Targeted next-generation sequencing identifies novel deleterious variants in ANK1 gene causing severe hereditary spherocytosis in Indian patients: expanding the molecular and clinical spectrum.

Hereditary Spherocytosis (HS) is a common cause of hemolytic anemia varying from mild to severe hemolysis due to defects in red cell membrane protein genes, namely ANK1, SPTB, SPTA1, SLC4A1, and EPB42. These genes are considerably very large spaning 40-50 exons making gene-by-gene analysis costly and laborious by conventional methods. In this study, we explored 26 HS patients harboring 21 ANK1 variants identified by next-generation sequencing (NGS), characteristics and spectrum of the detected ANK1variants were analyzed in this study. Clinically, all the HS patients showed moderate to severe transfusion-dependent hemolytic anemia, some requiring splenectomy. We identified 13 novel and 8 reported variants, mainly 9 frameshifts, 2 missense, 6 nonsense, and 4 splice site ANK1 variants, using NGS technology. Frameshifts were remarkably the most common variant type seen in Indian HS patients with ANK1 gene defects. We have also explored expression levels of red cell membrane ankyrin protein by flow cytometry in 14 HS patients with ANK1 gene defects and a significant reduction in ankyrin protein expression has been found. This report mainly illustrates the molecular and phenotypic heterogeneity of ANK1 variants causing HS in Indian patients. Ankyrin-1 mutations are a significant cause of loss of function in dominant HS in the Indian population. Comprehensive genetic and phenotypic evaluation assists in implementing the knowledge of genetic patterns and spectrum of ANK1 gene variants, providing molecular support for HS diagnosis.

Targeted next-generation sequencing identifies eighteen novel mutations expanding the molecular and clinical spectrum of PKLR gene disorders in the Indian population.

Pyruvate kinase deficiency (PKD) is an autosomal recessive condition, caused due to homozygous or compound heterozygous mutation in the PKLR gene resulting in non-spherocytic hereditary hemolytic anemia. Clinical manifestations in PKD patients vary from moderate to severe lifelong hemolytic anemia either requiring neonatal exchange transfusion or blood transfusion support. Measuring PK enzyme activity is the gold standard approach for diagnosis but residual activity must be related to the increased reticulocyte count. The confirmatory diagnosis is provided by PKLR gene sequencing by conventional as well as targeted next-generation sequencing involving genes associated with enzymopathies, membranopathies, hemoglobinopathies, and bone marrow failure disorders. In this study, we report the mutational landscape of 45 unrelated PK deficiency cases from India. The genetic sequencing of PKLR revealed 40 variants comprising 34 Missense Mutations (MM), 2 Nonsense Mutations (NM), 1 Splice site, 1 Intronic, 1 Insertion, and 1 Large Base Deletion. The 17 novel variants identified in this study are A115E, R116P, A423G, K313I, E315G, E318K, L327P, M377L, A423E, R449G, H507Q, E538K, G563S, c.507 + 1 G > C, c.801_802 ins A (p.Asp268ArgfsTer48), IVS9dsA-T + 3, and one large base deletion. In combination with previous reports on PK deficiency, we suggest c.880G > A, c.943G > A, c.994G > A, c.1456C > T, c.1529G > A are the most frequently observed mutations in India. This study expands the phenotypic and molecular spectrum of PKLR gene disorders and also emphasizes the importance of combining both targeted next-generation sequencing with bioinformatics analysis and detailed clinical evaluation to elaborate a more accurate diagnosis and correct diagnosis for transfusion dependant hemolytic anemia in a cohort of the Indian population.

Three novel mutations in CYB5R3 gene causing NADH-cytochrome b5 reductase enzyme deficiency leads to recessive congenital methaemoglobinemia.

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.

Mutation update: Variants of the CYB5R3 gene in recessive congenital methemoglobinemia.

