1) Glutaric Aciduria type 1 in South Africa
Glutaric Aciduria type 1 (GA 1) is a completely preventable and treatable inherited disorder of lysine and tryptophan catabolism that typically results in severe neurological impairment if not recognised. After the implementation of sensitive urine organic acid screening at our laboratory we identified an unusually large number of patients with GA 1 and initiated research into this area. In the first stage we gathered information about the disease in South Africa and investigated the clinical and biochemical features of 14 known patients with GA1. Twelve of these patients were unrelated black South Africans and strikingly, all those tested (n=11) were found homozygous for the same A293T mutation in the glutaryl-CoA dehydrogenase (GCDH) gene. Excretion of 3-hydroxyglutarate was > 30.1 μmol/mmol creatinine (reference range <2.5) in all cases but glutarate excretion varied with 5 patients considered low excretors (glutarate < 50 μmol/mmol creatinine). Fibroblast GCDH activity was very low or absent in all of five cases tested. Due to the apparent high frequency of a single mutation we determined the carrier frequency of this mutation in 750 unrelated black South African newborns. Heterozygosity for the A293T mutation was found 1 in 36 (95% CI; 1/54–1/24)) giving a predicted prevalence rate for GA 1 of 1 in 5184 (95% CI; 1 in 11664–1 in 2304) in this population. This suggests that GA1 may well be the most common unrecognised inherited metabolic disease in South Africa.
The second phase of this project is to develop and assess a low cost high throughput molecular screening test for the A293T GCDH mutation . In this regard a low cost PCR based method is being developed in collaboration with Dr Tricia Owen (Inherited Genetic Disease UCT Chem Path) that can be applied to dried blood spot samples.
The third phase of the project currently being initiated is to apply mutational screening for GA1 to selected populations to A) confirm the suspected high carrier frequency of the disease mutation and B) Increase the detection of misdiagnosed cases. This phase will be initiated as a joint project between the paediatric neurology divisions at UCT and the University of Stellenbosch and our group. Selelective screening for GA 1 will be applied in higher risk neurology patient groups.
The final goal is to assess, based on the data obtained, the feasibility of a newborn screening program for GA 1 and hopefully detect the first pre-symptomatic cases and follow up the outcomes of locally applied preventative treatment. This phase will run concurrently with phase 3 and will consist of high volume screening of approximately 20 000 blood samples obtained through the present newborn cord blood based thyroid screening program at Red Cross Childrens Hospital. At the conclusion of this phase we should have enough robust data to make cost/benefit and methodological recommendations as to the feasibility of rolling out newborn screening for this and other known higher prevalence inherited disorders in South Africa.
2) Effect of antiretroviral treatment (ART) on mitochondrial function in children with HIV
Nucleoside reverse transcriptase inhibitors (NRTIs) affect mitochondrial DNA polymerase gamma causing significant toxic effects, including fatal lactic acidosis. Little is known about mitochondrial DNA (mtDNA) in human immunodeficiency virus (HIV) infected children who face a lifetime exposure to these agents. This project consisted of a cross sectional observation of mtDNA levels in whole blood in a pediatric population to ascertain the relationship between mtDNA, NRTI regimens and parameters of HIV-infection severity using a light cycler based real-Time PCR method to quantify mitochondrial DNA. Twenty seven presumed HIV-negative, 89 HIV-infected, NRTI-treated and 62 HIV-infected treatment-naive children were studied. Depletion of whole blood mtDNA was demonstrated in all children with HIV, but interestingly the depletion demonstrated was associated independently with markers of HIV infection severity such as CD4% count and HIV-1 viral load. Furthermore we could not demonstrate an improvement in mtDNA with either stavudine or zidovudine based ART regimens despite effective virological control, suggesting that HIV itself also depletes mtDNA, a finding that has subsequently been shown in the adult literature. The long-term consequences of these combined potentially toxic effects on the mitochondrial genome remain a cause for concern as children with HIV will likely be exposed to these drugs for life. The methods developed during this study for mtDNA quantitation are currently being revised as they are applicable to detection of tissue mtDNA depletion that is associated with inherited mitochondrial disease.
Whole Blood Leukocyte mt:nuclear DNA Ratios
3) Development of Gas Chromatography Mass Spectrometry Methods
Methodological research was initiated into this area with the recent placement of two Agilent GC-MS instruments in the Red Cross Chemistry Laboratory as part of the Inherited Metabolic Disease diagnostic service offered through the division. Although routine methods for urine organic acids and fluid amino acid analysis make up the bulk of the workload we have developed and evaluated site specific methods for additional analytes.
a) Rapid Sensitive Measurement of Leukocyte Cystine by Isotope Dilution GCMS
Nephropathic cystinosis, a recessive disorder of lysosomal cystine transport is the single most common identifiable cause of renal Fanconi syndrome in children and a large number of patients are referred to the paediatric renal unit at Red Cross with this disorder. Currently the laboratory diagnosis and monitoring of treatment depends on the determination of leukocyte cystine (LC) by HPLC, tandem MS or competitive cystine binding protein assays. These methods are hampered by long run times and some have limited sensitivity, requiring large blood volumes that can be problematic with paediatric samples .We developed and evaluated the first sensitive and fast gas chromatographic-mass spectrometic (GCMS) method for LC quantitation. The method is based on a cationic resin amino acid exchange to remove cystine from a lysed leukocyte pellet. followed by amino and carboxy terminal derivation and GC-MS quantitation using an isotopic cystine standard. The method was evaluated against a large EQA scheme for this analyte and demonstrated comparable performance to other methods at a significantly lower cost with much reduced run times and sample volume requirements.
b) A fast Simple GC-MS method for urinary Homovanillic Acid
Urinary homovanillic acid (HVA) screening is an important screening test for neuroblastoma, the most common solid tumour of childhood. Although isotope based GC-MS methods for HVA are well described, this project was initiated to attempt to reduce the cost and run times of analysis compared to these methods. A micro solvent extraction method was developed whereby HVA was extracted and derivatised in 1mL tubes and prepared for GC-MS injection within 20 minutes. Costs were significantly reduced by utilizing a vanillic acid internal standard and running each sample with and without the internal standard to correct for endogenous vanillic acid. The method demonstrated acceptable performance characteristics with excellent accuracy when compared to internal and external QA scheme samples for this analyte based on HPLC methods.
4) Mechanisms of Assay Interference
a) Investigative research was initiated using a variety of techniques to elucidate the cause of positive interference in an unusual case of grossly elevated Free Thyroxin on an Advia centaur immunoassay platform. Using techniques such as radioimmunoprecipitation and immunoprecipitation of immunoglobulin with polyethylenegycol (PEG) the interference was ascribed to anti -T4 autoantibodies. Because of the efficacy of PEG in removing the interference on this platform we went further and evaluated the application of PEG precipitation as a tool for screening for antibody interference and developed a regression equation and predictive equation that could be employed in any routine lab would be able to reliably predict the presence of T4 autoantibodies on this platform without sophisticated reagents or equipment.
b) Investigation of an unusual paraprotein evident on Capillary Zone Electrophoresis of plasma in a patient after coronary angiography was initiated. This work involved various techniques such as agarose gel electrophoresis with various stains and immunofixations and UV wavelength scanning on spiked samples and arrived at the conclusion that iopamidol, a radiocontrast agent used during angiography was responsible for the false paraprotein .The term pseudoparaprotein was coined during this investigation.
