© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Clin Genet 2013 Printed in Singapore. All rights reserved
CLINICAL GENETICS doi: 10.1111/cge.12326
Short Report
Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India Warang P.P., Kedar P.S., Shanmukaiah C., Ghosh K., Colah R.B. Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India. Clin Genet 2013. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2013 We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH-cytochrome b5 reductase (NADH-CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice-site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three-dimensional (3D) structure of human erythrocyte NADH-CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH-CYB5R deficiency. Conflict of interest
The authors have no conflicts of interest.
Recessive congenital methemoglobinemia (RCM) is a rare disorder caused by NADH-cytochrome b5 reductase (NADH-CYB5R) deficiency, with an autosomal recessive inheritance. It is characterized by two distinct clinical phenotypes. In RCM type I, cyanosis is the only clinical presentation whereas in RCM type II, cyanosis is associated with severe mental retardation, neurological impairment and early death. NADHCYB5R exists in two isoforms, the membrane-bound and the RBC (soluble) form. They are encoded by the same CYB5R3 gene located on chromosome 22q13qter. This gene is 32 kb long with nine exons and eight
P.P. Waranga , P.S. Kedara , C. Shanmukaiahb , K. Ghosha and R.B. Colaha a National Institute of Immunohaematology, Indian Council of Medical Research, Mumbai, India and b Haematology Department, K.E.M Hospital, Mumbai, India
Key words: cyanosis – CYB5R3 gene – NADH-cytochrome b5 reductase deficiency – neurological impairment – recessive congenital methemoglobinemia Corresponding author: Dr Roshan B. Colah, PhD, Scientist ‘F’, Department of Haematogenetics, National Institute of Immunohaematology, Indian Council of Medical Research, 13th Floor, New Multistoried Building, K.E.M Hospital Campus, Parel, Mumbai 400012, India. Tel.: +91 22 24138518; fax: +91 22 24138521; e-mail:
[email protected] Received 24 September 2013, revised and accepted for publication 20 November 2013
introns (1–3). The Gene Bank reference sequences are: NC_000022.10 and NP_000389.1. Only 58 mutations causing NADH-CYB5R deficiency have been globally reported so far (http://www.biobase-international.com/). The molecular structure of NADH-CYB5R consists of two functional domains linked by a flexible hinge region: the N-terminal or FAD-binding domain (Thr31 to Ser146) and the C-terminal or NADH-binding domain (Ser174 to Phe301). These two domains are connected by a small linker domain (Gly147 to Lys173) consisting of a triple-stranded antiparallel β sheet, which is important for the maintenance of
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Warang et al. Table 1. Clinical, hematological and biochemical data of the patients with NADH-CYB5R deficiency
Case no. Age/sex RCM type I cases 1. 20 years/M 2. 7 years/M 3. 52 years/F 4. 5 months/M 5. 30 years /M 17 days/M 6.a 7. 31 days/M 8. 19 years/M 9. 11 years/M 10. 10 years/F 11. 18 months/F 12. 19 years /F 13. 14 years/M 14. 18 years/M 15. 33 years/M 16. 43 years/M 17. 49 years/M 18. 28 years/M RCM type II cases 19. 3 years/M 20. 2 1/2 years/F 21. 3 1/2 years/F 22. 3 years/F 23. 5 years/ F
Clinical Origin Consanguinity presentation
RBC × 106 /μl
HGB g/dl
HCT MCV % fl
MCH pg
MCHC g/dl
RDW %
Hi level NR: A c.530 T>C c.536G>A c.148C>T c.173G>A c.226G>A c.536G>A c.536G>A c.433C>T c.757G>A c.536G>A c.148C>T c.531G>A c.531G>A c.536G>A c.531G>A c.148C>T c.148C>T c.479G>C c.713C>T c.536G>A
p. Gly155Glu p.Ile178Thr p.Ala179Thr p.Arg50Trp p.Arg58Gln p.Gly76 Ser p.Ala179Thr p.Ala179Thr p.Pro145Ser p.Val253Met p.Ala179Thr p.Arg50Trp p.Met177 Ile p.Met177Val p.Ala179Thr p.Met177Val p.Arg50Trp p.Arg50Trp p.Arg160Pro p.Thr238Met p.Ala179Thr
Linker NADH binding NADH binding FAD binding FAD binding FAD binding NADH binding NADH binding FAD binding FAD binding NADH binding FAD binding NADH binding NADH binding NADH binding NADH binding FAD binding FAD binding Linker NADH binding NADH binding
Homozygous Homozygous Homozygous
3 3 8
c.226G>A c.226G>A c.708G>A
p.Gly76 Ser p.Gly76 Ser p.Trp236X
FAD binding FAD binding NADH binding
Mutation status
RCM type I cases 1. Gly154Glu 2. Ile177Thr 3. Ala178Thr/Arg49Trp
Homozygous Homozygous Compound Heterozygous
4.
Arg57Gln/Gly75 Ser
Compound Heterozygous
5. 6. 7. 8. 9.
Ala178Thr Ala178Thr Pro144Ser Val252Met Ala178Thr/Arg49Trp
Homozygous Homozygous Homozygous Homozygous Compound Heterozygous
10. Met176 Ile 11. Met176 Val 12. Ala178Thr 13. Met176Val 14. Arg49Trp 15. Arg49Trp 16. Arg159Pro 17. Thr237Met 18. Ala178Thr RCM type II cases 19. Gly75 Ser 20. Gly75 Ser 21. Trp235Ter
cDNA, complementary DNA; CYB5R, cytochrome b5 reductase; RCM, recessive congenital methemoglobinemia. a Novel mutations are shown in bold type. Two cases (no. 22 and 23 in Table 1) remained uncharacterized.
