Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects

https://doi.org/10.1016/j.ymgme.2008.12.003Get rights and content

Abstract

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype–genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

Section snippets

Patients

Eight young children with clinical and biological features consistent with a RC deficiency were included in this study after informed consent. Their main clinical phenotypes were hypertrophic cardiomyopathy and encephalopathy for patient 1, leukoencephalopathy for patients 2, 3, 7 and 8, and unspecified encephalomyopathy for patients 4, 5 and 6. Two other patients, who were previously shown to be deficient in complex I nuclear genes, were used as reference controls and were designated control 1

Results

Respiratory chain enzymes were measured by polarographic and spectrophotometric assays for the 8 patients in fibroblasts, and/or lymphocytes, skeletal muscle and liver biopsies. A complex I deficiency was concluded in the 8 patients. Results of pyruvate oxidation and complex I activity are reported in Table 2.

All cDNA fragments of controls 1 and 2 were sequenced and analysed to identify the wild-type fragments which were subsequently used for the heteroduplexes formation. Single nucleotide

Discussion

Screening of the complex I nuclear cDNAs mutations in complex I-deficient patients by Surveyor nuclease digestion allowed us a rapid detection of heterozygous or homozygous nucleotide changes which were confirmed by direct sequencing. In a total of 176 cDNA fragments screened, only 33 fragments (18.7%), harbouring homozygous or heterozygous transition, were sequenced. Missense mutations were detected by Surveyor screening and identified in 3/8 patients (37.5%). The skipping of the second exon

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