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.     

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.     

Human peritoneal macrophages synthesize leukotrienes B4 and C4

Macrophages were isolated from the dialysis fluid of patients undergoing continuous ambulatory peritoneal dialysis and separated by gradient centrifugation and purification on 50% Percoll. The cells were prelabeled with [14C]arachidonic acid for 1.5 h. The labeled cells were then incubated with calcium ionophore A23187 (1 microM), serum-treated zymosan (200 micrograms/ml), and a lipoxygenase inhibitor, nordihydroguairetic acid (1 X 10(-5) M). The arachidonate metabolites in the medium were separated on Sep-Pak columns, and finally purified by reverse-phase high-pressure liquid chromatography (HPLC). The labeled products co-chromatographed with authentic leukotriene B4 and leukotriene C4 standards. Serum-treated zymosan and A23187 significantly stimulated and nordihydroguairetic acid significantly inhibited leukotriene synthesis. Leukotriene D4 was not detected, which suggests that these cells contain low gamma-glutamyltranspeptidase or high dipeptidase activity. These results establish, for the first time, that human peritoneal macrophages synthesize the lipoxygenase products, leukotriene B4 and leukotriene C4.     

Gene organization of haplotypes expressing two different C4A allotypes

Summary The gene organization of C4 haplotypes expressing two different C4A allotypes with a C4B null allele (C4A3A2-BQ0 and C4A3A6BQO) was studied using Southern blot analysis with cDNA probes and restriction enzymes which give C4A and C4B locus-specific restriction fragments. These haplotypes were shown to have both a C4A and a C4B locus present, suggesting that the C4B locus expresses a C4A protein. The finding of a 21-OH A and a 21-OH B gene on the C4A3A6BQO haplotype further suggests that this haplotype has the common gene organization C4A, 21-OH A, C4B, 21-OH B. A model explaining C4 null alleles on haplotypes found to have two C4 loci is presented.     

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.     

Quantitative variation of C4 variant proteins associated with many MHC haplotypes

C4 protein variants were analyzed in 64 individuals, of which 51 were either homozygous or heterozygous for an extended major histocompatibility complex (MHC) haplotype (a fixed combination of MHC alleles). The relative amount of each C4 variant was measured by densitometric scanning of stained immunofixed electrophoretic patterns of neuraminidase- and carboxypeptidase-treated samples. The relative concentrations of C4 variants on any haplotype were stable and inherited in families. In five of the eight extended haplotypes investigated, the amount of one of the C4 variants relative to others in the same pattern was increased:[HLA-B8, SC01, DR3] and[HLA-B7, SC31, DR2] produced an approximately doubled amount of C4B1;[HLA-B18, S042, DR2] an increased amount of C4B2; and[HLA-B44, SC30, DR4] a double amount of C4A3. The extended haplotype[HLA-Bw57, SC61, DR7] gave rise to two to three times as much C4B1 as C4A6. In the extended haplotypes[HLA-B44, FC31, DR7] and[HLA-Bw62, SC33, DR4], the results did not clearly indicate differences in expression of the C4 isotypes. DNA analysis possibly supported an actual gene duplication only for the haplotype[HLA-B7, SC31, DR2]. The results suggest that, in addition to variation in the number of structural genes, other MHC-linked mechanisms may be involved in the regulation of the relative amounts of C4A or C4B protein specified by any haplotype.     

The molecular basis of complete complement C4A and C4B deficiencies in a systemic lupus erythematosus patient with homozygous C4A and C4B mutant genes

The disease course of a complete C4-deficient patient in the U.S. was followed for 18 years. The patient experienced multiple episodes of infection, and he was diagnosed with systemic lupus erythematosus at age 9 years. The disease progressed to WHO class III mild lupus nephritis and to fatal CNS vasculitis at age 23 years. Immunochemical experiments showed that the patient and his sibling had complete absence of C4A and C4B proteins and were negative for the Rodgers and Chido blood group Ags. Segregation and definitive RFLP analyses demonstrated that the patient and his sibling inherited two identical haplotypes, HLA A2 B12 DR6, each of which carries a defective long C4A gene and a defective short C4B gene. PCR and DNA sequencing revealed that the mutant C4A contained a 2-bp insertion in exon 29 at the sequence for codon 1213. The identical mutation was absent in the mutant C4B. The C4B mutant gene was selectively amplified by long range PCR, and its 41 exons were completely sequenced. The C4B mutant had a novel single C nucleotide deletion at the sequence for codon 522 in exon 13, leading to frame-shift mutation and premature termination. Thus, a multiplex PCR is designed by which known mutations in C4A and C4B can be elucidated conveniently. Among the 28 individuals reported with complete C4 deficiency, 75-96% of the subjects (dependent on the inclusion criteria) were afflicted with autoimmune or immune complex disorders. Hence, complete C4 deficiency is one of the most penetrant genetic risk factors for human systemic lupus erythematosus.     

