47. DR7 haplotype contains no deletion, but is immunologically identical to the CAH haplotype in both areas flanking the crossover region. We compared some of the genes in the MHC Class II and Class III regions in the Amish and CAH-linked haplotypes to define further the relationships between the two. The complement factor B (Bf) proteins differed, but no Bf RFLPs were identified. The complement factor 2 genes exhibited different BamHI RFLPs. Analyses of the tumor necrosis factor-α genes revealed the same Ncol restriction patterns. The RD genes contained microsatellites of the same size. Portions of the MHC Class II DR and DQ , and Class III CYP21 and C4 alleles were sequenced. The exon 2 sequences of DQ2 and DR7 were identical in the two haplotypes. In the Amish haplotype, both CYP21 and C4 gene pairs were present and functionally normal. The CAH haplotype had two sequence crossovers: from CYP21P to CYP21 in the 7th intron, and from C4A to C4B between codons 1106 (exon 26) and 1157 (exon 28). A model is proposed which accounts for the CAH-linked mutant haplotype arising from a nonmutant homologue via three crossings-over.     

C4 polymorphism and extended HLA haplotypes in Namibian San and Khoi and in South African Xhosa

We studied C4A and C4B polymorphisms and HLA-B and -DR associations in the San, Khoi and Xhosa. C4A and C4B alleles were determined using conventional protein allotyping methods. The C4A*3, C4B*1 haplotype had a high frequency (30–55%) in all populations. The frequency of C4A*3, C4B*Q0 was 7–19%. The C4A*Q0, C4B*1 haplotype was frequent (15%) in the Khoi but very rare in the San (P0.001). C4A*12 A*91, C4B*Q0 was frequent in the Xhosa (15%) but rare in the San and Khoi (P0.001). Alleles C4A*5 and C4A*6, and the C4B*2 B*92 duplication were only found in the Xhosa. C4A alleles A*4, A*45, A*58, A*12, A*14, A*19 and the C4A*3 A*91 duplication were only found in the San/Khoi population group. In the San, fourteen extended haplotypes were found in a relatively high frequency (2–7%). In the Xhosa, one extended haplotype (B42, C4A*12 A*91, C4B*Q0, DR18) was found in a very high frequency (13%) and was characteristic for this group; five other extended haplotypes were found with a low frequency (3%).     

A new duplication at the C4B locus associated with the HLA-Aw68, Cw8, Bw65 haplotype

A family with a cross-over between HLA-B and HLA-DR was analysed for its complement alleles. This allowed location of the cross-over between HLA-B and C4. Furthermore, the same family showed a previously undescribed duplication at the C4B locus (C4B* 2,2) that was associated with the HLA-Aw68, Cw8, Bw65, C2*1, Bf*S, C4A*2, DR7, DQw2 haplotype.     

C4 polymorphism and major histocompatibility complex haplotypes in IgA deficiency: association with C4A null haplotypes.

IgA-deficient individuals (n = 110) and six families comprising 9 cases of IgA deficiency were typed for HLA-A, -B, -DR, C4 and factor B. Phenotype frequencies were increased for HLA-B8 (p = 0.004), HLA-DR3 (p = 0.001) and homozygous C4AQ0 (p = 0.01) and decreased for HLA-B7 (p = 0.004), HLA-DR2 (p = 0.0001) and C4A3 (p = 0.00007) compared to controls. Homozygous C4A deficiency was found in 20% of IgA-deficient persons. As clearly suggested by investigation of families, the findings could be attributed to high prevalence of the extended major histocompatibility complex (MHC) haplotype [HLA-A1, B8, C4AQ0, C4B1, BfS, DR3] in IgA deficiency. All but 1 of the 9 IgA-deficient persons included in the family study carried this haplotype and 4 of them were homozygous. In the families, 3 persons with normal serum IgA concentrations had the same MHC haplotypes as their IgA-deficient relatives. The findings were also consistent with possible overrepresentation of other MHC haplotypes with aberrant C4 gene organization in IgA deficiency. As previously suggested, the presence of two MHC haplotypes associated with IgA deficiency appears to be a necessary but not sufficient requirement for manifestation of the condition. The putative existence of a recessive gene in the MHC with regulatory function with regard to IgA gene expression is consistent with the findings.     

