Abstract
Background
Methemoglobin is the reduced form of haemoglobin that is normally found in the blood in levels < 1%. Methemoglobinemia can occur as a congenital or acquired disease. Two types of recessive congenital methaemoglobinemia (RCM) are caused by the NADH-dependent cytochrome b5 reductase enzyme deficiency of the CYB5R3 gene. RCM-I is characterized by higher methaemoglobin levels (> 2 g/dL), causing only cyanosis, whereas RCM-II is associated with cyanosis with neurological impairment.
Methods
Routine haematological investigations were done by standard method. The methaemoglobin level was evaluated by the potassium ferricyanide assay. NADH-cytochrome b5 reductase (cytb5r) enzyme activities were measured by standard methods, and molecular analysis was performed by polymerase chain reaction (PCR) followed by DNA sequencing. The interpretation of mutation effect and the molecular modeling were performed by using specific software DEEP VIEW SWISS-PDB VIEWER and Pymol molecular graphics program.
Results
The present study discovered three novel homozygous pathogenic variants of CYB5R3 causing RCM I and II in four unrelated Indian patients. In patient-1 and patient-2 of RCM type I caused due to novel c.175C>T (p.Arg59Cys) and other reported c.469T>C (p.Phe157Ser) missense pathogenic variants respectively, whereas patient-3 and patient-4 presented with the RCM type II are related to developmental delay with cyanosis since birth due to a novel homozygous (g.25679_25679delA) splice-site deletion and novel homozygous c.824_825insC (p.Pro278ThrfsTer367) single nucleotide insertion. The CYB5R3 transcript levels were estimated by qRT-PCR in the splice-site deletion, which was 0.33fold of normal healthy control. The insertion of nucleotide C resulted in a frameshift of termination codon are associated with neurological impairment.
Conclusions
Molecular diagnosis of RCM can help to conduct genetic counselling for novel mutations and, subsequently, prenatal diagnosis of high-risk genetic disorders.
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Data availability
All data generated or analyzed during this study are included in this published article.
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Acknowledgements
We would like to thank patients and family members for their cooperation and participation in this study. This study was performed with the Indian Council of Medical Research New Delhi and the Council of Scientific and Industrial Research, India (CSIR) New Delhi for financial support.
Funding
This study was performed with the financial support of the Indian Council of Medical Research New Delhi and also supported by the Council of Scientific and Industrial Research (CSIR), India, Grant No. 27(0333)/18/EMR-II).
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PK: Conceived ideas and initiated the experimental work and supervision of work. AK: Contributed to the findings of the work. AD: Further experimental work and statistical analysis for expansion of idea were carried under the supervision of PK, AD, and PK: Manuscript was written by AD in consultation with PK.
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This study was approved by the Ethics Committee of the Institutional Ethical Committee of ICMR-National Institute of Immunohaematology, Mumbai. All procedures performed in studies involving human participants were as per the ethical standards of the institutional review board of the Institute and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Deorukhkar, A., Kulkarni, A. & Kedar, P. Three novel mutations in CYB5R3 gene causing NADH-cytochrome b5 reductase enzyme deficiency leads to recessive congenital methaemoglobinemia. Mol Biol Rep 49, 2141–2147 (2022). https://doi.org/10.1007/s11033-021-07031-3
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DOI: https://doi.org/10.1007/s11033-021-07031-3