| Abstract |
Vitamin B12 (cobalamin, Cbl) is an essential cofactor for two human
enzymes: methylmalonyl-CoA mutase (MUT) and methionine synthase (MTR). MUT
utilizes 5'-deoxyadenosylcobalamin (AdoCbl) to convert methylmalonyl-CoA
to succinyl-CoA in the mitochondria, whereas MTR utilizes methylcobalamin
(MeCbl) to convert homocysteine to methionine in the cytoplasm. To date,
eight complementation groups (cblA-G and mut), each the result of
mutations at a different gene, have been discovered to be involved in the
intracellular metabolism of cobalamin. A patient presented at birth,
following an abnormal newborn screen, with hypotonia, lethargy, poor
feeding and bone marrow suppression. There were elevated levels of
methylmalonic acid and homocysteine, suggestive of a defect in vitamin B12
metabolism. Studies of cultured fibroblast showed decreased function of
the cobalamin-dependent enzymes, MTR and MUT. There was increased uptake
of labelled cyanocobalamin (CNCbl) but decreased synthesis of the
cobalamin cofactors MeCbl and AdoCbl, with accumulation of "free" (i.e.
non-protein bound) CNCbl in the cells. The cellular phenotype mimicked
that of the cblF disorder caused by mutations in the LMBRD1 gene encoding
the lysosomal membrane protein LMBD1 that is thought to play a role in
transfer of cobalamin across the lysosomal membrane into the cytoplasm.
However, cells from the patient complemented those from all known
complementation groups, including cblF, and no mutations in LMBRD1 were
found. Whole-exome sequencing led to the identification of two mutations
in the ABCD4 gene: c.956A>G (p.Y319C) and c.1746_1747insCT (p.E583LfsX9).
Two additional patients with deleterious ABCD4 mutations were later found.
Transfection of patient fibroblasts with wild type ABCD4 led to rescue of
all abnormal cellular phenotypes. This thesis reports that this novel
disorder, named cblJ, is an autosomal recessive disorder caused by
mutations in ABCD4. The findings suggest that ABCD4, an ABC half-
transporter, is another essential component of intracellular cobalamin
metabolism.
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