Nβ2 and is located in the NADH-binding domain in close proximity to the adenosine moiety; it has been suggested that replacement by threonine would alter the binding of the NADH coenzyme, resulting in decreased catalytic activity and stability of the protein, providing an explanation for the severity of the clinical manifestations (2, 7). The p.Thr238Met mutation is localized at the C-terminal end of strand 10 of the NADH-binding domain, somewhat remote from the protein surface and relatively far away from the domain interface. In the protein structure, threonine at 238 is conservatively replaced by a less bulky hydrophobic residue methionine. Frequency distribution of CYB5R3 gene mutations in India
The allele distribution of all the reported mutations found worldwide and in Indian patients is shown in Fig. 2. (See Table S1, Supporting Information) (1, 8) Fifteen different putative disease-causing mutations were detected in 46 chromosomes in Indian patients including the ones described in two previous reports (9, 10). Nine of these mutations have been found exclusively in the Indian population. The p.Ala179Thr mutation was the most prevalent mutation in Indian NADHCYB5R deficient patients. Ten of 46 NADH-CYB5R
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deficient alleles (21.73%) having this mutation were associated with clinical features of type I/II RCM. The p.Arg50Trp novel mutation was the next most prevalent mutation (13.04%) in Indian NADH-CYB5R deficient patients. p.Gly76Ser is the third most common mutation (10.86%) found in the homozygous state in two cases (cases 19 and 20) as well as in the compound heterozygous state with another mutation (p.Arg58Gln) (case 4).
Discussion
Congenital methemoglobinemia due to NADH-CYB5R deficiency is a very rare autosomal recessive disorder. There are only a few cases report on NADH-CYB5R deficiency from India (11). Earlier the p.Gly144Asp and p.Leu217Pro novel mutations had been identified in an Indian patient and they were associated with type I methemoglobinemia (12) and the p.Trp236X mutation associated with type II methemoglobinemia had been reported in three Indian patients (10). RCM type II due to NADH-CYB5R deficiency associated with recurrent early pregnancy loss (REPL) was also encountered (13). Here, we report 13 different mutations among the 46 mutated alleles. The most frequent mutations in the
Clinical spectrum and molecular basis of RCM
Fig. 2. Allele distribution of CYB5R3 gene mutations worldwide and in Indian patients.
Indian population appear to be p.Ala179Thr followed by p.Arg50Trp and p.Gly76Ser. The p.Ala179Thr mutation has been reported earlier in two Asian Indian siblings (Kashmir Gujjar ethnicity) (14). This mutation was first reported in a Dutch Caucasian subject (15) and then in other individuals (1, 8). We identified the p.Arg50Trp novel mutation in the homozygous state and compound heterozygous state with another common mutation (p.Ala179Thr) leading to the clinically less severe RCM type I phenotype. The p.Arg50Gln mutation was reported in the same codon in a Spanish family (7). Another homozygous novel mutation p.Thr238Met was seen in a patient with 30% methemoglobinemia associated with type I RCM. p.Val253Met is the most common mutation reported globally associated with type I and II RCM (1,8). It was identified in a 19-year-old male patient in the homozygous form in our study. This patient did not have mental retardation. The second common mutation globally (p.Ala179Thr) was also identified in a 17day-old male child, who presented with severe anemia (Hb – 6.7 g/dl) associated with cyanosis (case 6). His three elder siblings also had cyanosis and died at an early age (4 days, 6 months and 4 months). This patient had a methemoglobin level of 20.0% with markedly reduced NADH-CYB5R activity. The p.Arg50Trp novel mutation identified in six alleles concerns a nonconservative region located near the protein surface in the first β-strand of the FAD domain. In the protein structure, arginine forms a strong hydrogen bond with the carbonyl oxygen of the conserved region. From this observation and the low levels of NADH-CYB5R in these patients, we conclude that a positively charged residue at position 50 in human NADH-CYB5R is
important for the structural integrity of the FADbinding domain by forming optimal intra domain contacts. We found the p.Gly76Ser mutation in the homozygous state in two cases as well as in the compound heterozygous state with another mutation (p.Arg58Gln). Interestingly, both the homozygous cases showed cyanosis associated with severe mental retardation. The first case was a 3-year-old boy born of a nonconsanguineous marriage (case 19) and the second case had a methemoglobin level of 72% in his erythrocytes with very low NADH-CYB5R activity (case 20). In this family, there was a strong history of severe neurological impairment. Both his siblings had severe mental retardation and one sibling had died at the age of 3 years. This p.Gly76Ser mutation was previously reported in type I and II RCM (16). In all our patients, treatment with methylene blue (0.5 mg/kg body weight) resulted in rapid reduction of the methemoglobin levels. In RCM type II, cyanosis was associated with severe mental retardation and neurological impairment without possible treatment. Its prevention by prenatal diagnosis in early pregnancy is therefore warranted. RCM type I seems to be associated with missense mutations only, whereas RCM type II seems to be associated with nonsense mutations and splice-site mutations. An important practical application of characterization of the mutations is the possibility of reliable prenatal diagnosis (17). This is the first comprehensive report on molecular characterization of NADHCYB5R deficiency from India where 13 different mutations have been identified and the structural implications of amino acid substitutions on CYB5R3 studied.
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Warang et al. These investigations contribute to a better understanding of genotype–phenotype relationships and to explain the mechanism of severity of the disease. Supporting Information
7.
8.
The following Supporting information is available for this article: Table S1. Update of mutations in the CYB5R3 gene associated with recessive hereditary methemoglobinemia type I and II. Additional Supporting information may be found in the online version of this article.
Acknowledgements This work was supported by the Indian Council of Medical Research, New Delhi. We thank all the clinicians who referred these patients to us for diagnosis.
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