NADH-cytochrome b5 reductase 3 deficiency is an important genetic cause of recessive congenital methemoglobinemia (RCM) and occurs worldwide in autosomal recessive inheritance. In this Mutation Update, we provide a comprehensive review of all the pathogenic mutations and their molecular pathology in RCM along with the molecular basis of RCM in 21 new patients from the Indian population, including four novel variants: c.103A>C (p.Thr35Pro), c.190C>G (p.Leu64Val), c.310G>T (p.Gly104Cys), and c.352C>T (p.His118Tyr). In this update, over 78 different variants have been described for RCM globally. Molecular modeling of all the variants reported in CYB5R3 justifies association with the varying severity of the disease. The majority of the mutations associated with the severe form with a neurological disorder (RCM Type 2) were associated with the FAD-binding domain of the protein while the rest were located in another domain of the protein (RCM Type 1).

Mechanosensitive Piezo1 ion channel protein (PIEZO1 gene): update and extended mutation analysis of hereditary xerocytosis in India.

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.

Glucose phosphate isomerase (GPI) Tadikonda: Characterization of a novel Pro340Ser mutation.

After a thirty-year lag, we serendipitously reestablished contact with a patient with glucose phosphate isomerase deficiency and hydrops fetalis first reported in 1987. We now provide a clinical update and provide results of mutation analysis in this patient, from Southern India. The patient now an adult female of 36 years of age has moderate anemia but requires no transfusions except with some intercurrent illnesses. Exome sequencing studies showed a homozygous c.1018C>T (Pro340Ser) mutation in exon 12 of the glucose phosphate isomerase gene and later confirmed by direct sequencing. This mutation has not been previously described. To our knowledge, this is also the first known homozygous mutation in the hydrophobic core of the protein and is a highly deleterious mutation by in silico analysis and by clinical history in the family. Flow cytometry studies of band 3 content with eosin maleimide showed a unique tail of red cells on histograms, reflecting the dense red cells (presumably ATP depleted) seen on blood smears; similar findings were seen in patients with pyruvate kinase and phosphoglycerate kinase deficiency.

Molecular and clinical heterogeneity in pyruvate kinase deficiency in India.

We studied the PK-LR gene in 10 unrelated Indian patients with congenital haemolytic anemia associated with erythrocyte pyruvate kinase deficiency. The patients had a variable presentation ranging from a very mild compensated hemolysis to severe anemia. Nine different mutations were detected among the 20 mutated alleles identified: one deletion (c.1042-1044del) p.Lys348del and eight single-nucleotide (nt) substitutions resulting in amino acid exchanges c.397A>G (p.Asn133Asp), c.992A>G (p.Asp331Gly), c.1072G>A (p.Gly358Arg), c.1076G>A (p.Arg359His), c.1219G>A (p.Glu407Lys), c.1241C>T (p.Pro414Leu), c.1436G>A (p.Arg479His) and c.1529G>A (p.Arg510Gln) were identified. Although all the exons, the flanking regions and the promoter region were sequenced in all cases, we failed to detect the second expected mutation in two subjects. Two mutations [c.397A>G; c.1241C>T] were novel. These novel missense mutations involved highly conserved amino acids. Two mutations were identified for the first time in the homozygous state globally (c1042-1044del; c.1072G>A) and two other mutations were identified for the first time in our population (c.1076G>A; c.1529G>A). This study along with our earlier report suggests that the most frequent mutations in India would appear to be c.1436G>A (18.33%), followed by c.992A>G (11.66%) and c.1456C>T (11.66%). Structural implications of amino acid substitutions were correlated with the clinical phenotypes seen.

Development of High-Resolution Melting Curve Analysis for rapid detection of SEC23B gene mutation causing Congenital Dyserythropoietic Anemia type II in Indian population.

