Severe mental retardation and recessive congenital methemoglobinemia in three Indian patients: Compound heterozygous for NADH‐cytochrome b5 reductase gene mutations
Authors: Prabhakar S. Kedar, Prashant Warang, Kanjaksha Ghosh, Roshan B. Colah
Journal: American Journal of Hematology (2010)

The diagnosis of Type II congenital methemoglobinemia has been established in three Indian patients in a single family based on persistent cyanosis and neurological manifestations with severe enzyme deficiency in erythrocytes. Clinical evaluation showed very severe encephalopathy, microcephaly, generalized dystonia, and mild cyanosis. Molecular studies demonstrated compound heterozygosity for two mutations in the reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) gene. One was a novel mutation 705G→A, which leads to the substitution of a Trp by a stop codon at residue 235 within exon 8 and the other was a previously reported missense mutation 608G→A leading to a replacement of Cys-203 (TGC) by Try (TAC) in exon 7. Although both amino acid substitutions are located in the NADH-binding domain, the whole protein structure, especially the region between the flavin adenine dinucleotide and NADH-binding domains, is disturbed. The presence of a premature stop codon results in the production of truncated b5R and should explain the severe enzyme deficiency seen in these cases. In healthy individuals, less than 1% of hemoglobin is present in the oxidized form called methemoglobin [1, 2]. The major pathway for the reduction of methemoglobin to functional hemoglobin in human erythrocytes involves a NADH dependent methemoglobin reductase system. In addition to NADH, this system requires the presence of cytochrome b5 reductase (b5R), also named diaphorase and cytochrome b5 [3]. Recessive congenital methemoglobinemia (RCM) (MIM 250800) is a very rare disorder caused by deficiency of b5R. Two forms of this enzyme are known, a membrane-bound form mainly found in microsomes and the endoplasmic reticulum of all tissues and a soluble form present in erythrocytes. These two forms are generated from the same gene by a combination of transcriptional and translational mechanisms. The enzyme deficiency is usually restricted to the red cell soluble b5R (RCM Type I), whereas in 10–15% of cases, the enzyme defect is generalized to all tissues, involving both soluble and microsomal forms of the enzyme (RCM Type II) [4]. Patients with RCM Type I have cyanosis but hardly any systemic symptoms when the methaemoglobin level is less than 25%. In RCM Type II, cyanosis is associated with severe progressive neurological disability. The clinical picture is very similar in most cases, with severe encephalopathy, microcephaly, generalized dystonia, movement disorders, and mild cyanosis [5]. RCM Type I and II are caused by a defect in a single gene (DIAl), which has nine exons and eight introns and is located on chromosome 22 (locus DIAl; q13.31-qter). Recent literature focuses on genetic aspects and mutation analysis [6]. Here, we emphasize the severe neurological outcome of RCM Type II, which is usually associated with mental retardation and neurological impairment because of cytb5r enzyme deficiency due to a novel stop codon mutation. The propositus was a 6-year-old female belonging to the Sahu community from Bihar state in eastern India, who was referred to us with complaints of bluish discoloration of lips and fingers noted since birth. On examination, she had slate grey pigmentation of the skin and mucous membranes, complaints of breathlessness, fever along with mental retardation, and neurological abnormalities. There was no history of cardiac illness as indicated by her normal 2D ECHO report. She had no history of exposure to drugs and chemicals. There was no history of consanguinity in the family. Two other siblings also presented with clinical symptoms of RCM Type II. The clinical picture showed severe encephalopathy, microcephaly, generalized dystonia, movement disorders and mild cyanosis. The diagnosis of RCM Type II in the proband was established at 5 years because of persistent cyanosis and a neurological disorder associated with a severe enzyme deficiency in erythrocytes. Her methemoglobin was elevated to 51%, (normal < 1.5%). After intravenous administration of methylene blue (0.5 mg/kg body weight), the methemoglobin levels reduced to normal. Subsequently, she was on vitamin-C 500 mg twice a day. During this time, her methemoglobin was within normal limits, but as soon as she stopped taking vitamin C, she again developed cyanosis. The two other affected siblings also had similar problems. Both her parents and one sister had mild cyanosis without any complaints of breathlessness while one sister was normal (Table I). We describe an Indian family with Type II disease because of deficiency of cytochrome b5 reductase enzyme. The pedigree of the family along with the mutations in DIA-I gene are shown