Complex approach to prenatal diagnosis of cytochromec oxidase deficiencies

Abstract

Different severe disorders of cytochrome c oxidase (COX) have been described in children, but only the defects with autosomal inheritance are suitable for prenatal diagnosis. To perform prenatal diagnosis of fatal infantile COX deficiency a complex approach has been used which combined determination of the genetic origin of the defect, and detailed analysis of the function, content and subunit composition of the enzyme in cultured fetal cells. The tissues and cultured fibroblasts of the patient with Leigh's syndrome showed a COX deficiency of systemic character. The decrease of COX activity to 5–11 per cent was accompanied by proportionally decreased content of the assembled COX enzyme. With the help of transmitochondrial cybrids derived from patient fibroblasts it was proven that the COX defect was of nuclear origin. In a successive pregnancy, the function of oxidative phosphorylation (OXPHOS) was analysed in cultured amniocytes by substrate‐stimulated ATP production and COX activity was compared with the activity of citrate synthase. The amount and composition of OXPHOS complexes was estimated by two‐dimensional (Blue Native/SDS) polyacrylamide gel electrophoresis and was verified immunochemically with specific antibodies. Three independent lines of evidence provided us with reliable data on the function of COX and OXPHOS in fetal cells which were sufficient to rule out the expected enzymatic defect within three weeks after amniocentesis. Copyright © 1999 John Wiley & Sons, Ltd.

Prenatal Diagnosis of Disorders of Energy Metabolism

“…A recent prenatal diagnosis for COX with Leigh disease was performed on amniocytes by demonstrating low enzyme activity and failure of assembly of the cytochrome oxidase complex. 16 mtDNA Depletion Syndrome While this syndrome entails gradual loss of mtDNA from liver or muscle, the gene responsible is a nuclear one and probably is responsible for some critical function in mitochondrial maintenance. The defect is only sometimes expressed in cultured cell systems, which means that looking at amniocytes or chorionic villous cells may not be reliable.…”

Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis

“…The approach allows screening for the presence or absence of these subunits in patients, as was first shown by Klement et al [4] in cultured human fibroblasts, and later by Bentlage et al [5] who applied the technique in a study of patients with different forms of mitochondrial encephalomyopathies. Houstek et al [6] used the method for the diagnosis of cytochrome c oxidase deficiency at the prenatal level, showing a significant decrease of all complex IV subunits. Others applied the method in the analysis of patients with diseases that are now generally accepted to be caused by mitochondrial defects, such as Parkinson's disease [7,8].…”

Determination of enzyme activities for prenatal diagnosis of respiratory chain deficiency

“…Prenatal diagnosis of respiratory chain de®ciency has been previously carried out, including for complex I de®ciency (Table 3, Ruitenbeek et al, 1988Ruitenbeek et al, , 1992Merante et al, 1993;Wanders et al, 1996;Houstek et al, 1999). Expression of the disease in cultured skin ®broblasts of the proband was not consistently required.…”

“…For these reasons, the molecular prenatal diagnosis of these conditions is seldom feasible and the only alternative option is based on determination of enzymes activities in fetal tissues (Ruitenbeek et al, 1996). This goal has been occasionally achieved (Ruitenbeek et al, 1988Merante et al, 1993;Wanders et al, 1996;Houstek et al, 1999) but its reliability remains questionable. Here, we report on 21 prenatal diagnoses in 10 families and emphasize the (1) paramount importance of taking skin biopsies of affected individuals for future prenatal diagnosis and (2) pitfalls in interpreting results during pregnancy.…”