Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency

Abstract

Objective

Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family.

Methods

The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure.

Results

By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids.

Conclusion

Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.

Keywords

glucose-6-phosphate isomerase deficiency / whole-exome sequencing / compound heterozygous variants / genetic characterization / hydrogen bond

References

[1]	Kugler W, Lakomek M. Glucose-6-phosphate isomerase deficiency. Baillieres Best Pract Res Clin Haematol, 2000,13(1):89–101
[2]	Baughan MA, Valentine WN, Paglia DE, et al. Hereditary hemolytic anemia associated with glucosephosphate isomerase (GPI) deficiency—a new enzyme defect of human erythrocytes. Blood, 1968,32(2):236–249
[3]	Zu Y, Wang H, Lin W, et al. Hereditary nonspherocytic hemolytic anemia caused by glucose-6-phosphate isomerase (GPI) deficiency in a Chinese patient: a case report. BMC Pediatr, 2022,22(1):461
[4]	Sampagar A, Gosavi M, Kedar P, et al. Clinical, laboratory, and mutational profile of children with glucose phosphate isomerase deficiency: a single centre report. Int J Hematol, 2022,115(2):255–262
[5]	Mojzikova R, Koralkova P, Holub D, et al. Two novel mutations (p.(Ser160Pro) and p.(Arg472Cys)) causing glucose-6-phosphate isomerase deficiency are associated with erythroid dysplasia and inappropriately suppressed hepcidin. Blood Cells Mol Dis, 2018,69:23–29
[6]	Kugler W, Breme K, Laspe P, et al. Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency. Hum Genet, 1998,103(4):450–454
[7]	Fermo E, Vercellati C, Marcello AP, et al. Clinical and molecular spectrum of glucose-6-phosphate isomerase deficiency. Report of 12 new cases. Front Physiol, 2019,10:467
[8]	Climent F, Roset F, Repiso A, et al. Red cell glycolytic enzyme disorders caused by mutations: an update. Cardiovasc Hematol Disord Drug Targets, 2009,9(2):95–106
[9]	Manco L, Bento C, Victor BL, et al. Hereditary nonspherocytic hemolytic anemia caused by red cell glucose-6-phosphate isomerase (GPI) deficiency in two Portuguese patients: clinical features and molecular study. Blood Cells Mol Dis, 2016,60:18–23
[10]	Kedar PS, Dongerdiye R, Chilwirwar P, et al. Glucose phosphate isomerase deficiency: high prevalence of p.Arg347His mutation in Indian population associated with severe hereditary non-spherocytic hemolytic anemia coupled with neurological dysfunction. Indian J Pediatr, 2019,86(8):692–699
[11]	Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009,25(14):1754–1760
[12]	Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res, 2010,38(16):e164
[13]	Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature, 2021,596(7873):583–589
[14]	Huang CC, Meng EC, Morris JH, et al. Enhancing UCSF Chimera through web services. Nucleic Acids Res, 2014,42(Web Server issue):W478–W484
[15]	Song Z, Wang K, Arthur DT, et al. Case report: glucose 6-phosphate-isomerase deficiency combine with avascular necrosis of bilateral femoral head. Front Pediatr, 2022,10:909752
[16]	Huang P, Tang L, Wang AP, et al. Glucose-6-phosphate isomerase deficiency caused by GPI gene mutation: a case report and literature review. J Clin Pediatr, 2020,38(10):785–788
[17]	Huang K, Wu RH, Yao JF, et al. Glucose phosphate isomerase gene mutation causing severe hemolytic anemia with residual neurological symptoms. Zhonghua Er Ke Za Zhi (Chinese), 2019,57(2):153–155
[18]	See WQ, So CJ, Cheuk DK, et al. Congenital hemolytic anemia because of glucose phosphate isomerase deficiency: identification of 2 novel missense mutations in the GPI gene. J Pediatr Hematol Oncol, 2020,42(7):e696–e697
[19]	Kanno H, Fujii H, Hirono A, et al. Molecular analysis of glucose phosphate isomerase deficiency associated with hereditary hemolytic anemia. Blood, 1996,88(6):2321–2325
[20]	Beutler E, West C, Britton HA, et al. Glucosephosphate isomerase (GPI) deficiency mutations associated with hereditary nonspherocytic hemolytic anemia (HNSHA). Blood Cells Mol Dis, 1997,23(3):402–409
[21]	Xu W, Beutler E. The characterization of gene mutations for human glucose phosphate isomerase deficiency associated with chronic hemolytic anemia. J Clin Invest, 1994,94(6):2326–2329
[22]	Burger NCM, van Wijk R, Bresters D, et al. A novel mutation of glucose phosphate isomerase (GPI) causing severe neonatal anemia due to GPI deficiency. J Pediatr Hematol Oncol, 2019,41(3):e186–e189
