: MOLECULAR STUDY OF RED CELL NADH-CYTOCHROME B5 REDUCTASE DEFICIENCY IN 21 INDIAN PATIENTS WITH RECESSIVE HEREDITARY METHEMOGLOBINAEMIA
Author(s): ,
Prabhakar Kedar
Affiliations:
Haematogenetics,National Institute of Immunohaematology (ICMR),Mumbai,India
,
Vinod Gupta
Affiliations:
Haematogenetics,National Institute of Immunohaematology (ICMR),Mumbai,India
,
Anuja Kulkarni
Affiliations:
Haematogenetics,National Institute of Immunohaematology (ICMR),Mumbai,India
,
Prashant Warang
Affiliations:
Haematogenetics,National Institute of Immunohaematology (ICMR),Mumbai,India
,
Rati Devendra
Affiliations:
Haematogenetics,National Institute of Immunohaematology (ICMR),Mumbai,India
,
Ashish Chiddarwar
Affiliations:
Haematogenetics,National Institute of Immunohaematology (ICMR),Mumbai,India
Manisha Madkaikar
Affiliations:
Director,National Institute of Immunohaematology (ICMR),Mumbai,India
EHA Library. Kedar P. Jun 15, 2019; 266818; PS1201
Prabhakar Kedar
Prabhakar Kedar
Contributions
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Abstract

Abstract: PS1201

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Recessive congenital methemoglobinemia (RCM) due to NADH–cytochrome b5 reductase 3 (cyb5r3) deficiencies is an autosomal recessive disorder that occurs sporadically worldwide. Over seventy six different mutations have been described for RCM.

Aims
The aim of this study molecular basis of RCM in 21 patients and prenatal diagnosis in 4 cases with severe mental retardation and neurological disabilities along with a comprehensive review of all the pathogenic mutations in cyb5r3 and their molecular pathology in RCM.

Methods

Twenty-five cases were analyzed amongst which 21 cases were diagnosed with NADH-CYB5R deficiency based on significantly reduced NADH-CYB5R activity while 4 were referred for prenatal screening.  The study has been approved by the National Institute of Immunohematology Ethical committee and as per protocol, written informed consents were obtained from all the patients or their parents.   Complete blood counts were measured by automated hematology analyzer XN   (Sysmex, Kobe,   Japan). Methemoglobin estimation, G6PD, and NADH-Cyb5R enzyme assay were performed as described by Beutler et al. method. DNA was extracted from the Blood/CVS using Qiagen DNA extraction kit according to the manufacturer’s instructions.PCR exon-specific primers were utilized and PCR and Sanger’s sequencing was performed by a standard method. Molecular modeling of cyb5r3 was done using the PYMOL and SWISS-PDB viewer tool.

Results

In the 21 patients analyzed in this study, all index cases from twenty unrelated families were referred for the cause of cyanosis. All patients showed mild to moderate cyanosis and few patients had mental retardation or any neurologic abnormalities. Methemoglobin levels were increased, varying from 8.5% to 65%. NADH-Cyb5r3 activity was seen to be in the range of 6 to 19%. In the molecular analysis, eighteen mutations (fourteen missense, one deletion, one-stop codon, and 1 splice-site mutation) were observed (Figure 1) in this study. Amongst fourteen missense mutations 5 were novel mutations [c.105A>C (p.Thr35Arg), c.192C>G (p.Leu64Val), c.312G>T (p.Gly104Cys), c.354C>T (p.His118Tyr) and c.470T>G (p.Phe157Cys)]. Over seventy-six different mutations have been described for RCM globally (Figure 1). Same mutations are associated with the severe form with a neurological disorder (RCM type 2) were associated with the FAD-binding domain of the protein while rests were located in the other domains of the protein (RCM Type 1). One of the prenatal analyses showed splice-site mutation p.Gly76Ser at nucleotides 226 in exon 3 of the CYB5R3 gene in the homozygous state while in another one it was found in the heterozygous state. In the third case stop codon mutation p.Gln77X at nucleotide position 229 in exon 4 was seen in the heterozygous condition. In the fourth case, normal wild type genotype for the CYB5R3 gene was found.

Conclusion
This study presents the results of the first molecular study of a large number of cases of NADH cytochrome b5 reductase deficiency in India Molecular modeling of all the mutations reported in CYB5R3 justifies the association of the mutations with the varying severity of the disease. Knowing the profile of mutations in families with severe neurological disorders allowed us to offer a prenatal diagnosis of RCM Type 2 for the first time in the Indian population. Thus severity and recurrence risk of type II RCM merit could be prevented by prenatal diagnosis in affected families.

Session topic: 28. Enzymopathies, membranopathies and other anemias

Keyword(s): Clinical data, Genetic, Genomics, Mutation analysis

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