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Biomedical subjects

A Orren

Publications and source records attributed to A Orren.

At least 19 recordsLinked to original sources
PubMed1996

Molecular bases of combined subtotal deficiencies of C6 and C7: their effects in combination with other C6 and C7 deficiencies.

Combined subtotal deficiency of C6 and C7, in which both proteins are expressed at very low levels, has been observed in homozygous form in two families. A defect at the 5' splice donor site of intron 15 of the C6 gene explains the low molecular weight of the C6 protein and is probably responsible for its low expressed concentration. The C7 defect is more enigmatic: the protein is of normal molecular weight, low circulating concentration, and altered isoelectric point. An Arg > Ser codon substitution in exon 11 is the only molecular alteration within the mature C7 protein. These defects are associated with a characteristic set of polymorphic DNA markers in the C6/C7 region, forming a distinct haplotype. The haplotype has been found in combination with a number of other haplotypes containing defective genes that lead either to C6 or C7 deficiency, but with different consequences. Where it is combined with a C6-deficient gene, the serum C7 levels can be surprisingly high, possibly because there is no C6 generating C56 to consume the C7. In contrast, where the C7 genes are both defective (but still partially functional), there may be a profound deficit of circulating C7 because there is ample C6 to produce C56 and consume the already small amount of C7. Each molecular defect has also been found in isolation and has the expected effect.

Amino Acid Sequence↗
PubMed1996

How partial C7 deficiency with chronic and recurrent bacterial infections can mimic total C7 deficiency: temporary restoration of host C7 levels following plasma transfusion.

An apparently completely complement C7-deficient patient with refractory otitis media and two episodes of meningococcal disease was given therapeutic plasma transfusions in 1992 and 1994. Following these transfusions unexpected changes were found in C7 levels. Immediately after transfusion the serum C7 levels failed to rise to the expected levels but then rose to 5-10% of the normal mean during the next 5 days and remained at that level for more than 2 weeks before eventually returning to zero. The patient's DNA genotyped C7 M, and therefore C7 N donor plasma was selected for the second transfusion to allow identification of the source of the C7 circulating post-transfusion. This C7 phenotyped C7 M, demonstrating it to be of recipient origin. Therefore, the apparently completely C7-deficient patient was able to secrete some C7. By a combination of DNA typing and isoelectric focusing of the C7 appearing after transfusion, it was demonstrated that the patient was heterozygous for combined subtotal C6/C7 deficiency (inherited from his father) and a different, so far uncharacterized, subtotal C7 deficiency (inherited from his mother). The low amount of C7 secreted appeared to be constantly consumed, probably by generation of C5b6 as a result of his chronic infection. He had been shown to have circulating C5b6 most of the time, and thus only when sufficient exogenous C7 was given to consume the free C5b6 did his own C7 appear in circulation.

Bacterial Infections↗
PubMed1996

A new intronic polymorphism in the C7 gene 36 bp from the common expressed C7 M/N polymorphism.

We report a new polymorphism in the complement C7 gene that results from an A-C transversion in intron 12, 27 bp upstream of exon 13 (C712.-27) and 36 bp upstream of the point mutation that underlies the C7 M/N antigenic polymorphism. The C7 12.-27 polymorphism subdivides C7 M haplotypes, but not C7 N. It also sheds light on the evolution of the various types of deficiency genes at the adjacent C6 locus.

Base Sequence↗
PubMed1995

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.

Alleles↗
PubMed1995

Molecular basis of subtotal complement C6 deficiency. A carboxy-terminally truncated but functionally active C6.

Individuals with subtotal complement C6 deficiency possess a C6 molecule that is 14% shorter than normal C6 and present in low but detectable concentrations (1-2% of the normal mean). We now show that this dysmorphic C6 is bactericidally active and lacks an epitope that was mapped to the most carboxy-terminal part of C6 using C6 cDNA fragments expressed as fusion proteins in the pUEX expression system. We thus predicted that the abnormal C6 molecule might be carboxy-terminally truncated and sought a mutation in an area approximately 14% from the carboxy-terminal end of the coding sequence. By sequencing PCR-amplified products from this region, we found, in three individuals from two families, a mutation that might plausibly be responsible for the defect. All three have an abnormal 5' splice donor site of intron 15, which would probably prevent splicing. An in-frame stop codon is found 17 codons downstream from the intron boundary, which would lead to a truncated polypeptide 13.5% smaller than normal C6. This result was unexpected, as earlier studies mapped the C5b binding site, or a putative enzymatic region, to this part of C6. Interestingly, all three subjects were probably heterozygous for both subtotal C6 and complete C6 deficiency.

