Ann Orren

The effects of sex and age on serum IgE concentrations in three ethnic groups.

Serum IgE concentrations were measured using a radio-radial immunodiffusion method in 4,440 blood donors resident in the Western Cape Province, South Africa, and the following differences were observed. Firstly, in all three ethnic groups studied, that is the Whites, the Cape Coloureds and the Africans, there was a tendency for males to have higher serum IgE concentrations than females. This differences was highly significantly in Whites. Secondly, in the Whites, serum IgE concentrations tended to be lower with advancing age. Thirdly, previous reports of elevated serum IgE concentration in indigenous African population were confirmed. No clearly defined cause for these differences emerged rom this study.

Complete deficiency of the sixth complement component (C6Q0), susceptibility to Neisseria meningitidis infections and analysis of the frequencies of C6Q0 gene defects in South Africans

Complete complement component 6 deficiency (C6Q0) is a co‐dominant genetic disease presenting as increased susceptibility to invasive Neisseria meningitidis infections. Affected individuals have two affected alleles which can be homozygous or compound heterozygous for the particular gene defects they carry. This disorder has been diagnosed relatively frequently in Western Cape South Africans. Affected patients are prescribed penicillin prophylaxis. In 2004 we commenced a clinical follow‐up study of 46 patients. Of these, 43 had family age‐matched C6 sufficient controls. Participants were classified as either (i) well, or (ii) having a serious illness (SI) or died (D). An SI was a long‐term illness that did not allow the performance of normal daily activities. Among 43 patients, 21 were well and 22 were SI/D, while among 43 matched controls, 35 were well and eight were SI/D. This difference is highly significant. Among all 46 C6Q0 patients, those who had had recurrent infection had significantly more SI/D than those who had suffered none or one infection. Thus, this work demonstrates the long‐term serious outcome of repeated meningococcal disease (MD) episodes. We investigated the frequencies of four C6Q0 pathogenic mutations known to affect Cape patients (828delG, 1138delC, 821delA and 1879delG) in 2250 newborns. A total of 103 defective alleles (2·28%) and three affected C6Q0 individuals were detected. For all defects combined, 5·24 affected subjects (C6Q0) are expected among 10 000 individuals. What is still unknown is the number of C6Q0 individuals who suffer MD or other infectious diseases.

Effects of allergy, intestinal helminthic infestation and sex on serum IgE concentrations and immediate skin hypersensitivity in three ethnic groups.

Serum IgE concentrations were measured in 232 blood donors resident in the Western Cape, South Africa. Subjects were interviewed for histories of allergy and skin tested with a variety of common allergens. Faecal specimens were collected for microscopic examination. Results confirmed that Cape Coloureds and Africans tend to have elevated serum IgE concentrations and showed that African males have a high prevalence of immediate skin hypersensitivity. Cape Coloureds and Africans had high prevalences of helminthic infestation and relatively low prevalence of allergic symptoms. Only in Whites were elevated serum IgE concentrations associated with positive allergic histories; in this group, results suggested that the sex difference in serum IgE concentrations observed previously is owing to differences in the IgE responses of allergic males and females.

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.

A complement C5 gene mutation, c.754G>A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases

Patients with genetically determined deficiency of complement component 5 are usually diagnosed because of recurrent invasive Neisseria meningitidis infections. Approximately 40 individual cases have been diagnosed worldwide. Nevertheless, reports of the responsible genetic defects have been sporadic, and we know of no previous reports of C5 deficiency being associated with a number of independent meningococcal disease cases in particular communities. Here we describe C5 deficiency in seven unrelated Western Cape, South African families. Three different C5 mutations c.55C>T:p.Q19X, c.754G>A:p.A252T and c.4426C>T:p.R1476X were diagnosed in index cases from two families who had both presented with recurrent meningococcal disease. p.Q19X and p.R1476X have already been described in North American Black families and more recently p.Q19X in a Saudi family. However, p.A252T was only reported in SNP databases and was not associated with disease until the present study was undertaken in the Western Cape, South Africa. We tested for p.A252T in 140 patients presenting with meningococcal disease in the Cape Town area, and found seven individuals in five families who were homozygous for the mutation p.A252T. Very low serum C5 protein levels (0.1-4%) and correspondingly low in vitro functional activity were found in all homozygous individuals. Allele frequencies of p.A252T in the Black African and Cape Coloured communities were 3% and 0.66% and estimated homozygosities are 1/1100 and 1/22,500 respectively. In 2012 we reported association between p.A252T and meningococcal disease. Molecular modelling of p.A252T has indicated an area of molecular stress in the C5 molecule which may provide a mechanism for the very low level in the circulation. This report includes seven affected families indicating that C5D is not rare in South Africa.

