Elizabeth P. Owen

Myasthenia gravis in South Africans: Racial differences in clinical manifestations

We present data on the phenotypic variation in myasthenia gravis of 205 subjects from a multi-racial South African cohort. Consecutive subjects seen more than twice from 1996 to 2006, were included. Documented observational data included a myasthenia gravis and extra-ocular eye muscle score. Results showed Black subjects were more likely than Whites to develop treatment-resistant complete ophthalmoplegia and ptosis (18% vs. 2%; p=0.041). Of the 14 patients with this phenotype, 13 had generalised disease and positive AChR antibodies. Despite similar sized cohorts, White subjects were more likely than Blacks to develop generalised myasthenia poorly responsive to therapy (p=0.005). There were no significant racial differences in the time between diagnosis to initiation of therapy, or the performance and timing of thymectomy. The racial variation in some phenotypic features of myasthenia gravis and outcome to therapy, highlights the need to study biological factors in different subgroups to develop a more rational approach to immuno-suppressive therapy.

Hyperhomocysteinemia - A risk factor for abruptio placentae

Recurrent early spontaneous abortion has been linked to hyperhomocysteinaemia (Steegers-Theunissen et al 1992; Wouters et al 1993). Homocysteine is derived from the essential amino acid methionine. It can be remethylated to methionine by betaine-homocysteine methyltransferase or by methionine synthase, which requires 5-methyltetrahydrofolate as the methyl donor and vitamin B 12 as cofactor. Excess homocysteine can be irreversibly trans-sulphurated via cystathionine into cysteine, in which the first reaction is catalysed by cystathionine β-synthase which is dependent on the cofactor pyridoxal 5-phosphate (PLP, a form of vitamin B 6 ). Heterozygotes for cysta-thionine β-synthase deficiency have been shown to have hyperhomocysteinemia and have been estimated to occur with a frequency between 1:100 and 1:200 in the normal popu-lation (Mudd et al 1989). However, Steegers-Theunissen et al (1992) reported normal cystathionine β-synthase activity (control range 1.8-18.9nmol/h per mg protein in cultivated fibroblasts) in patients with hyperhomocysteinemia and a history of early spontaneous abortion, so elevated levels of serum homocysteine do not necessarily imply the presence of a defective cystathionine β-synthase gene. We undertook in this study to investigate the relationship between total homocysteine levels and cystathionine β-synthase activity in patients with abruptio placentae in order to try to resolve that discrepancy.

Glutaric aciduria type 1 in South Africa-high incidence of glutaryl-CoA dehydrogenase deficiency in black South Africans.

Glutaric Aciduria type 1 (GA 1) is an inherited disorder of lysine and tryptophan catabolism that typically manifests in infants with acute cerebral injury associated with intercurrent illness. We investigated the clinical, biochemical and molecular features in 14 known GA 1 patients in South Africa, most of whom were recently confirmed following the implementation of sensitive urine organic acid screening at our laboratory. Age at diagnosis ranged from 3days to 5years and poor clinical outcome reflected the delay in diagnosis in all but one patient. Twelve patients were unrelated black South Africans of whom all those tested (n=11) were found homozygous for the same A293T mutation in the glutaryl-CoA dehydrogenase (GCDH) gene. Excretion of 3-hydroxyglutarate (3-OHGA) was >30.1μmol/mmol creatinine (reference range <2.5) in all cases but glutarate excretion varied with 5 patients considered low excretors (glutarate <50μmol/mmol creatinine). Fibroblast GCDH activity was very low or absent in all of five cases tested. Heterozygosity for the A293T mutation was found 1 in 36 (95% CI; 1/54 - 1/24) unrelated black South African newborns (n=750) giving a predicted prevalence rate for GA 1 of 1 in 5184 (95% CI; 1/11664 - 1/2304) in this population. GA 1 is a treatable but often missed inherited disorder with a previously unrecognised high carrier frequency of a single mutation in the South African black population.

The R563Q mutation of the epithelial sodium channel beta-subunit is associated with hypertension.

Background A high prevalence of the R563Q mutation of the epithelial sodium channel β-subunit has been reported in South African hypertensives compared with unrelated normotensive controls. To delineate the effects of this mutation against a more uniform genetic background, this study investigated the association of the mutation with hypertension within affected kindreds. Methods Forty-five index patients and members of their kindreds were studied. Blood pressure, serum potassium and the presence of the R563Q mutation were determined. Results Of the 136 individuals studied, 89 were heterozygous for the R563Q mutation and 47 homozygous RR. The mean arterial pressure was significantly higher in the R563Q heterozygous group (p = 0.005) after adjusting for gender, race, age and kindred membership. Of the R563Q heterozygous subjects, 71 (80%) had hypertension, while 17 (36%) of the R563Q homozygous RR subjects were hypertensive. Six R563Q heterozygous subjects had hypokalaemia and one R563Q homozygous RR subject had hypokalaemia, but the difference was not statistically significant. Two heterozygous patients had Liddle’s syndrome, both occurring during pregnancy. Conclusion The R563Q mutation of β-ENaC is associated with hypertension within affected kindreds, but does not usually cause the full Liddle’s syndrome phenotype.

