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Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia.

ABSTRACT: Isovaleric acidemia (IVA) is an autosomal recessive inborn error of the leucine metabolism that is caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). Recent application of tandem mass spectrometry to newborn screening has allowed a significant expansion of the recognition of individuals with IVD deficiency. Although many patients have been reported worldwide, there are no genetically confirmed patients in Korea. This study characterizes IVD mutations in seven Korean IVA patients from six unrelated families. Bi-directional sequencing analysis identified two novel variations affecting consensus splice sites (c.144+1G>T in intron 1 and c.457-3_2CA>GG in intron 4) and three novel variations altering coding sequences (c.149G>T; Arg21Leu, c.832A>G; Ser249Gly, and c.1135T>G; Phe350Val). Five patients from four families were found to be compound heterozygotes while two unrelated patients were homozygous for the c.457-3_2CA>GG variation. Reverse-transcription polymerase chain reaction confirmed that both intron variations cause aberrant splicing. Furthermore, analysis of cultured lymphocyte extracts of the seven patients showed no detectable enzyme activity and reduced levels of IVD protein (<10.0% of control) in all samples. These results confirm IVD mutations in Korean patients with IVA and reveal that the mutation spectrum is different from previously reported patients.

SUBMITTER: Lee YW

PROVIDER: S-EPMC4136440 | biostudies-literature | 2007 Sep-Oct

REPOSITORIES: biostudies-literature

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Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia.

Lee Yong-Wha YW Lee Dong Hwan DH Vockley Jerry J Kim Nam-Doo ND Lee You Kyoung YK Ki Chang-Seok CS

Molecular genetics and metabolism 20070618 1-2

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of the leucine metabolism that is caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). Recent application of tandem mass spectrometry to newborn screening has allowed a significant expansion of the recognition of individuals with IVD deficiency. Although many patients have been reported worldwide, there are no genetically confirmed patients in Korea. This study characterizes IVD mutations in seven Korean IVA patients from ...[more]

PMID: 17576084

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Project description:Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Project description:IntroductionClinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS.MethodsAn IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity.Resultsc.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants.ConclusionsWe have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.

Dataset Information

Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia.

Publications

Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia.

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