Amino Acid Sequence↗
PubMed1994

Hereditary deficiency of the seventh component of complement and recurrent meningococcal infection: investigations of an Irish family using a novel haemolytic screening assay for complement activity and C7 M/N allotyping.

Terminal complement component deficiency predisposes to meningococcal infection and is inherited in an autosomal co-dominant manner. An Irish family is described, in which 2 of 3 brothers had recurrent meningococcal infection. A novel screening assay was used to investigate for terminal complement deficiency and the 2 affected brothers were found to be completely deficient in the seventh component of complement (C7). Enzyme-linked immunosorbent assay for C7 revealed lower than normal levels in the remaining brother and parents. C7 M/N protein polymorphism allotyping, used to investigate the segregation of the C7 deficiency genes, showed that the apparently complement sufficient brother was heterozygous C7 deficient and a carrier of one of the deficiency genes. Complement screening should be carried out in any individual suffering recurrent meningococcal infection or infection with an uncommon meningococcal serogroup. Identification of complement deficient patients allows the implementation of strategies to prevent recurrent infection.

Adolescent↗
PubMed1994

C6 haplotypes: associations of a Dde I site polymorphism to complement deficiency genes and the Msp I restriction fragment length polymorphism (RFLP)

Complement C6 has a common charge polymorphism designated A and B with gene frequencies of 0.65 and 0.35. The probable molecular basis for this is a Glu (C6A) for Ala (C6B) substitution at amino acid position 98, and is detected by digestion with the restriction enzyme Dde I of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA. C6A was found to be Dde I-positive and C6B corresponds to Dde I-negative. We have applied our Dde I A/B polymorphism genotyping method to the investigation of C6-deficient individuals with complete (C6Q0) and sub-total deficiency (C6SD) protein phenotypes, including members of four families. We have also investigated the RFLP detected by digestion of genomic DNA with the enzyme Msp I, which is due to a polymorphic site located in the 5' section of the gene, the variable sequence of which has yet to be determined. Sixteen out of seventeen unrelated C6Q0 subjects were found to be genotypically Dde I B/Msp I-negative; the remaining subject was heterozygous at both the loci under investigation. The C6SD phenotype was found to be associated with the Dde I A/Msp I-positive genotype in two families with combined C6/C7 subtotal deficiency and two with C6SD. It can be concluded that the two forms of C6 deficiency, C6Q0 and C6SD, arose independently on two different C6 allelic backgrounds. These associations have allowed the genotyping of the rare families that contain both types of deficiency. We have also defined a number of normal C6 Dde I/Msp I haplotypes in Caucasians and Cape Coloured populations.

Adult↗
PubMed1994

Characterization of strains of Neisseria meningitidis recovered from complement-sufficient and complement-deficient patients in the Western Cape Province, South Africa.

Complement deficiency has been associated with increased susceptibility to meningococcal disease. In order to determine whether special meningococcal strains caused disease in complement-deficient (CD) patients, 17 Neisseria meningitidis strains recovered from patients in the Western Cape Province, South Africa, known to be CD were compared with 124 routine isolates obtained from patients living in the same area. Serogrouping of the strains from the CD subjects revealed that the common serogroups, particularly serogroup B, predominated. However, the prevalence of rare serogroups among isolates from CD subjects was significantly higher than that found among isolates from the control group. Sero- and subtyping of the class 1 and class 2 or 3 outer membrane proteins showed no significant difference between isolates from CD subjects and the routine clinical isolates. Multilocus enzyme electrophoresis of the 141 isolates revealed six clusters of electrophoretic types (ETs) and two unrelated ETs. The same degree of genetic diversity existed in ETs of isolates from CD subjects and the control group. However, the ET-5 complex, which is known to be associated with epidemic disease, was found in 22 (18%) of the routine clinical isolates but in none of the isolates from the CD subjects. This difference was marginally significant. What was highly significant was the finding that 8 of the 17 isolates from CD subjects were in one ET cluster, cluster F, which comprised a total of 20 isolates. Thus, our results show a difference in the clonal compositions of the strains that infect CD subjects in comparison with the clonal compositions of those that cause clinical infections in the population at large.

Adolescent↗
PubMed1994

Paradoxical reconstitution of complement activity following plasma transfusion of an individual with deficiency of the seventh component of complement.