Short communication: Association of pre‐eclampsia with the R563Q mutation of the β‐subunit of the epithelial sodium channel

The R563Q mutation of the β‐subunit of the epithelial sodium channel (ENaC) is associated with hypertension in black and mixed ancestry (MA) men and women in South Africa. The frequency of the R563Q mutation in black and MA women with pre‐eclampsia (n= 230) and in controls (n= 198) was studied. The R563Q mutation was found in 7.8% of the women with pre‐eclampsia and in 2.6% of controls (P= 0.014). This remained significant if the black women were analysed separately (P= 0.031). We have demonstrated that a genetic variant of the ENaC is associated with pre‐eclampsia. This has implications for understanding the pathogenesis and treatment of pre‐eclampsia.

Recessive congenital methaemoglobinaemia type II, a new mutation which causes incorrect splicing in the NADH-cytochrome b5 reductase gene

Recessive congenital methaemoglobinaemia (RCM, McKusick 250800) is due to a deficiency in NADH-cytochrome b5 reductase (b5R, EC 1.6.2.2). There are two types of RCM. Type I presents with mild cyanosis due to a significant deficiency of b5R in red cells only and is usually caused by enzyme instability. Type II is characterized by cyanosis, severe mental retardation with neurological manifestations, and a generalized deficiency of b5R (Jaffe 1989). Two forms of the enzyme are known. The membrane-bound isoform of b5R is localized to the endoplasmic reticulum and outer mitochondrial membrane and the soluble form is found in erythrocytes. The patient was a 4-year-old boy who was previously reported to have dystonic athetoid cerebral palsy with mental retardation and microcephaly. Cyanosis had been noted previously but had not been investigated. On this admission (for a chest infection) he was found to have 60% methaemoglobinaemia which was persistent but responded to ascorbic acid treatment. b5R activity in red cells, cultured fibroblasts and lymphoblastoid cells was 16%, 8% and 21% of control activity, respectively. Sequencing of PCR-amplified b5R cDNA demonstrated an in-frame deletion of exon 6 which resulted in the deletion of 28 amino acids (codon 155→182). Sequencing of the intron 5 splice acceptor showed an A→C transversion of the invariant AG dinucleotide (CCCCAG to CCCCCG) which would be consistent with skipping of exon 6. The patient appears to be homozygous for the mutation since only a single sequence was found on direct sequencing of genomic DNA across the splice junction of exon 6. These results are consistent with RCM type II. To our knowledge nine mutations (types I and II) have appeared in the literature (Shirabe et al 1994; Vieira et al 1995). This is the second description of abnormal splicing in this enzyme and the first one defined in genomic DNA.

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.

An OTC deficiency 'phenocopy' in association with Klinefelter syndrome

SummaryLate-onset urea cycle disorder in a 20-month-old boy is unusually associated with Klinefelter syndrome with a 47XXY karyotype. We record the typical clinical and biochemical findings of ornithine transcarbamylase (OTC) deficiency in a young boy with a short history of recurrent vomiting, self mutilating behaviour, lethargy, ataxia and seizures. Laboratory studies showed hyperammonaemia and orotic aciduria, with normal citrulline and other urea cycle amino acids. Unfortunately, a liver biopsy for OTC activity measurement was refused by the parents. A rapid reversal of phenotype was seen on the introduction of a low-protein diet with accompanying benzoate and phenylbutyrate administration. Linkage studies suggested the inheritance of two X chromosomes, which was confirmed by karyotype analysis. Sequencing of all exons and immediate splice site regions revealed no sequence alterations in these sections of the OTC gene. A search for skewing of X-inactivation in the liver was not possible but we did show a random pattern of X-inactivation in leukocytes. The possibility of maternal X chromosome iso-disomy in our patient was discounted by microsatellite analysis, which revealed the inheritance of two independent X chromosomes. Mutation analysis in the OTC gene has shown that approximately 20% of patients with liver biopsy confirmed OTC deficiency do not have mutations in the coding or immediate splice-site sequences of this gene. Their classification as OTC phenocopies remains speculative, awaiting clarification of the underlying DNA alteration. We report on the novel association of OTC deficiency and Klinefelter syndrome with the additional interest of a probable unusual genetic defect underlying the OTC abnormality.