Steroid 21-hydroxylase deficiency and the major histocompatibility complex

Steroid 21-hydroxylase (21-OHase) deficiency is an HLA-linked recessive disorder of cortisol biosynthesis that can occur in several forms which differ in severity. Because they are in genetic linkage disequilibrium with different HLA antigens, the inheritance of these forms is consistent with the existence of several alleles at a single locus. When severe 21-OHase deficiency occurs in association with the HLA haplotype A3;Bw47:DR7, there is a simulataneous null allelle at one of the C4 loci. This was hypothesized to result from a single deletion or rearrangement affecting the 21-OHase and C4 loci and perhaps the HLA-B gene as well. To test this hypothesis and identify the 21-OHase gene, a cDNA clone was isolated that encoded the cytochrome P450 specific for steroid 21-hydroxylation in the bovine adrenal gland. This clone hybridized to two genese in normal human DNA, but to only one gene in DNA from an individual homozygous for A3;Bw47;DR7. All individuals heterozygous for A3;Bw47;DR7 carry a heterozygous deletion of a cytochrome P450 gene. Cosmid clones have been used to locate the 21-OHase genes both in man and mouse. It both species, there are two 21-OHase genes, each located immediately 3′ of one of the two C4 genes, and oriented in the same direction as the C4 genes. In man, the gene located 3′ of the C4B gene is deleted in 21-OHase deficiency on the Bw47 haplotype, but the gene 3′ of the C4A gene is deleted in hormonally normal individuals on the A1;B8;C4AQ0, C4B1;DR3 haplotype. Thus the 21-OHase B gene is normally active in man, but the 21-OHase A gene is not.     

Some disease-associated ancestral haplotypes carry a polymorphism of TNF

We describe here an Nco I restriction fragment length polymorphism of tumor necrosis factor carried by the 8.1 (HLA-A1,B8,BfS,C4AQ0,C4B1,DR3) and the 44.1 (HLA-B44,BfS,C4A3,C4BQ0,DR4) ancestral haplotypes associated with complications of rheumatoid arthritis. By examining multiple examples of these and other ancestral haplotypes it was seen that 8.1 and 44.1 ancestral haplotypes yield fragments of approximately 5.5 kb while many other ancestral haplotypes carry fragments of approximately 10.5 kb. The polymorphism is associated with the ancestral haplotype rather than the HLA-B or -DR allele defined by conventional serology.     

Extended HLA haplotypes in families with insulin-dependent diabetes mellitus in Northern Finland

Haplotypes including HLA, Bf and C4 loci were analyzed in a material comprising 55 families with diabetic children. One hundred and ten haplotypes found in IDDM patients were compared with 101 haplotypes present only in healthy family members. Two complotypes, BfSC4A3B3 and SC4A0B1 . were significantly more common (P <0.05) in the diabetic haplotypes, and these were in most cases found in haplotypic combinations with HLA-B15,Dw4,DR4 and HLA-B8.Dw3,DR3 genes, respectively. The B8/DR3 haplotype was better conserved, as 72% included the BfSC4A0B1 complotype as compared with only 35% of the B15/DR4 haplotypes with "high risk" C4A3B3 complement alleles (p <0.05). DR3 was found in 26% of the diabetic haplotypes and DR4 in 43%. DR4 associated with the Dw4 in 69% of cases and with D w14 in 26% of the diabetic haplotypes. Our results confirm that the two phenotypes found earlier to be associated with IDDM in Northern Finland, e.g. "B15, BfS, C4A3B3, Dw4, DR4" and "B8, BfS, C4A0B1, Dw3, DR3" are inherited as haplotypes.     

