Five Novel Hemizygous Variants in the PGK1 Gene Associated with Neurological Dysfunction and Mild Hemolytic Anemia in Five Indian Patients

Abstract

Introduction Phosphoglycerate kinase (ATP: 3-phosphoglycerate 1-phosphotransferase; EC 2.7.2.3; PGK) is the crucial enzyme of the glycolytic pathway, catalyzing1, 3 bis-phosphoglycerate to 3-Phosphoglycerate, generating one molecule of ATP [1]. PGK enzyme is encoded by the PGK1 gene located at Xq13.3, measures 23kb in size, and spans into 11 exons. PGK deficiency is an X-linked disorder associated with a very rare cause of hereditary non-spherocytic hemolytic anemia (HNSHA). PGK1 gene is ubiquitously expressed but the patients exhibit three major symptoms -1) haemolytic anemia 2) myopathy (rhabdomyolysis), 3) mental retardation and various neurological manifestations[1]. The severity of symptoms varies among patients with PGK deficiency. PGK deficiency is reported worldwide, although to our knowledge no case with Indian ancestry has been reported. Method Genomic DNA was extracted from the EDTA blood sample and processed for targeted Next Generation Sequencing (t-NGS) as per the manufacturer's protocol. The t-NGS panel broadly consists of 80 genes of the spectrum of the disorder including congenital hemolytic anemia, bone marrow failure disorders, and iron deficiency anemia. The libraries were sequenced to >80-100X mean coverage on the Illumina sequencing platform. The variants were classified based on ACMG guidelines. The impact of non-synonymous variations was estimated using PolyPhen-2, SIFT, MutationTaster2, CADD Phred, and Mutpred2. In silico effect of the variant was studied by PyMol. Latter the enzyme deficiency was confirmed by standard protocol and parental segregation analysis was performed. Relative quantification (qPCR) was evaluated based on the comparative CT method ( 2 − ΔΔCT). Result We report five male children of <4 years of age with PGK deficiency from Indian origin. Clinically, all of them manifest global development delay and no speech. Autism spectrum disorder is observed in two of the cases, incidence of seizure and convulsion are present in two cases. None except one, presented with haemolytic anaemia and was given one transfusion post an episode of seizure at the age of 2 years. Additionally, a family history of haemolytic anemia with developmental delay and seizure was present in only one case although the patient himself required no transfusion requirement to date. All the patients are routinely followed up for any evidence of a haemolytic crisis, transfusion requirement, or any other clinical symptoms. Detailed phenotypes of all the patients are listed in Table 1. t-NGS revealed five novel hemizygous variants in the PGK1 gene - p. Cys50Trp, p. Gly137Trp, p. Val140Ile, p. Asp159Asn, and p. Arg330Trp. These variants have not been reported in the literature or available databases. DNA Sanger sequencing confirmed the heterozygous state in the mothers. In silico tools confirmed the pathogenicity of the variants. The PGK enzymatic activity was markedly reduced. The PGK1 transcript levels were reduced to 60% in patients as compared to the healthy controls. Discussion To date, only 34 mutations are known to be associated with PGK enzyme deficiency. Amongst them, 50% of variants exhibit symptoms of anaemia whereas the remaining show neurological and /or myopathy. Figure 1 illustrates all 39 mutations reported with PGK deficiency. Only four mutations p.E120K, p.G158R, p.A354P, and p.I371K are known to exhibit all three categories of symptom (PGK-anaemia-neurological-myopathy)[2]. Conclusion NGS allows for the identification of novel genetic variants that may contribute to the development of rare disorders, expanding our knowledge of their underlying mechanisms. This advanced technology also enables researchers to analyze multiple genes simultaneously, increasing the efficiency and speed of diagnosis in unexplained cases of HNSHA.