BACKGROUND: Congenital dyserythropoietic anemias (CDAs) are a very rare and heterogeneous group of disorders characterized by ineffective erythropoiesis. CDA II is caused by mutations in the SEC23B gene. The most common mutation reported in India is c.1385 A > G, p.Y462C. There is no simple and cost-effective confirmatory diagnostic test available for CDA, and therefore, many patients remain undiagnosed. High-resolution melting curve (HRM) analysis is a polymerase chain reaction (PCR) based technique applied to identify genetic differences and scan nucleic acid sequences. HRM can be used to rapidly screen the common mutation causing CDA II in the Indian population. Thus, we studied the use of High-Resolution Melting Curve Analysis to detect common mutation causing CDA II in the Indian population. METHOD: 11 patients having SEC23B (Y462C) mutation causing CDA II are considered for this study. HRM was used to check the presence of Y462C mutation. To verify the accuracy of the HRM analysis, we compared HRM results with the results of Sanger sequencing. This helped us to confirm the diagnosis. RESULTS: We have described the clinical, hematological, and genetic data of eleven patients suffering from CDAII. According to HRM and Sanger sequencing, a homozygous SEC23B (Y462C) mutation was present in all patients, whereas a heterozygous Y462C mutation was present in their parents. CONCLUSION: Our data showed that High-Resolution Melting (HRM) analysis could be used to rapidly screen common SEC23B mutation that causes CDA II in the Indian population.

Unravelling the genetic and phenotypic heterogeneity of SPTA1 gene variants in Hereditary Elliptocytosis and Hereditary Pyropoikilocytosis patients using next-generation sequencing.

Hereditary Elliptocytosis (HE) and Hereditary Pyropoikilocytosis (HPP) are clinically and genetically heterogeneous red cell membranopathies that result from the defects in the horizontal linkage between RBC (red blood cell) membrane and cytoskeletal proteins affecting its mechanical stability and deformability thereby reducing its lifespan. The principal defect in HE and HPP is due to dysfunction or deficiency of RBC cytoskeletal proteins namely, α-spectrin (SPTA1), ß-spectrin (SPTB) and protein 4.1R (EPB41R). This study reports the genetic and phenotypic heterogeneity of 10 Indian patients (5 with HE and 5 with HPP)harboringSPTA1 gene variants. We used targeted next-generation sequencing (t-NGS) to characterize the causative genetic variants in 10 HE/HPP suspected patients and studied the correlation between the identified variants with their corresponding phenotypic features.t-NGS detected 12 SPTA1 variants, out of which 8 are novel. Nearly all of the detected variants have a damaging effect on the protein stability and function, as shown by the insilico analysis. The possible effect of the detected variants on the protein structure was studied using the HOPE software and DynaMut tools wherever possible. To the best of our knowledge, this is the first report on HE/HPP cases confirmed by a genetic study from India. To conclude, HE is caused by monoallelic mutations while HPP, the more severe form, is typically caused by biallelic (homozygous or compound heterozygous) mutations justifying the phenotypic heterogeneity associated with patients. Moreover, analysis at the molecular level by NGS permits diagnosis in these disorders with highly variable heterogeneity requiring regular transfusions and may facilitate prognostic contemplations.

Targeted next-generation sequencing revealed a novel homozygous mutation in the LRBA gene causes severe haemolysis associated with Inborn Errors of Immunity in an Indian family.

OBJECTIVES: LPS-responsive beige-like anchor protein (LRBA) deficiency abolishes LRBA protein expression due to biallelic mutations in the LRBA gene that lead to autoimmune manifestations, inflammatory bowel disease, hypogammaglobulinemia in early stages, and variable clinical manifestations. MATERIALS AND METHODS: Mutational analysis of the LRBA gene was performed in Indian patients using targeted Next Generation Sequencing (t-NGS) and confirmed by Sanger sequencing using specific primers of exons 53. Then, bioinformatics analysis and protein modeling for the novel founded mutations were also performed. The genotype, phenotype correlation was done according to the molecular findings and clinical features. RESULTS: We report an unusual case of a female patient born of a consanguineous marriage, presented with severe anaemia and jaundice with a history of multiple blood transfusions of unknown cause up to the age of 5 yrs. She had hepatosplenomegaly with recurrent viral and bacterial infections. Tests for hemoglobinopathies, enzymopathies, and hereditary spherocytosis were within the normal limits. The t-NGS revealed a novel homozygous missense variation in exon 53 of the LRBA gene (chr4:151231464C > T; c.7799G > A) (p.C2600Y), and the parents were heterozygous. The further immunological analysis is suggestive of hypogammaglobulinaemia and autoimmune haemolytic anaemia. The bioinformatics tools are suggestive of deleterious and disease-causing variants. CONCLUSION: This study concludes the importance of a timely decision of targeted exome sequencing for the molecular diagnostic tool of unexplained haemolytic anaemia with heterogeneous clinical phenotypes.

