The terminal components of the complement system (C6-C9) are related proteins, differing in size and complexity. They seem to be typical mosaic proteins, composed of modules which are homologous with parts of other proteins. Individual elements in a mosaic protein are often bounded by introns in the gene, and where they are duplicated within a polypeptide, partial gene duplication within the gene is responsible. It is often found in such genes that the intron/exon boundaries are of the class 1 type. We have examined the boundaries of 17 of the 18 exons of C6 and five of C7. When considered with published data for C9, only one of the protein elements appears to follow the conventional pattern. These data suggest a more complex evolutionary history for the genes of the terminal complement components than had been anticipated and challenge the notions both that discovery of a recognized protein module is of predictive value in relation to gene structure and that these genes evolved from the simple to the complex.

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 -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. (J. Clin. Invest. 1995. 95:1877-1883.) Key words: antigenic determinants complement membrane attack complex * immunologic diseases -meningococcal infections * RNA splicing

The results of the present (VIIth Complement Genetics Workshop and Conference, Mainz, May 1998) and past reference typing workshops for the terminal complement components C6, C7 and C9 are compiled and discussed both on the protein level and on the DNA level. This report also focusses on the molecular bases of expressed and silent polymorphisms and reviews the molecular bases of subtotal and complete deficiencies of these proteins and their associations with protein and DNA markers. The results of the protein typing for C6 are published in the following paper of this issue.

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The linked C6 and C7 loci are rich in genetic markers, both at the protein and DNA levels. There are now seven common DNA polymorphisms distributed over about 300 kbp of chromosome 5p12-14. We report a new TaqI RFLP for C7 and a method for typing a C7 variant (T368S) hitherto known only from cDNA clones. We have re-investigated the published RFLPs to provide information on their frequency in North European Caucasian (predominantly British and Irish) subjects and have revised some of the published parameters, especially the sizes of polymorphic restriction fragments. Their precise locations within the genes are also reported: the three markers for C6 are in exon 3, intron 3 and adjacent to exon 17 and the four markers for C7 are in introns 15 and 13 and in exons 13 and 9. The gene frequencies of the second commonest allele of all seven markers lie in the range 0.2 to 0.37, except C6 A/B in the Japanese, where the frequencies of both common alleles are about 0.45. We have estimated the gene frequencies for the DNA polymorphisms which correlate with C7 M/N phenotype and for the C6 A/B phenotype and find them to be the same as the phenotypic estimates in Caucasians and in the Japanese respectively. The markers provide the possibility of defining 128 haplotypes, many (28) of which have been observed. Allelic associations in these genes are generally surprisingly weak.

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.

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.

Human deficiencies of terminal complement components are known to be associated with increased susceptibility to Neisseria meningitidis infection. Polymorphic DNA marker studies in complement deficient investigations allow identification of haplotypes associated with the deficiency and enable the possible identification of heterozygote carriers of the defect. We report studies of an Irish family in which the index case had suffered recurrent meningococcal disease and was found to be deficient in the seventh component of complement (C7). The availability of all family members enabled us to determine the segregating haplotypes. The defects in the family segregated with two very closely related C6 and C7 DNA haplotypes, one of which is known to be associated with the large Irish C7 DNA deletion defect. The index case and two C7 deficient siblings were found to be homozygous for this defect, a deletion that spans approx. 6.8 kbp and encompasses exons 7 and 8. The deletion defect of exons 7 and 8 of C7 has been found in homozygous form in another C7 deficient Irish individual, and is present in heterozygous form in C7 deficient members of a third Irish family. Therefore, this deletion defect occurs in five of the six deficient chromosomes of these three unrelated Irish families, raising the interesting question of how prevalent this defect may be within the Irish community.