in Fig. 1A. Three of five siblings in this family with no history of consanguinity were affected with a severe disease. The propositus had a methemoglobin level of 51%. Erythrocyte NADH-b5R activity was very low (6.0 IU/g Hb). She had developmental delay, severe tetraspastic cerebral palsy, profound cognitive impairment, and impressive secondary microcephaly. Two of her elder brothers also presented with clinical symptoms of RCM Type II. The clinical picture was very similar in all cases with severe encephalopathy, microcephaly, generalized dystonia, movement disorders, and mild cyanosis. Their enzyme activity was 6.21 IU/g Hb and 9.60 IU/g Hb, respectively. The patient's mother and father and one sister (SS) had intermediate enzyme levels (26.56, 22.47, and 24.02 IU/g Hb, respectively), whereas her younger sister (AS) was absolutely normal. (Normal range of NADH-b5R 30.50–40.50 IU/g Hb).Their hematological, biochemical, and clinical manifestations are shown in Table I. Glucose-6-phosphate dehydrogenase (G6PD) activity was normal in all the members of the family. The cyanosis was treated with methylene blue, which stimulates the production of the reduced NADPH substrate of b5R via the hexose monophosphate pathway of red cells [7]. There is an important caveat to the use of methylene blue in countries like India, as deficiency of G6PD is prevalent varying from 5.7% to 27.9% in the different population groups in India [8]. (A) Pedigree of the family with mutations in the DIA-I gene. (B) Ribbon diagram of the 3D model of the human cytb5R was generated from the atomic coordination of the protein data bank entry 1UMK using Pymol program; mutated residue 203 Cys→Try is shown in red color whereas the stop codon position is shown in blue color. FAD binding domain is drowning in cyan (residue 86–135), while NADH binding domain is shown in yellow color (residue 171–195). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] SSCP gel electrophoresis showed altered mobilities in exon 7 and 8. Both these exon were directly sequenced. The propositus showed two mutations in exon 7 and 8, respectively. The first was a missense mutation 608G→A leading to a replacement of TGC in codon 203 by TAC in exon 7, which would cause a change of cysteine to tyrosine (Supporting Information Fig. 2A). This mutation was inherited from the mother. The second was a novel mutation 705G→A that leads to the substitution of a TGG codon (Trp) by a TGA codon (stop codon) at codon 235 within exon 8 (Supporting Information Fig. 2B), which was inherited from the father. The two elder brothers were also compound heterozygous for these two mutations with similar clinical manifestations, while one of the sisters was heterozygous and the other sister was normal (Fig. 1A). The novel allelic mutation identified in this patient at codon 235 is in helix 5, which has a helix–helix interaction with helix 9 in the NADH binding domain. This mutation substitutes the non-polar neutral tryptophan for a basic positively charged arginine in helix 7. This is likely to disrupt the secondary structure and the stability of the enzyme consistent with a Type II recessive congenital methemoglobinemia (RCM) phenotype with 25% b5R enzyme activity in red blood cells. The presence of a premature stop codon results in the production of truncated NADH-b5R and should explain the severe enzyme deficiency observed in this patient (Fig. 1B). A model of Cys203Try mutation was generated by replacing the wild type residue with the mutant one in the human b5R structure. The Cys-203, located in the P2-sheet of the NAD domain is one of four cysteines (Cys-203, Cys-273, Cys-283, and Cys-297) found in the last third of the b5R protein at the COOH-terminus. The Cys203Try mutation introduces a positive charge and may eventually abolish a thiol group critical to enzyme functioning. A decrease in solvent accessibility for residue C203 was observed in our model, indicating that this residue is buried deeper in the hydrophobic pocket. According to the model, a slightly wider hydrophobic cavity is predicted to form in the mutant enzyme, leading to increased entry of water molecules and disruption of conserved hydrogen bonds (Fig. 1B). Previously, two cases of RCM Type-I with novel mutations have been reported from India [9, 10], and another novel mutation in exon 8 of the cytochrome b5 reductase gene changing codon 217 from Leu to Pro (L217P) has been identified in an Indian patient causing Type I methemoglobinemia [11]. Characterizing the mutations in Indian patients with this rare enzymopathy will help to understand the genotypic and phenotypic variability and to offer prenatal diagnosis if required. This is the first report of mental retardation because of the novel mutation, along with a second mutation in the NADH-b5R gene in an Indian family with recessive congenital methemoglobinemia Type II. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.