Clinical, laboratory, and mutational profile of children with glucose phosphate isomerase deficiency: a single centre report.

Glucose phosphate isomerase (GPI) deficiency is an autosomal recessive condition with mutations in the GPI gene on chromosome 19q13.1. Patients present with congenital non-spherocytic hemolytic anemia, and occasionally intellectual disability. In this study, we describe the clinical, hematological and biochemical parameters in the largest single-center cohort consisting of 17 GPI-deficient cases. Demographic and clinical data were noted, and red cell enzyme activity levels were estimated. Mutation analysis was done by single-stranded-conformation polymorphism, restriction-fragment length polymorphism and Sanger's sequencing of exon 12 of the GPI gene. The male-to-female ratio was 0.7:1, median age at diagnosis was 5.0 years, 82.3% of patients had severe neonatal jaundice, and 13.3% had subtle neurological manifestations. Median Hb and MCV levels were 6.3 g/dl and 130.2 fl. Splenectomized patients required fewer transfusions. Sixteen of 17 patients had the pathogenic c.1040G > A (p.Arg347His) homozygous mutation in exon12 of the GPI gene, and one had the pathogenic c.1414C > T(p.Arg472Cys) homozygous mutation in exon 16. In summary, we report that neonatal jaundice, macrocytosis and high prevalence of p.Arg347His variant were predominant in GPI deficiency with prominent lack of neurological manifestations, and we emphasize the benefits of splenectomy and the need for genetic counseling.

Successful management of Methemoglobinemia and G6PD deficiency in a patient posted for surgical excision of branchial cyst.

A 27-year-old female patient who came for branchial cyst excision was found to have cyanosis and a saturation gap during preanesthetic check-up and hence she was referred to haematology for further workup. She had a Hb of 9 gm% with all other baseline tests as normal. Blood samples were sent for methaemoglobin estimation and related work up to the National Institute of Immunohematology (NIIH) Mumbai. She was diagnosed as a case of Methemoglobinemia with a methaemoglobin level of 68.7% with NADH cytochrome B5 reductase activity of 10.82 IU/g Hb. The drug of choice for treatment is Methylene blue and hence G6PD deficiency had to be ruled out prior to initiating therapy. She was found to have a concurrent existence of G6PD deficiency. The blood sample was further sent to NIIH for genetic confirmation. We avoided methylene blue and other precipitating factors that could trigger a haemolysis. She was further consulted by the Patient blood management team to optimize her erythropoiesis and avoid unnecessary transfusions. Anaesthetic consultation and planning were done to avoid drugs that could induce haemolysis. She was started on Vitamin C, Niacin, hematinic and advised to follow up after a month. She was symptomatically better. Cyanosis had reduced, and Hb improved to 12 gm%. She was taken up for surgery with all precautions. The surgery and the post-operative period were uneventful. She was discharged on postoperative day 4 with an advice to continue Vitamin C & Niacin and to follow-up in Haematology OPD after a month.

Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis.

We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na(+) efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na(+) efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl(-) transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl(-) transport activity, but R730 substitution with I, E, or H inactivated Cl(-) transport. AE1 R730C expression substantially increased endogenous oocyte Na(+)-K(+)-ATPase-mediated (86)Rb(+) influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH(o)) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH(o)-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH(o) insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl(-) influx by acid pH(o). Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb(+) and Li(+) influxes. Selective rescue of acid pH(o)-stimulated sulfate uptake and conferral of acid pH(o)-stimulated Cl(-) influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.

Genotypic analysis of SLC4A1 A858D mutation in Indian population associated with distal renal tubular Acidosis (dRTA) coupled with hemolytic anemia.