A subject deficient in the seventh component of complement (C7) was plasmapheresed with 660 ml C7-sufficient plasma. The expected reconstitution of C7 activity, followed by exponential decay, was not observed. During day 1, serum haemolytic C7 and total haemolytic complement were undetectable and C7 levels were very low by C7 ELISA. However, low levels of circulating fluid phase terminal complement complex (TCC) were detected. On day 2 about microgram C7/ml serum was detected and this rose to 6 micrograms/ml by day 17. Functional complement activity was also present. At day 28 the serum C7 and total haemolytic complement had dropped to pretransfusion levels. A low level of C5b6 was present in pretransfusion serum and this increased markedly immediately following transfusion when the patient's serum also acquired C7 consuming activity. Throughout the study low levels of anti-C7 antibodies were present but there was no evidence that antibody was directly responsible for the C7 consumption. Nevertheless antibody-antigen interactions could have generated circulating C5b6. C5b6 has been shown previously to have the capacity to inhibit C7 activity in vitro. Investigations of the C7 circulating on days 2-17 demonstrated normal molecular weight, functionally active C7. The donor sera and the recirculating C7 allotyped C7-1 by isoelectric focusing; however, the recirculating C7 showed additional weak bands with C7 functional activity, suggesting a possible genetic or acquired abnormality. Although the disappearance of C7 immediately post-transfusion may be explained by the presence of C5b6, there is no satisfactory explanation for the rising C7 levels on days 2-17 and we cannot exclude temporary C7 secretion by the patient.

Adult↗
PubMed1993

Association of a 12.5-kilobase allele of the MspI restriction fragment length polymorphism of the C6 gene in patients with total deficiency of the sixth component of complement.

The distribution of MspI restriction fragment length polymorphism (RFLP) alleles was investigated using the C6-PVX probe of the sixth component of complement (C6) and DNA from lymphocytes of 11 patients with homozygous C6 deficiency (C6Q0), 18 of their family members, 3 patients with subtotal C6 deficiency (C6SD) and 28 normal C6-sufficient controls. A biallelic polymorphism of 12.5- and 8.2-kb RFLP alleles was observed, and co-dominant inheritance of the two alleles was demonstrated in family studies. All 11 C6Q0 patients were homozygous for the 12.5-kb allele; this includes 8 unrelated propositi. The gene frequencies for both the 12.5- and 8.2-kb alleles in control subjects were 0.5, and the association of C6*Q0 with the 12.5-kb allele was found to be highly significant (p = 0.0001). Family studies in a C6Q0 family demonstrated that the MspI polymorphism may be used to trace C6*Q0 heterozygous carriers. Studies in families with C6SD, when considered with the results of C6 and C7 allotyping, showed definite co-segregation of C6*SD with the MspI 8.2-kb allele in one family and very probable co-segregation in another. All 11 South African C6Q0 subjects were homozygous for the C6Q0/MspI 12.5-kb/C7 M haplotype. Our data describe new associations of C6 deficiency genes which may assist in the future identification of the molecular defects.

Alleles↗
PubMed1992

Meningococcal septicaemia in a C6-deficient patient and effects of plasma transfusion on lipopolysaccharide release.

Patients whose blood is deficient in the terminal component of complement have an increased susceptibility to meningococcal infection. However, mortality from meningococcal infection is lower in these patients than in immunocompetent subjects. We studied a C6-deficient patient with meningococcal sepsis who received fresh frozen plasma (FFP). The patient's initial plasma endotoxin, C6, and terminal-complement-complex concentrations were low, but rose sharply after treatment with FFP. Samples of the patient's serum taken shortly after admission did not cause endotoxin release from Escherichia coli J5 in vitro, but endotoxin-releasing activity was restored in serum samples taken after infusion of FFP. It is possible that C6-deficient patients have reduced mortality from meningococcal infection because their serum cannot cause acute release of endotoxin from the invading organism and extensive tissue damage is thus avoided.

Adult↗
PubMed1992

C7 M/N protein polymorphism typing applied to inherited deficiencies of human complement proteins C6 and C7.

C7 M/N typing, the determination of the complement component C7 M/N phenotypes, was successfully used in family studies to trace haplotypes bearing C7 deficiency genes. Furthermore, it was shown to be preferable to C7 allotyping based on isoelectric focusing (IEF) since it distinguishes two common alleles (C7*M and C7*N), whereas one common C7 IEF allele (C7*1) predominates in most populations. It is also the more sensitive method, as it enabled detection of very low amounts of abnormal C7 molecules in the third generation of a combined subtotal C6/C7-deficient subject and thus confirmed that this partial deficiency gene is not silent in heterozygotes. In this respect C7 M/N typing is even more informative than DNA restriction fragment length polymorphism typing which will assess the presence but not necessarily the functional status of a gene. C6 and C7 genes are tightly linked and therefore C7 M/N typing was also applied to tracing C6 deficiency genes in families. C6/C7 haplotype analysis of South African C6-deficient (C6Q0) subjects revealed a strong allelic association of C6*Q0 and C7*M.