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     

The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes

The structure of the human immunoglobulin heavy chain constant region (IGHC), on chromosome 14q32, comprises nine CH genes and two pseudogenes, all originating from multiple duplication events. Continuing evolution of the region is demonstrated by the finding of various types of duplicated and deleted haplotypes, which together add up to 6%. Here we provide molecular and genetic evidence that the G4 gene is duplicated in 44% of IGHC haplotypes in the Italian population. The duplication spans about 20 kb of genomic DNA and probably originated through unequal crossing over. Refined characterisation of the genomic region downstream from the G4 gene improves our knowledge of the evolutionary history of CH genes. Received: 4 December 1996 / Accepted: 10 February 1997     

Human B and T lymphocytes convert leukotriene A4 into leukotriene B4

Incubation of human tonsillar B lymphocytes and peripheral blood T lymphocytes with leukotriene A4 led to the formation of leukotriene B4. The purity of these cell suspensions was more than 99%, containing less than 0.5% monocytes. Incubation of purified B or T lymphocytes with the calcium ionophore A23187 did not lead to the formation of any detectable amounts of leukotrienes. Several established cell lines of B and T lymphocytic origin were also found to convert leukotriene A4 into leukotriene B4, showing that monoclonal lymphocytic cells possess leukotriene A4 hydrolase activity.     

Determining the one,two, three,or four long and short loci of human complement C4 in a major histocompatibility complex haplotype encoding C4A or C4B proteins

The complex genetics of human complement C4 with unusually frequent variations in the size and number of C4A and C4B, as well as their neighboring genes, in the major histocompatibility complex has been a hurdle for accurate epidemiological studies of diseases associated with C4. A comprehensive series of novel or improved techniques has been developed to determine the total gene number of C4 and the relative dosages of C4A and C4B in a diploid genome. These techniques include (1) definitive genomic restriction-fragment-length polymorphisms (RFLPs) based on the discrete duplication patterns of the RCCX (RP-C4-CYP21-TNX) modules and on the specific nucleotide changes for C4A and C4B isotypes; (2) module-specific PCR to give information on the total number of C4 genes by comparing the relative quantities of RP1- or TNXB-specific fragments with TNXA-RP2 fragments; (3) labeled-primer single-cycle DNA polymerization procedure of amplified C4d genomic DNA for diagnostic RFLP analysis of C4A and C4B; and (4) a highly reproducible long-range-mapping method that employs PmeI-digested genomic DNA for pulsed-field gel electrophoresis, to yield precise information on the number of long and short C4 genes in a haplotype. Applications of these vigorously tested techniques may clarify the roles that human C4A and C4B gene-dosage variations play in infectious and autoimmune diseases.     

A region flanking H-2K is duplicated to a distant site in most mouse t haplotypes

Mouse t haplotypes contain at least one inversion, which encompasses the major histocompatibility complex, relative to their wild-type counterparts. A DNA probe for a single copy sequence which flanks the H-2K region in inbred strains was found to have undergone further rearrangements in the t haplotypes. In most t haplotypes, this sequence is duplicated at a distant site, and the two regions show 1 % recombination. The length of homology shared by the two sites is likely to be at least 10–15 kb. Three different alleles, as defined by restriction fragment length polymorphisms, were found for each of the two sites among different t haplotypes. These may reveal evolutionary relationships among these chromosomes.     

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.     

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.     

High incidence of ace-1 duplicated haplotypes in resistant Culex pipiens mosquitoes from Algeria

The status of genes conferring resistance to organophosphate and carbamate insecticides has been examined in Culex pipiens pipiens mosquitoes sampled in Algeria. Presence of overproduced esterases was sporadic, but acetylcholinesterase-1 resistant alleles were observed in almost all samples. We focused our study on the AChE1 G119S substitution characterized in almost all samples, mostly at the heterozygous state. A genetic test revealed the presence of ace-1 duplication associating a susceptible and a resistant ace-1 copy. Molecular characterization showed a high occurrence of ace-1 duplication with six distinct duplicated alleles out of four samples. The inferred frequency of duplicated allele suggests that it is replacing the single resistant G119S allele. Finally, we discuss the mechanism at the origin of these duplicated haplotypes and their consequences on the management of insecticide resistance.     

Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction

Low protein levels and copy number variation (CNV) of the fourth component of human complement (C4A and C4B) have been associated with various diseases. High-throughput methods for analysing C4 CNV are available, but they commonly do not detect the most common C4A mutation, a silencing CT insertion (CTins) leading to low protein levels. We developed a SYBR? Green labelled real-time quantitative polymerase chain reaction (qPCR) with a novel concentration range approach to address C4 CNV and deficiencies due to CTins. This method was validated in three sample sets and applied to over 1600 patient samples. CTins caused C4A deficiency in more than 70% (76/105) of the carriers. Twenty per cent (76/381) of patients with a C4A deficiency would have been erroneously recorded as having none, if the CTins had not been assessed. C4A deficiency was more common in patients than a healthy reference population, (OR?=?1.60, 95%CI?=?1.02-2.52, p?=?0.039). The number of functional C4 genes can be straightforwardly analyzed by real-time qPCR, also with SYBR? Green labelling. Determination of CTins increases the frequency of C4A deficiency and thus helps to elucidate the genotypic versus phenotypic disease associations.     

Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.