Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene

Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535G→A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757G→A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379A→G transition (M127V). The second allele carried a sequence difference at the invariant 3′ splice‐acceptor dinucleotide of intron 4 (IVS4‐2A→G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)+ RNA was analyzed by RT‐PCR and sequencing. The results show that RNA is made from the allele harboring the 3′‐splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001. © 2001 Wiley‐Liss, Inc.