Multiple carboxylase deficiency is a clinical condition caused by defects in the
enzymes involved in
biotin metabolism, holocarboxylase
synthetase (HLCS) or
biotinidase.
HLCS deficiency is a potentially fatal condition if left untreated, although the majority of patients respond to oral supplementation of 10-20 mg/day of
biotin. Patients who display incomplete responsiveness to this
therapy have a poor long-term prognosis. Here we investigated cell lines from two such HLCS-deficient patients homozygous for the c.647T>G p.L216R allele. Growth of the patients' fibroblasts was compromised compared with normal fibroblasts. Also the patient cells were not sensitive to
biotin-depletion from the media, and growth rates could not be restored by re-administration of
biotin. The molecular basis for the
HLCS deficiency was further investigated by characterisation of the p.L216R
protein. The HLCS
mRNA was detected in MCD and normal cell lines. However,
protein and
enzyme activity could not be detected in the patients' cells. In vitro kinetic analysis revealed that
enzyme activity was severely compromised for recombinantly expressed p.L216R and could not be increased by additional
biotin. Furthermore, the turn-over rate for the
mutant protein was double that of wildtype HLCS. These results help provide a molecular explanation for the incomplete
biotin-responsiveness of this p.L216R form of HLCS.