The implementation of the molecular characterisation of 3-methylcrotonyl-CoA carboxylase deficiency in South Africa

[摘要] The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that morethan 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase(MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase thatparticipates in the fourth step of the leucine catabolism is defective. Tandem massspectrometry (MS/MS) based screening programmes in North America, Europe and Australia,showed that MCC deficiency is the most frequent organic aciduria detected, with an averagefrequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in theseregions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yetknown. However, one 48 year old male Caucasian individual (HGS) was diagnosed sufferingfrom mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3-hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine.Several groups are currently working on various aspects of this emerging disease with the focuson the molecular characterisation of MCC deficiency. In the RSA no molecular baseddiagnostic method which complements MS/MS screening programmes have yet beenimplemented. Therefore, the aim of this study was to implement the necessary techniques forthe molecular characterisation of MCC deficiency, the determination of the sequence of theopen reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) arepresent in the South African MCC deficient patient.For the implementation of the molecular characterisation, a two-pronged approached was usedto characterize MCC of a MCC non-deficient individual (CFC). This approach included thereverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of theassociated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected(genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 andmccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated withMCC deficiency in Caucasians.The sequence analyses produced surprising results of the amplified ORFs (CFCmccA andCFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotidepolymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al.,2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listedin GenBank was observed in the amplified CFCmccB ORF. No significant novel variations ordescribed mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5,mccB6 and mccB5-6.The implemented molecular approach was used to characterise MCC of our MCC deficientpatient (HGS). The patient did not have any mutation in the four selected exons mccA8,mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of bothORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons ofthe expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includesall 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17).The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficientindividual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS.The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and9-17 was sequenced but no described or novel mutations were identified. The lack of sequencedata of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiencyin HGS.In conclusion, the basic methods and techniques for the molecular characterisation of MCCdeficient patients have been implemented locally. A few additional sequencing primers need tobe designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands ofeach MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need tobe further refined to ensure better specificity.

[发布机构] North-West University