Michael J. Hobart

The molecular basis of C6 deficiency in the western Cape, South Africa

Deficiency of the sixth component of human complement (C6) has been reported in a number of families from the western Cape, South Africa. Meningococcal disease is endemic in the Cape and almost all pedigrees of total C6 deficiency (C6Q0) have been ascertained because of recurrent disease. We have sequenced the expressed exons of the C6 gene from selected cases and have found three molecular defects leading to total deficiency: 879delG, which is the common defect in the Cape and hitherto unreported, and 1195delC and 1936delG, which have been previously reported in African-Americans. We also show that the 879delG and 1195delC defects are associated with characteristic C6/C7 region DNA marker haplotypes, although small variations were observed. The 1936delG defect was observed only once in the Cape, but its associated haplotype could be deduced. The data from the haplotypes indicate that these three molecular defects account for the defects in all the 38 unrelated C6Q0 individuals we have studied from the Cape. We have also observed the 879delG defect in two Dutch C6-deficient kindreds, but the 879delG defect in the Cape probably did not come from the Netherlands.

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 patients 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.

Study of the in vitro activation of the complement alternative pathway by Echinococcus granulosus hydatid cyst fluid

In the present study we have investigated the fluid phase activation of the complement (C) alternative pathway by Echinococcus granulosus sheep hydatid cyst fluid (SHCF) and its higher molecular weight fraction (SHCF‐I) by quantitating the formation of both the terminal C intermediary C5b6 complex and the terminal C complex (TCC). Our results show that in vitro C activation progresses beyond the C5 step suggesting that potentially lytic complexes may be generated in vivo. In addition, SHCF and SHCF‐I glucidic moieties are probably involved in C activation since 80% and 86% of SHCF and SHCF‐I activity respectively was destroyed by period‐ate oxidation. Furthermore, partial deglycosylation with Peptide N‐Glycosidase F of SHCF‐I which had been digested with Pronase E, released an active fraction (MW <14KDa) which bound to Soybean agglutinin, suggesting that N‐linked oligosaccharides containing α‐or β‐linked N‐acetyl galactosamine play a role in C activation by SHCF.,

Complement C7 deficiency: seven further molecular defects and their associated marker haplotypes

Seven further molecular bases of C7 deficiency are described. All these new molecular defects involve single-nucleotide events, deletions and substitutions, some of which alter splice sites, and others codons. They are distributed along the C7 gene, but predominantly towards the 3′ end. All were found in compound heterozygous individuals. The C6/C7 marker haplotypes associated with most C7 defects are tabulated.

Close linkage between mouse genes determining the two forms of complement component C6 and component C7, and cis action of a C6 Regulatory Gene

Two forms of mouse complement component C6, with molecular weights (Mrs) of 90 and 100 kilodaltons (kd), are present in the sera from certain inbred strains such as the CBA strain; other strains, such as the BALB/c and DBA/2 strains, have only the 90 kd C6A form. The present work was undertaken to determine whether the two Mr forms were the products of genes coding at separate loci. We screened sera from mice from a number of inbred strains by isoelectric focusing and found one strain, AKR, exhibiting allotypic structural variations of C6 forms. To distinguish the various types, we designated the 90 kd types from CBA and AKR mice C6A1 and C6A2, respectively, and the corresponding 100 kd types C6B 1 and C6B2, respectively. Mice possessing only one Mr form were all typed as C6A1. Results of breeding experiments strongly suggested that the two Mr forms of C6 are coded for at two closely linked loci. Sera from a number of inbred strains were also screened for a complement C7 polymorphism by means of isoelectric focusing and functional overlay. C7 from all strains, excepting the AKR strain, produced identical C7 band patterns. AKR C7 produced a unique band pattern, and results of breeding experiments with AKR and BALB/c mice showed the C6 and C7 loci to be closely linked. In addition, we identified a regulatory gene for C6 production. The gene apparently requires androgen to facilitate C6 production in the majority of strains. In these strains C6 activity is virtually absent from female sera. However, we observed moderate levels of C6 activity in sera from IS/Cam females, indicating that, in this strain, male physiological androgen levels are not necessary for C6 production. IS/Cam possess one form of circulating C6 which appears identical with BALB/c C6A1, and therefore IS/Cam mice differ from AKR mice at both the C6 structural and regulatory loci. These two strains were thus suitable for use in breeding experiments to determine the manner of action of the regulatory gene. Results showed that it acted in a cis manner.