Alleles↗
PubMed1992

Antibodies to meningococcal class 1 outer membrane proteins in South African complement-deficient and complement-sufficient subjects.

Inhibition assays were used to investigate human serum antibodies to the meningococcal class 1 outer membrane proteins. We adapted the whole-cell enzyme-linked immunosorbent assay technique to determine the ability of sera to inhibit the binding of murine subtyping monoclonal antibodies. Serum samples from 33 South African subjects with a deficiency in the sixth component of complement as well as serum samples from various groups of complement-sufficient subjects were investigated. Subjects were subdivided according to whether they were (i) convalescent from Neisseria meningitidis infections, (ii) nonconvalescent, or (iii) controls. Preliminary subtyping investigations had shown that P1.2 was present on 36% of meningococcal clinical isolates from Cape Province, South Africa. Assays with the anti-P1.2 antibodies showed the presence of high antibody levels in many deficient sera and moderately elevated levels in some sera from the complement-sufficient convalescent patients. P1.2, P1.4, P1.15, and P1.16 are epitopes situated on loop 4 of the class 1 outer membrane proteins, whereas P1.7 is on loop 1. Inhibition assays showed that human sera that inhibited binding by P1.2 monoclonal antibodies tended to inhibit the other monoclonal antibodies directed to loop 4 epitopes. This suggests that the epitopes recognized by the human antibodies are not exactly the same as the epitopes recognized by the murine monoclonal antibodies and raises the possibility of the importance of other epitopes.

Antibodies, Bacterial↗
PubMed1992

Properties of a low molecular weight complement component C6 found in human subjects with subtotal C6 deficiency.

A sensitive ELISA assay was used to quantitate serum complement component C6 concentrations. Levels in the range 0.3-3 micrograms/ml were measured in samples from eight individuals (four separate pedigrees) and two subjects with subtotal combined C6/C7 deficiency who have been reported previously. We defined C6 levels in this range as subtotal C6 deficiency (C6SD). In contrast, C6 deficiency with levels below 0.03 micrograms/ml was defined as C6Q0. C6Q0 has been found in 29 unrelated cases which have already been reported. Investigations of the properties of the C6 found in the C6SD subjects showed it to be haemolytically active and able to incorporate into the terminal complement complex. The protein had a relative molecular weight (Mr) of approximately 86% of normal C6 and this Mr was identical to that of the C6 of one combined deficient subject. The Mr of the C6 of the other combined deficient subject was previously estimated as 79% of the Mr of normal C6. Isoelectric focusing (IEF) analysis with band development by haemolytic overlay revealed that all C6SD samples produced an identical weak C6 band pattern anodal to normal C6A bands. The C7 IEF patterns of the two combined deficient subjects were identical, and the C6 IEF patterns of both were identical to those of the C6SD subjects. Thus the C6 of the combined deficient subjects is probably the same abnormal protein found in the C6SD individuals. None of the C6SD or combined deficient subjects have had meningococcal disease and it may be that low C6 levels afford some protection.

Adult↗
PubMed1992

Inherited deficiencies of the terminal components of human complement.

The particularly frequent occurrence of terminal complement deficiencies in patients with Neisserial infections suggests that the cytolytic activity of the complement system is important in resistance to Neisseria meningitidis. There are, however, geographical differences in the prevalence of terminal complement deficiency in patients with meningococcal disease. The data available suggest that either recurrent infection or infection with uncommon serogroups should alert the clinician in Western countries whereas recurrent disease is the important indicator in high risk endemic or epidemic areas. An association of terminal complement deficiencies with susceptibility to autoimmune diseases or non-Neisserial infections is doubtful. For a better understanding of complement deficiencies in relation to disease more accurate characterization of the defects involved will be helpful. Sensitive ELISA techniques and molecular biological assays will be needed. Thus it has been established that two types of deficiencies exist (at least for C6, C7 and C8): one with low but detectable amounts of the component and the other with a complete absence of the protein in question. The subtotal variety appears to show less association with Neisserial infection. Low amounts of functional terminal complement activity may be sufficient for many of its biological functions, suggesting that there is a wide "safety margin".

Autoimmune Diseases↗
PubMed1991

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals.

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.

Antibodies, Monoclonal↗