A novel nine base deletion mutation in NADH-cytochrome b5 reductase gene in an Indian family with recessive congenital methemoglobinemia-type-II

Recessive hereditary methemoglobinemia (RCM) associated with severe neurological abnormalities is a very rare disorder caused by NADH- cytochrome b5 reductase (cb5r) deficiency (Type II). We report a case of 11 month old male child who had severe mental retardation, microcephaly and gross global developmental delay with methemoglobin level of 61.1%. The diagnosis of NADH-CYB5R3 deficiency was made by the demonstration of significantly reduced NADH-CYB5R3 activity in the patient and intermediate enzyme activity in both the parents. Mutation analysis of the CYB5R gene revealed a novel nine nucleotide deletion in exon 6 leading to the elimination of 3 amino acid residues (Lys173, Ser174 and Val 175). To confirm that this mutation was not an artifact, we performed PCR-RFLP analysis using the restriction enzyme Drd I. As the normal sequence has a restriction recognition site for Drd I which was eliminated by the deletion, a single band of 603-bp was seen in the presence of the homozygous mutation. Molecular modeling analysis showed a significant effect of these 3 amino acids deletion on the protein structure and stability leading to a severe clinical presentation. A novel homozygous 9 nucleotide deletion (p.K173–p.V175del3) is shown to be segregated with the disease in this family. Knowing the profile of mutations would allow us to offer prenatal diagnosis in families with severe neurological disorders associated with RCM — Type II

Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis

We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na+ efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na+ efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl− transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl− transport activity, but R730 substitution with I, E, or H inactivated Cl− transport. AE1 R730C expression substantially increased endogenous oocyte Na+-K+-ATPase-mediated 86Rb+ influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pHo) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pHo-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pHo insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl− influx by acid pHo. Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb+ and Li+ influxes. Selective rescue of acid pHo-stimulated sulfate uptake and conferral of acid pHo-stimulated Cl− influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1