Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients

Abstract

<jats:p>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&gt;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&gt;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.</jats:p>

Clinical and Molecular Spectrum of Glucose-6-Phosphate Isomerase Deficiency. Report of 12 New Cases

“…The present cohort of GPI deficient patients represents the largest series so far described in a single study, collecting retrospective information and follow-up data over a median period of 18 years. All the cases were never reported before, consistently increasing the number of GPI patients reported in literature (Kugler and Lakomek, 2000; Clarke et al, 2003; Repiso et al, 2006; Warang et al, 2012; Adama van Scheltema et al, 2015; Zhu et al, 2015; Manco et al, 2016; Jamwal et al, 2017; Zaidi et al, 2017; Burger et al, 2018; Kedar et al, 2018; Mojzikova et al, 2018).…”

“…Despite the fact that GPI deficiency is considered the second most frequent RBC enzymopathy of anaerobic glycolysis after pyruvate kinase, the exact frequency of this disorder is not known and a diagnosis is often difficult to reach; this may be due to the lack of availability of the enzymatic assay, performed only in a few specialized centers, or because of the lack of knowledge about some rare disorders for which specific tests are not considered during laboratory investigations (Bianchi et al, 2018; Kedar et al, 2018; Sonaye et al, 2018). Moreover, due to the similarity in clinical presentation with other congenital hemolytic anemias, an exact diagnosis is often delayed.…”

“…The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons (Walker et al, 1995). So far, about 60 cases of GPI deficiency have been described, and more than 40 mutations have been reported at the nucleotide level (Kugler and Lakomek, 2000; Clarke et al, 2003; Repiso et al, 2006; Zhu et al, 2015; Manco et al, 2016; Jamwal et al, 2017; Zaidi et al, 2017; Kedar et al, 2018; Mojzikova et al, 2018). Missense mutations are the most common, but non-sense and splicing mutations have also been observed.…”

Exome sequencing for diagnosis of congenital hemolytic anemia

“…Different NGS strategies have been developed over the last few years for CHA (Additional file 1, Supplementary Table S1). They included targeted gene panels (comprising 28 to 76 genes) with a success rate for diagnosis varying from 38 to 90%, depending on proband numbers (ranging from 2 to 62) and subtypes of anemia [15][16][17][18][19][20][21][22][23], and whole-exome sequencing (WES) in a few studies (limited to 1 to 7 probands, except two studies focusing on 38 Chinese cases of HS [24] and 24 cases of autosomal recessive HS [25]), with success rates between 29 and 100% [14,[24][25][26][27][28][29][30][31][32]. We used WES to explore 40 CHA patients: 20 with suspected HS and 20 with unexplained hemolysis (UH) despite available phenotype exploration.…”

Exome sequencing for diagnosis of congenital hemolytic anemia

“…They included targeted gene panels (comprising 28 to 76 genes) with a success rate for diagnosis varying from 38% to 90%, depending on probands numbers (ranging from 2 to 62) and subtypes of anemia [15][16][17][18][19][20][21][22][23], and whole-exome sequencing (WES) in a few studies (limited to 1 to 7 probands, except two studies focusing on 38 Chinese cases of HS [24] and 24 cases of autosomal recessive HS [25]), with success rates between 29 and 100% [14,[24][25][26][27][28][29][30][31][32]. We used WES to explore 40 CHA patients: 20 with suspected HS and 20 with unexplained hemolysis (UH) despite available phenotype exploration.…”