[23]	Baronciani L, Zanella A, Bianchi P, et al. Study of the molecular defects in glucose phosphate isomerase-deficient patients affected by chronic hemolytic anemia. Blood, 1996,88(6):2306–2310
[24]	Repiso A, Oliva B, Vives-Corrons JL, et al. Red cell glucose phosphate isomerase (GPI): a molecular study of three novel mutations associated with hereditary nonspherocytic hemolytic anemia. Hum Mutat, 2006,27(11):1159
[25]	Walker JI, Layton DM, Bellingham AJ, et al. DNA sequence abnormalities in human glucose 6-phosphate isomerase deficiency. Hum Mol Genet, 1993,2(3):327–329
[26]	De la Morena-Barrio ME, Ballesta-Martínez MJ, López-Gálvez R, et al. Genetic predisposition to fetal alcohol syndrome: association with congenital disorders of N-glycosylation. Pediatr Res, 2018,83(1–1):119–127
[27]	Jamwal M, Aggarwal A, Palodhi A, et al. Next-generation sequencing-based diagnosis of unexplained inherited hemolytic anemias reveals wide genetic and phenotypic heterogeneity. J Mol Diagn, 2020,22(4):579–590
[28]	Clarke JL, Vulliamy TJ, Roper D, et al. Combined glucose-6-phosphate dehydrogenase and glucose-phosphate isomerase deficiency can alter clinical outcome. Blood Cells Mol Dis, 2003,30(3):258–263
[29]	Kedar PS, Gupta V, Dongerdiye R, et al. Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients. J Clin Pathol, 2019,72(1):81–85
[30]	Zaidi AU, Kedar P, Koduri PR, et al. Glucose phosphate isomerase (GPI) Tadikonda: characterization of a novel Pro340Ser mutation. Pediatr Hematol Oncol, 2017,34(8):449–454
[31]	Huppke P, Wünsch D, Pekrun A, et al. Glucose phosphate isomerase deficiency: biochemical and molecular genetic studies on the enzyme variants of two patients with severe haemolytic anaemia. Eur J Pediatr, 1997,156(8):605–609
[32]	Zhu X, Petrovski S, Xie P, et al. Whole-exome sequencing in undiagnosed genetic diseases: interpreting 119 trios. Genet Med, 2015,17(10):774–781
[33]	Park H, Haller J, Smith F, et al. Attenuation of hemolysis due to glucose-6-phosphate isomerase deficiency with ketogenic diet–a case report. Hemasphere, 2020,4 (1):e328
[34]	Ceyhan-Birsoy O, Murry JB, Machini K, et al. Interpretation of genomic sequencing results in healthy and ill newborns: results from the BabySeq Project. Am J Hum Genet, 2019,104(1):76–93
[35]	Lakomek M, Winkler H. Erythrocyte pyruvate kinase- and glucose phosphate isomerase deficiency: perturbation of glycolysis by structural defects and functional alterations of defective enzymes and its relation to the clinical severity of chronic hemolytic anemia. Biophys Chem, 1997,66(2–3):269–284
[36]	Warang P, Kedar P, Ghosh K, et al. Hereditary nonspherocytic hemolytic anemia and severe glucose phosphate isomerase deficiency in an Indian patient homozygous for the L487F mutation in the human GPI gene. Int J Hematol, 2012,96(2):263–267
[37]	Jamwal M, Aggarwal A, Das A, et al. Next-generation sequencing unravels homozygous mutation in glucose-6-phosphate isomerase, GPIc.1040G>A (p.Arg347His) causing hemolysis in an Indian infant. Clin Chim Acta, 2017,468:81–84
[38]	Kedar PS, Harigae H, Ito E, et al. Study of pathophysiology and molecular characterization of congenital anemia in India using targeted next-generation sequencing approach. Int J Hematol, 2019,110(5):618–626
[39]	Sussman RG, Yan AP, Baker JM. Glucose 6 phosphate isomerase deficiency, a rare hemolytic anemia misdiagnosed as hereditary spherocytosis. J Pediatr Hematol Oncol, 2023,45(1):41–43
[40]	Gurney ME, Heinrich SP, Lee MR, et al. Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons. Science, 1986,234(4776):566–574
[41]	Schr?ter W, Eber SW, Bardosi A, et al. Generalised glucosephosphate isomerase (GPI) deficiency causing haemolytic anaemia, neuromuscular symptoms and impairment of granulocytic function: a new syndrome due to a new stable GPI variant with diminished specific activity (GPI Homburg). Eur J Pediatr, 1985,144(4):301–305
[42]	Nabi IR, Watanabe H, Raz A. Autocrine motility factor and its receptor: role in cell locomotion and metastasis. Cancer Metastasis Rev, 1992,11(1):5–20
[43]	Watanabe H, Takehana K, Date M, et al. Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res, 1996,56(13):2960–2963
[44]	Yanagawa T, Funasaka T, Tsutsumi S, et al. Novel roles of the autocrine motility factor/phosphoglucose isomerase in tumor malignancy. Endocr Relat Cancer, 2004,11(4):749–759