Functional identification of serum complement components following electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate

A method is described for detecting the active complement components C6 and C7 after polyacrylamide gel electrophoresis (PAGE) of whole serum in the presence of sodium dodecyl sulphate (SDS). The method involves the removal of SDS by washing with non-ionic detergent followed by the application of an erythrocyte/agarose gel to detect haemolytic activity. Two forms of human C6 with apparent molecular weights of approximately 121,000 daltons and 114,000 daltons were observed. Major activity resided in the 121,000 dalton species. The 2 forms of human C6 were not related to known genetic polymorphisms for this component. Analysis of sera from different animal species showed that not all possessed the 2 forms of C6 and that there were interspecies differences in C6 molecular weights. These are most marked in the case of human and murine C6; the major form of murine C6 had a molecular weight approximately 20,000 daltons less than the major human form. One form of human C7 with an apparent molecular weight of 104,000 daltons was seen. The molecular weights of C7 from the various animal sera tested did not differ significantly from this. Studies with reducing agents and metabolic inhibitors showed that both C6 and C7 required intact disulphide bonds and sulphydral groups for functional activity.

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.

Characterization of a large genomic deletion in four Irish families with C7 deficiency.

Inherited deficiency of the seventh complement component (C7) is associated with increased susceptibility to Neisseria meningitidis infections. The disease is rare in most Western countries. Here we report new investigations of a large, but incompletely characterized genomic deletion of exons 8 and 9 [c.739-?_1093+?del], previously identified in three unrelated Irish families with C7 deficiency. We have analysed DNA from one individual, who is homozygous for the deletion, by PCR using primers progressively proximal to the deleted exons. Thus we were able to map the deletion boundaries. Amplification across the breakpoint and sequencing revealed an indel mutation that included a 6.4kb deletion together with an insertion of a novel 8bp sequence [c.739+1262_1270-2387delinsGCAGGCCA]. We demonstrated the same defect in the C7 deficient patients from each family and developed a duplex PCR method to enable the detection of alleles containing the deletion in heterozygotes. A member of a fourth family was found to be homozygous for the deletion defect. Thus, the deletion defect may be a more commonly distributed cause of C7 deficiency in Ireland.

Complement component C6 deficiency and susceptibility to Neisseria meningitidis infections.

May 2004, Vol. 94, No. 5 SAMJ Meningococcal disease remains one of the most serious bacterial infections in both Western and developing countries. Despite recent advances in treatment the mortality rate remains at about 12%. There is a group of South Africans who are particularly vulnerable to this disease. They are individuals with genetically determined deficiencies of individual terminal complement proteins, in particular of the sixth component of complement (C6).

Allotypes of mouse complement component C6 in inbred strains and some wild populations

This collaborative work was undertaken to resolve discrepancies in reports of the number of forms of complement component C6 present in the circulation of mice from various inbred strains. Plasma C6 was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by isoelectric focusing (IEF), and C6 band patterns were developed by electroblotting and immunoprobing. Results of C6 allotyping of mice from 36 strains confirmed that while 20 strains (prototype strain BALB/c) possessed only one relative mass (Mr) form which typed C6A1 on IEF, the other 16 strains all possessed more than one C6 Mr form. Moreover, IEF analysis demonstrated additional polymorphic differences; among these 16 strains, 11 typed C6A l B l like the prototype strain CBA, the AKR and RF/J strains typed C6A2B2, and the Japanese MOM strain as well as the C57BR/cdJ and C57L/J strains possessed two forms with IEF mobilities intermediate between C6A1B1 and C6A2B2. These will now be referred to as C6A3B3. Thus, a total of four different mouse C6 haplotypes have been identified.Testing C6 allotypes in a limited number of wild mice revealed that haplotypes found in inbred strains of Western or Eastern origin tend to reflect haplotypes of the wild mice from Europe or Japan, respectively.