Common mutation causes cystinosis in the majority of black South African patients.

BACKGROUND: The mutations responsible for cystinosis in South African patients are currently unknown. A pertinent question is whether they are similar to those described elsewhere in the world. METHODS: Children who were being managed for cystinosis in the Western Cape Province of South Africa between 2002 and 2013 were studied. All underwent molecular analysis to detect sequence variations in the cystinosis gene. RESULTS: This cohort study included 20 patients, 13 of whom were Xhosa-speaking black South Africans and seven were Cape Coloureds (mixed race); none were Caucasian. All had nephropathic infantile-type cystinosis with evidence of proximal tubulopathy, with glycosuria and renal phosphate wasting. Diagnosis was confirmed in 19 cases by demonstrating an elevated cystine concentration in leukocytes. Molecular analysis of the cystinosin gene revealed that 19 patients had a G > A mutation in intron 11 (CTNS-c.971-12G > A p.D324AfsX44) which caused an out-of-frame 10-bp insertion. Of these 19 patients, 16 were homozygous for this mutation, which was the most frequent mutation identified in the alleles of the black South African and Cape Coloured patients (96 and 71 %, respectively). CONCLUSION: We recommend that black South African and Cape Coloured patients presenting with cystinosis be tested for CTNS-c.971-12G > A in the first instance, with the possibility of prenatal testing being offered to at-risk families.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

RATIONALE: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. OBJECTIVES: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. METHODS: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. MEASUREMENTS AND MAIN RESULTS: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. CONCLUSIONS: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

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.

Association between apolipoprotein E4 genotype and human immunodeficiency virus-associated dementia in younger adults starting antiretroviral therapy in South Africa.

It is not known whether the apolipoprotein E (ApoE) ε4 allelic variant is associated with human immunodeficiency virus (HIV)-associated dementia (HAD) in a South African population, where HIV clade C is predominant. ApoE genotyping was performed on 144 participants in a larger study of HIV-associated neurocognitive disorders (HAND). There was a lower frequency of the ε2 and ε3 alleles in the HIV-positive group, compared to a group of 300 community-based newborn infants. There were no differences in ApoE genotype across different categories of HAND. The ε4 allelic variant was less common in individuals with HAD than in those without HAD. These findings suggest that the ε4 allelic variant in HIV-positive individuals is not associated with the development of HAD in Southern Africa.

Autosomal recessive hypercholesterolaemia: discrimination of ARH protein and LDLR function in the homozygous FH phenotype.

BACKGROUND: Phenocopies of homozygous familial hypercholesterolemia (hoFH) having autosomal recessive inheritance, were recently found to arise from defects in the LDL receptor (LDLR) adapter protein, called ARH, which facilitates the clearance of circulating LDL. Discrimination between the two causes of the phenotype at a clinical level may not be possible when parents display moderate hypercholesterolaemia. An effective strategy is thus required to identify the appropriate mechanism for the disorder. METHODS: Fibroblast LDL uptake studies were coupled with Western blotting for ARH protein in cell extracts, to identify the defective gene before DNA studies were initiated. Two subjects with the hoFH phenotype, but with indeterminate dyslipidaemia in their parents, were fully worked up. RESULTS: Defective LDL metabolism was established in both patients by functional and protein studies and further confirmed by detecting deleterious mutations, in the LDLR and ARH genes. The ARH patient is the first subject of Negroid identity to be described and records a specific mutation in this racial grouping. CONCLUSION: This study highlights the occasional complexity and uncertainty of a clinical diagnosis of hoFH and presents Western blotting of leucocyte extracts for ARH protein, as a rapid strategy for the detection of ARH before sequencing the gene for mutation(s). This strategy may be particularly useful in populations where founder mutations for ARH and LDLR defects are rare or co-exist.

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.

The clinical and molecular spectrum of galactosemia in patients from the Cape Town region of South Africa.

BACKGROUND: The objective of this study was to document the clinical, laboratory and genetic features of galactosemia in patients from the Cape Town metropolitan region. METHODS: Diagnoses were based on thin layer chromatography for galactosuria/galactosemia and assays of erythrocyte galactose-1-phosphate uridyltransferase (GALT) and galactokinase activities. Patients were screened for the common S135L and Q188R transferase gene mutations, using PCR-based assays. Screening for the S135L mutation in black newborns was used to estimate the carrier rate for galactosemia in black South Africans. RESULTS: A positive diagnosis of galactosemia was made in 17 patients between the years 1980 to 2001. All had very low or absent galactose-1-phosphate uridyltransferase (GALT) activity, and normal galactokinase levels. The mean age at diagnosis was 5.1 months (range 4 days to 6.5 months). A review of 9 patients showed that hepatomegaly (9/9), and splenomegaly, failure to thrive, developmental delay, bilateral cataracts (6/9) were the most frequent features at diagnosis. Six had conjugated hyperbilirubinemia. Four experienced invasive E. coli infection before diagnosis. Ten patients were submitted to DNA analysis. All 4 black patients and 2 of mixed extraction were homozygous for the S135L allele, while all 3 white patients were homozygous for the Q188R allele. The remaining patient of mixed extraction was heterozygous for the Q188R allele. The estimated carrier frequency of the S135L mutation in 725 healthy black newborns was 1/60. CONCLUSIONS: In the absence of newborn screening the delay in diagnosis is most often unacceptably long. Also, carrier frequency data predict a galactosemia incidence of approximately 1/14 400 for black newborns in the Cape Metropole, which is much higher than the current detection rate. It is thus likely that many patients go undetected.

Tuberculosis of the knee.

Fifty-two children with tuberculosis of the knee treated from 1979 to 1999 were reviewed retrospectively. The radiologic appearance of the joint at presentation was predictive of the outcome. Ninety-two percent of the patients had Stage 1 or Stage 2 involvement (synovitis) with or without bony erosions, but had a normal joint space. Treatment with antituberculous chemotherapy without synovectomy had an excellent or good result in all patients with Stage 1 or Stage 2 disease, and there was no difference in outcome whether the knee was immobilized or mobilized. Patients with Stage 3 and Stage 4 disease who had a narrow joint space (arthritic) at presentation had a fair or poor result. In patients with monoarthritis of the knee with nonspecific histologic features and a negative culture, the differential diagnosis between tuberculosis and pauciarticular juvenile rheumatoid arthritis is problematic. The histologic evaluations of biopsy specimens of the synovium of 25 knees from 25 patients were reviewed for synovial lining hyperplasia. The sensitivity for the 17 knees that subsequently were diagnosed as having juvenile rheumatoid arthritis was only 53%. Deoxyribonucleic acid from 13 consecutive joints was subjected to polymerase chain reaction for Mycobacterium tuberculosis infection with only 40% sensitivity for tuberculosis.