INTRODUCTION: Although distinctive, distal renal tubular acidosis (dRTA) and Hereditary Spherocytosis (HS) shares a common protein, the anion exchanger1 (AE1) encoded by SLC4A1gene. In spite of this, the co-existence of dRTA and HS has rarely been observed. To date, 23 mutations have been identified in SLC4A1 gene causing both autosomal recessive (AR) and autosomal dominant (AD) forms of dRTA. METHODS: We have assessed the applicability of the High Resolution Melting curve (HRM) method for the detection of SLC4A1 (A858D) mutation in 12 Indian families having AR dRTA coupled with HS. The reliability of the HRM analysis was verified by comparing the results of the HRM method with those of conventional methods such as Polymerase Chain Reaction-Restriction Fragment-Length Polymorphism (PCR-RFLP) and Sanger sequencing thereby confirming the diagnosis. RESULTS: We here described the clinical, hematological and genetic data of 16 individuals from 12 families having AR dRTA coupled with HS. All patients carried homozygous SLC4A1 (A858D) mutation, whereas their family members had heterozygous A858D obtained by HRM analysis and confirmed by RFLP and Sanger sequencing. CONCLUSION: Our data indicates that a missense mutation of A858D in SLC4A1 gene is the most common cause of dRTA coupled with HS in the Indian population. HRM analysis can be used as a rapid screening method for common SLC4A1 mutations that cause AR dRTA in the Indian population.

Rare hereditary nonspherocytic hemolytic anemia caused by a novel homozygous mutation, c.301C > A, (Q101K), in the AK1 gene in an Indian family.

BACKGROUND: Adenylate kinase (AK) deficiency is a rare red cell enzymopathy associated with moderate to severe congenital nonspherocytic hemolytic anemia, along with mental and psychomotor retardation (in exceptional cases). Only ten mutations have been detected in the AK1 gene to date. In this study, we aimed to diagnose the unexplained issue of haemolytic anaemia and offer antenatal screening to the family. METHODS: Genomic DNA was isolated from whole blood by a standard protocol. Targeted next-generation sequencing (t-NGS) was performed to identify pathogenic variants in the patient and control samples. A chronic villus sample was collected at 11 weeks of gestation from the mother, and molecular testing was performed. Genetic confirmation was concluded by Sanger DNA sequencing. Bioinformatics tools predicted the pathogenicity of the variant. RESULTS: t-NGS revealed a homozygous variant (c.301C > A, p. Gln101Lys) in the AK1 gene in the patient and heterozygosity in the fetus and parental samples. The prediction tools SIFT, Polyphen2, Provean, PMUT, Mutation taster, and Mutation Assessor, confirmed the damaging effect of the variant on the AK1 protein structure CONCLUSION: We have presented a novel mutation in the AK1 gene (p. Gln101Lys) associated with adenylate kinase deficiency. It is the first prenatal diagnosis of AK deficiency in India, where heterogeneity is exceptionally high.

Novel pathogenic variant c.2714C>A (p. Thr905Lys) in the HK1 gene causing severe haemolytic anaemia with developmental delay in an Indian family.

Hexokinase (EC 2.7.1.1, Adenosine Tri Phosphate (ATP): D-hexose-6-phosphotransferase) is a crucial regulatory enzyme of the glycolytic pathway (Embden-Meyerhof pathway). Hexokinase deficiency is associated with chronic non-spherocytic haemolytic anaemia (HA) with some exceptional cases showing psychomotor/mental retardation and fetus death. The proband is a four-and-half-year-old female child born of a four-degree consanguineous marriage hailing from South India with autosomal recessive congenital HA associated with developmental delay. She was well till 3 months of her age post an episode of diarrhoea when she was noted to be severely anaemic and requiring regular transfusions. The common causes of HA, haemoglobinopathies, red cell membranopathies and common red cell enzymopathies (G6PD, GPI, PK and P5N) were ruled out. Targeted analysis of whole exome sequencing (WES) using an insilico gene panel for hereditary anaemia was performed to identify pathogenic variants in the patient. Next-generation sequencing revealed a novel homozygous variant in hexokinase gene c.2714C>A (p. Thr905Lys) in exon-18. The pathogenic nature of the variant p. Thr905Lys in the HK1 gene was confirmed collectively by biochemical and molecular studies. Insilico analysis (PolyPhen-2, Provean, Mutation Taster) predicted the variant to be severe disease causing. Multiple sequence alignment demonstrated the conservation of p. Thr905 across the species. The impact of the mutation on the protein structure was studied by PyMOL and Swiss Protein databank viewer.