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