NO EVIDENCE FOR ASSOCIATION OF MULTIPLE SCLEROSIS WITH THE COMPLEMENT FACTORS C6 AND C7

Four genome screens in multiple sclerosis have been completed and each has identified evidence for linkage in the pericentromeric region of chromosome 5. This region encodes a number of candidate genes including those for the complement components C6, C7 and C9. We have used a multiplexed oligoligation assay (OLA) to test single nucleotide polymorphisms (SNPs) from the C6 and C7 genes for evidence of association with multiple sclerosis in our sibling pair families. There was no statistically significant difference in the allele frequencies of these polymorphisms in the index cases from our families when compared with locally derived controls. No evidence for transmission distortion was seen with any of the polymorphisms, or with the haplotype built from the three SNPs from the C7 gene. Despite offering themselves as potential candidates these complement genes appear not to confer susceptibility to multiple sclerosis.

Difficulties in the ascertainment of C9 deficiency: lessons to be drawn from a compound heterozygote C9-deficient subject

A group of patients with long‐surviving mismatched kidney allografts were investigated for complement function using haemolytic assays in agarose gels. One patient was found to have no alternative pathway activity but a low normal classical pathway. Surprisingly, investigation revealed that the patient’s complement was normal for all components except C9, which was functionally absent. The patient was shown to be heterozygous for DNA markers in the C6, C7 and C9 region of chromosome 5 and therefore appears to be a compound heterozygote for two uncharacterized C9 deficiency genes. Serological analysis by ELISA revealed that he has trace concentrations of a non‐functional C9 molecule. Western blot analysis was not sufficiently sensitive to permit detection of this molecule. We hypothesize that the patient is heterozygous for a complete deficiency of C9 and for a gene directing hyposynthesis of a defective C9. We also suggest that C9 deficiency may be more common among Caucasians than has been reported.

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 out Dde I A/B polymorphism genotyping method to the investigation of C6‐delicient 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 haptotypes in Caucasians and Cape Coloured populations.

Physical linkage and orientation of the human complement C8α and C8β genes on chromosome 1p32

Abstract Human C8 is one of five components (C5b, C6, C7, C8, C9) of the cytolytic C5b-9 complex of complement. It consists of three nonidentical subunits (C8α, C8β, C8γ), which are encoded in separate genes. Genetic linkage and chromosomal localization studies previously established that C8α and C8β are closely linked on chromosome 1p32. In this study, clones with inserts containing genes for both C8α and C8β were isolated from a yeast artificial chromosome (YAC) human genomic DNA library and characterized in an effort to determine intergenic distance and orientation. One clone with a ∼330-kb insert yielded restriction digest patterns for C8α and C8β that agreed with those obtained previously from digests of human genomic DNA, thereby confirming the presence of intact copies of both genes. A second clone with a ∼280-kb insert yielded similar results; however, it was truncated at the 5′ end of the C8α gene. Restriction digests of both clones were subjected to PFGE and Southern blot analysis using probes specific for the terminal exons of C8α (exons 1 and 11) and C8β (exons 1 and 12). Results indicate the genes are physically linked (< 23 kb) and in a 3′– 3′ orientation. This is the same orientation as the ancestrally related C6 and C7 genes, which are also physically linked on chromosome 5p13.

Five New Polymorphisms in the Complement C7 Gene and Their Association with C7 Deficiency

Five new polymorphisms in the C7 gene are described: 2 in intron 1, and 1 each in introns 7, 8 and 15. Four of these are single nucleotide exchanges, while the fifth is a T insertion at 10 sequential Ts. Allele frequency data are presented for intervening sequence (IVS)1+ 55 in 6 normal population groups. We present new and updated data in these populations on a previously described C7 polymorphism in exon 13 (cDNA 1792 A/T). We also report the extended haplotypes associated with C7 deficiency for which marker investigation is a useful, and in some cases vital, adjunct to the identification of the gene defects. Almost without exception, a particular haplotype is associated with a particular mutation causing the deficiency state. Haplotyping is especially useful where polymerase chain reaction failure on one chromosome could be a cause for difficulties in detecting a molecular defect due to heterozygosity for large deletions or unidentified variations at the locations of the primers.