Complement component C6 and C7 haplotypes associated with deficiencies of C6

Both complete C6-deficiency (C6*Q0) and subtotal C6-deficiency (C6*SD) have been described as simple recessive traits and C6*SD has been described in combination with subtotal deficiency of the C7 coded at an adjacent locus. The trace of C6 protein found in both C6*SD traits is phenotypically indistinguishable, being smaller than normal C6 and having different isoelectric properties. A defect has been found in the C6 gene which plausibly explains the C6*SD phenotype, and this defect is also common to both C6*SD traits. We present data from seven DNA markers of the C6 and C7 genes which show that although at least four haplotypes are associated with C6*Q0, most South African C6*Q0 patients carry a common defective haplotype. The most common haplotype associated with C6*Q0 has been observed only once among unaffected haplotypes of relatives. In one family, the cases of C6*SD share a complete haplotype with both cases of combined deficiency and are probably heterozygous for this condition and complete deficiency of C6. In another family, the C6*SD is on a slightly different haplotype and C7 is normally expressed. Thus, the C6 defect is not sufficient on its own to explain the C7 deficiency in the combined deficient haplotype. The haplotype associated with the combined deficiency is found not only in normal control subjects, but also in one case of complete C6 deficiency. In this case the molecular defect seen in combined or C6*SD cases is absent.     

Molecular basis of complete C4 deficiency. A study of three patients

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes, C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes and is also remarkable by the high frequency of the null alleles, C4A*Q0 and C4B*Q0. Complete C4 deficiency is exceptional because this condition appears only in homozygotes for the very rare double-null haplotype C4AQ0,BQ0. This condition in most cases gives rise to systemic lupus erythematosus and an increased susceptibility to infections. The molecular basis for complete C4 deficiency has not yet been established. Therefore we studied the DNA of three previously described C4 deficient patients belonging to unrelated families by restriction fragment length polymorphism analysis using C4 and 21-OH probes. These studies revealed a deletion of the C4B and 21-OHA genes in two patients and no deletion at all in the third patient. Therefore, complete C4 deficiency as a result of homozygosity for the C4AQ0, BQ0 haplotype is not a consequence of a deletion of the C4 genes. The molecular basis of this genetic abnormality is certainly very complex and may vary also from one case to another.     

Haplotypes bearing HLA-A, -B, and -DR: Bf and C4 genes in rheumatoid arthritis families

We have compared haplotypes bearing HLA-A, -B, -DR; Bf and C4 genes in 54 rheumatoid arthritis (RA) and 24 control families. There was no statistically significant differences in C4A or C4B gene frequencies between RA and control groups, although there were trends for C4B*Q0 to be reduced and C4B2 to be increased in DR4 positive RA compared with DR4 positive controls. The lack of any strong association between C4 variants and RA overall makes it unlikely that the association between RA and genes within the MHS represents a direct effect of variants within the C4A or C4B loci themselves. On comparison of DR4-bearing haplotypes, the haplotype B15-BfS-DR4 was increased fourfold and the B44-Bfs-DR4 haplotype was less frequent in the RA group. When C4 variants were also considered, the haplotype B44-C4B*Q0-C4A3-BfS-DR4 was nine times less frequent in RA patients than in controls. The observation that different DR4 bearing haplotypes may confer either increased or decreased susceptibility to RA suggests either that it is unlikely that DR4 itself is involved in the disease process or that specific haplotypic combinations are important. Thirty-two RA patients were HLA-DR4 negative. No single DR4 negative haplotype was found to confer significantly increased susceptibility to RA.     

C4 complement allotypes in juvenile dermatomyositis

Twenty probands with juvenile dermatomyositis and their relatives were studied to determine the inherited segregation patterns of class I, II, and III HLA region markers including C4A, C4B, Bf, and C2 complement polymorphisms. The extended haplotype B8, DR3, C4A*Q0, C4B*1, C2*C, and Bf*S was present in 13 of the 20 probands. Three other probands also carried a haplotype with a null allele for C4A and two further probands carried a null allele for C4B; only two probands had no detectable C4 null allele. These data confirm previous studies showing high frequencies of B8 and DR3 in patients with juvenile dermatomyositis, but show that there is a higher association with null alleles of C4. This suggests that the C4 genes are either themselves the disease-susceptibility genes or are in very strong linkage disequilibrium with such genes.     

