| dc.description.abstract | The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more
than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase
(MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that
participates in the fourth step of the leucine catabolism is defective. Tandem mass
spectrometry (MS/MS) based screening programmes in North America, Europe and Australia,
showed that MCC deficiency is the most frequent organic aciduria detected, with an average
frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these
regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet
known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering
from 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 focus
on the molecular characterisation of MCC deficiency. In the RSA no molecular based
diagnostic method which complements MS/MS screening programmes have yet been
implemented. Therefore, the aim of this study was to implement the necessary techniques for
the molecular characterisation of MCC deficiency, the determination of the sequence of the
open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are
present in the South African MCC deficient patient.
For the implementation of the molecular characterisation, a two-pronged approached was used
to characterize MCC of a MCC non-deficient individual (CFC). This approach included the
reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the
associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected
(genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and
mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with
MCC deficiency in Caucasians.
The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and
CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide
polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al.,
2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed
in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or
described 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 deficient
patient (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 both
ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of
the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes
all 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-deficient
individual), 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 and
9-17 was sequenced but no described or novel mutations were identified. The lack of sequence
data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency
in HGS.
In conclusion, the basic methods and techniques for the molecular characterisation of MCC
deficient patients have been implemented locally. A few additional sequencing primers need to
be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of
each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to
be further refined to ensure better specificity. | |
