Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing congenital haemolytic anaemia (CHA). Diagnosis of GPI deficiency by the biochemical method is unpredicted. Molecular diagnosis by identifying genetic mutation is the gold standard method for confirmation of disease, but causative genes involved in CHA are numerous, and identifying a gene-by-gene approach using Sanger sequencing is also cumbersome, expensive and labour intensive. Recently, next-generation targeted sequencing is more useful in the diagnosis of unexplained haemolytic anaemia. We used targeted next-generation sequencing (NGS) clinical panel for diagnosis of unexplained haemolytic anaemia in two Indian patients which were pending for a long time. All possible causes of haemolytic anaemia were found within normal limit. NGS by clinical exome panel revealed homozygous novel missense mutation in exon 12, c.1009G>A (p.Ala337Thr) in both patients. We further confirm by measuring red blood cell GPI activity in the patients and showed deficiency whereas parents were having intermediate activity. c.1009G>A mutation was also confirmed by Sanger sequencing of exon 12 of GPI gene. The structural–functional analysis by bioinformatics software like Swiss PDB, PolyPhen-2 and PyMol suggested that this pathogenic variant has a direct impact on the structural rearrangement at the region near the active site of the enzyme. This rapid and high-performance targeted NGS assay can be configured to detect specific CHA mutations unique to an individual defect, making it a potentially valuable method for diagnosis of unexplained haemolytic anaemia.
- hereditary Non-spherocytic haemolytic anaemia (HNSHA)
- neurological disorders
- molecular modeling. next-generation sequencing
- Glucose-6-phosphate isIsomerase (GPI) deficiency
- Molecular modeling
- Next-generation sequencing
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Handling editor Mary Frances McMullin.
Contributors VG, RD, AC, PW and PSK performed the experiments. PW and PSK collected and analysed data. PSK wrote the manuscript. MRM gave technical support and conceptual advice. All authors read and approved the final manuscript.
Funding This study was funded by the Indian Council of Medical Research and Department of Biotechnology, Ministry of Science and Technology (grant no. BT/PR20782/MED/12/737/16).
Competing interests None declared.
Patient consent Obtained.
Ethics approval NIIH Institutional Ethical Committee Mumbai.
Provenance and peer review Not commissioned; externally peer reviewed.
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