Immunoglobulin deficiencies and susceptibility to infection among homozygotes and heterozygotes for C2 deficiency

About 25% of C2-deficient homozygotes have increased susceptibility to severe bacterial infections. C2-deficient homozygotes had significantly lower serum levels of IgG2, IgG4, IgD, and Factor B, significantly higher levels of IgA and IgG3 and levels of IgG1 and IgM similar to controls. Type I (28 bp deletion in C2 exon 6 on the [HLA-B18, S042, DR2] haplotype or its fragments) and type II (non-type I) C2-deficient patients with increased susceptibility to bacterial infection had significantly lower mean levels of IgG4 (p < 0.04) and IgA (p < 0.01) than those without infections (who had a higher than normal mean IgA level) but similar mean levels of other immunoglobulins and Factor B. Of 13 C2-deficient homozygotes with infections, 85% had IgG4 deficiency, compared with 64% of 25 without infections. IgD deficiency was equally extraordinarily common among infection-prone (50%) and noninfection-prone (70%) homozygous type I C2-deficient patients. IgD deficiency was also common (35%) among 31 type I C2-deficient heterozygotes (with normal or type II haplotypes), but was not found in 5 type II C2-deficient heterozygotes or 1 homozygote. Thus, C2 deficiency itself is associated with many abnormalities in serum immunoglobulin levels, some of which, such as in IgG4 and IgA, may contribute to increased susceptibility to infection. In contrast, IgD deficiency appears not to contribute to increased infections and appears to be a dominant trait determined by a gene or genes on the extended major histocompatibility complex (MHC) haplotype [HLA-B18, S042, DR2] (but probably not on type II C2-deficient haplotypes) similar to those previously identified on [HLA-B8, SC01, DR3] and [HLA-B18, F1C30, DR3].     

Studies of a C2 DNA polymorphism in RA, Felty's and normal subjects

A restriction fragment length polymorphism at the C2 locus was studied in rheumatoid arthritis (RA), Felty's and control subjects. No association was found between any C2 variant and either RA itself or within the rheumatoid population with Felty's syndrome. The C2 DNA polymorphism can be used to subdivide Bf*S- as well as Bf*F-bearing haplotypes. The 2.65-kb C2 DNA allele showed allelic association with HLA-B44 and C4B*Q0 and may help to further characterize the haplotype B44-Bf*S-C4A*3-C4B*Q0-DR4, which has previously been described in Felty's syndrome.     

Sequence, genetics and serology of a new HLA-B allele: B*4903

A new HLA-B allele - B*4903 - was detected by the polymerase chain reaction using sequence-specific priming (PCR-SSP), in a Caucasoid bone marrow panel donor, that differs from B*4901 by 8 nucleotides at positions 141, 142, 144, 165, 167, 193, 206 and 213 in exon 2. These substitutions all occur in HLA-B*51 and B*52 alleles and encode 4 amino acid substitutions at positions 24 (Thr to Ala), 32 (Leu to Gln), 41 (Thr to Ala) and 45 (Lys to Thr). This suggests that B*4903 occurred following a gene conversion-like event involving B*4901 and probably a B*51 allele. HLA-B*4903 was identified on a haplotype with: HLA-A*0201; Cw*07; DRB1*1302/34; DRB3*0301; DQA1*0102; DQB1*0604; BfS; C4A3; C4BQ0 and encodes a unique serological specificity which was characterised by the reactivity of 55 antisera directed towards at least four predicted epitopes. No further examples of B*4903 were found in 15,796 consecutive HLA PCR-SSP typed donors from the Welsh Bone Marrow Donor Registry, indicating that this allele has a phenotype frequency of <0.01% and a gene frequency of <0.00004.     

Polymorphism of C4 and factor B in type I diabetes

The haplotypic frequencies of the fourth component of complement (C4) and factor B (Bf) have been determined in 44 Alsatian type 1 diabetics. An increased frequency of the rare allele Bf F1 (9.1% vs 1.5%) and of the silent alleles of C4 (C4 AQO: 21.6% vers 15.5% -C4 BQO: 29.6% vs 16.0%) was observed in diabetics in comparison to the general population of the same geographic area. A complete HLA haplotype determination has been obtained in 24 type 1 French diabetics. Three haplotypes were associated with the diabetic susceptibility: HLA-A30 CW5 B18 BfF1 C4A3BQO DR3 (18.75% vs 0.86%), HLA-A1 CW7 B8 BfS C4AQOB1 DR3 (15.58% vs 4.17%), HLA-A2 CW3 BW62 BfS C4A3B3 DR4 (6.25% vs 0.45%). The authors suggest that the silent alleles of C4 could modulate the expression of the diabetic susceptibility genes by lowering of the serum C4 hemolytic activity.