Molecular characterisation of suspected heterozygotes of trimethylaminuria

Thesis, MSc, Potchefstroom University for Christian Higher Education 2002.

Trimethylaminuria (McKusick 602079) or Fish odour syndrome is inherited recessively as a defect in hepatic nitrogen-oxidation of dietary derived trimethylamine (TMA), which causes excess excretion of trimethylamine such that affected individuals have a body odour reminiscent of rotten fish (Zhang et al., 1995). Trimethylaminuria is a result of either partial or total incapacity to oxygenate trimethylarninuria to its oxide, trimethylamine oxide (TMAO), by an enzyme known as the flavin-containing monooxygenase 3 (FM03). Mutations in the gene of the major human liver enzyme isoform, FM03, are responsible for causing trimethylaminuria (Akerman et al., 1999a and Dolphin et al., 1997b ). Clinical symptoms of this disorder of metabolism include fish-like to garbage-like odour of urine (trimethylaminuria), sweat (ishthyhidrosis) and breath (halitosis) as well as psycho-clinical symptoms such as depression (Akerman et al., l999a). To establish the percentage of homo- and heterozygous trimethylaminuric individuals, a screening programme was introduced for the Potchefstroom first year students. Evaluation of the screening results through the liquid chromatograph-mass spectrometer, which is based on the accurate determination of the TMA:TMAO ratio, showed a 1.46% of mild trimethylaminuria individuals. In this study, clinical symptoms induced by the loading test prior to urine evaluation of the TMA:TMAO ratio is described. This was followed by isolation of the FM03 gene from the blood of suspected individuals and subsequent amplification using the PCR. Amplification was succeeded by restriction fragment length polymorphism analysis for the determination of known common mutations throughout the different exons of the FM03 gene. Single stranded conformation polymorphism and heteroduplex analysis were performed to validate their applicability towards screening the FM03 gene. Preliminary work was also done towards establishing the usage of the denaturing gel gradient gel electrophoresis to screen the FM03 gene for aberrant sequences. xvi Results obtained through restriction fragment length polymorphism indicated the possible presence of the A52T mutation in ex on 3 of both patients that showed symptoms of mild trimethylaminuria. The A52T mutation may be the most prevalent in the South African population although more research still have to be done to investigate this possibility. The main objective of this study was to establish the suitability of different methods towards mutation screening of the FM03 gene. The methods attempted so far include polymerase chain reaction, restriction fragment length polymorphism, single stranded conformation polymorphism, heteroduplex analysis as well as denaturing gradient gel electrophoresis. All methods were applicable, although to different extents and with different limitations and resolutions. This study was a preliminary evaluation for a bigger study, which will include family members of the suspected heterozygotes. In the subsequent study, all nucleotide sequence fragments suspected of having mutations will be sequenced to confirm the presence and the type of the mutation present. xvii

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