A NOVEL POINT MUTATION IN A 3'-SPLICE-SITE OF THE NADH-CYTOCHROME-B(5) REDUCTASE GENE RESULTS IN IMMUNOLOGICALLY UNDETECTABLE ENZYME AND IMPAIRED NADH-DEPENDENT ASCORBATE REGENERATION IN CULTURED FIBROBLASTS OF A PATIENT WITH TYPE-II HEREDITARY METHEMOGLOBINEMIA

Hereditary met hemoglobinemia with generalized deficiency of NADH-cyto chrome b(5) reductase (b(5)R) (type II) is a rare disease characterize d by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of th is condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activiti es of the endoplasmic reticulum-located reductase. However, the functi ons of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G-->T transversion at the spli ce-acceptor site of the 9th exon, which results in the complete absenc e of immunologically detectable b(5)R in blood cells and skin fibrobla sts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reducta se activities were severely reduced. The latter activity is known to b e due to b(5)R located on outer mitochondrial membranes. Thus, our res ults demonstrate that the reductase in its two membrane locations, end oplasmic reticulum and outer mitochondrial membranes, is the product o f the same gene and suggest that a defect in ascorbate regeneration ma y contribute to the phenotype of hereditary methemoglobinemia of the g eneralized type.