G6PD Akdeniz mutasyonu olan hemolitik anemili bireylerde gen ekspresyonlarının karşılaştırılması

Year 2023, Volume: 48 Issue: 2, 351 - 360, 02.07.2023

Başak Günaştı , Abdullah Tuli

https://doi.org/10.17826/cumj.1226150

Abstract

Amaç: Çalışmamızda, Gd-Akdeniz’li bireylerde hemolitik anemisi olan ve olmayan olguların Glukoz-6-fosfat dehidrogenaz (G6PD) enzimi kinetiği ve G6PD geninin mRNA ekspresyon seviyeleri arasındaki ilişkinin irdelenerek bu duruma açıklık getirmeyi hedefledik.
Gereç ve Yöntem: Çalışma grubu, Gd-Akdeniz mutasyonu bulunan 30 olgu ve enzim aktivite düzeyi referans aralığında olan 30 olgudan oluşmaktadır. G6PD aktivitesi Beutler yöntemi ile tayin edilmiştir. G6PD enzimi DE-52 anyon değiştirici reçine ile kısmi olarak saflaştırılarak kinetik özellikleri çalışılmıştır. MboII enzim kesimi ve sekans analizi ile Gd-Akdeniz mutasyonu genotiplendirilmiştir. G6PD geninin ekspresyon seviyesi 2-ΔΔCt formülüne göre hesaplanmıştır.
Bulgular: Çalışmamızda Gd-Akdeniz mutasyonu bulunan olguların KmNADP+ ve KmG6P değerleri ile G6PD enzim aktivitesi referans değerlerde olan olgular arasında anlamlı düzeyde fark bulundu. Hemolitik anemisi olan ve olmayan Gd-Akdeniz mutasyonuna sahip olguların ise KmNADP+ ve KmG6P değerleri arasında anlamlı fark bulunamadı. Hemolitik anemisi olmayan 18 hastanın gen ekspresyon sonuçları, hemolitik anemisi olan 12 hastaya göre anlamlı derecede yüksek olduğu görüldü. Ayrıca enzim aktivite düzeyi referans aralığında olan olgularla da bu değişkenler kıyaslandığında yine anlamlı farklılık olduğu belirlendi
Sonuç: Gd-Med mutasyonu olan olgularda enzimin substrat bağlanma bölgesinin post-transkripsiyonel veya post-translasyonel modifikasyonlara uğramış olması ve bu nedenle gen ekspresyonunun değişmesi olası bir açıklama olabilir. İleri çalışma olarak Gd-Akdeniz mutasyonuna sahip hemolitik anemisi bulunan olguların gen ekspresyonlarındaki azalmayı geninin promotör bölgesi değerlendirilerek açıklık getirilebilir.

Keywords

Enzim Kinetiği, Gen Ekspresyonu, Glukoz-6-fosfat Dehidrogenaz, Hemolitik Anemi

Supporting Institution

Çukurova Üniversitesi Bilimsel Araştırma Projeleri Birimi

Project Number

TF2013YL2

Thanks

Çalışmayı TF2013YL2 No’lu proje ile maddi olarak destekleyen Çukurova Üniversitesi Bilimsel Araştırma Projeleri Birimi’ne teşekkür ederim.

Comparison of gene expressions in individuals with G6PD Mediterranean mutation and hemolytic anemia

Abstract

Purpose: In our study, we aimed to clarify the relationship between Glucose-6-phosphate dehydrogenase (G6PD) enzyme kinetics and mRNA expression levels of the G6PD gene in Gd-Med patients with and without hemolytic anemia.
Materials and Methods: The study group consisted of 30 cases with Gd-Med mutation and 30 cases with enzyme activity levels in the reference range. G6PD activity was determined by the Beutler method. G6PD enzyme was partially purified with DE-52 anion exchange resin, and its kinetic parameters were studied. Gd-Med mutation was genotyped by MboII enzyme digest and sequence analysis. The expression level of the G6PD gene was calculated according to the 2-ΔΔCt formula.
Results: In our study, a significant difference was found between the KmNADP+ and KmG6P values of the cases with Gd-Med mutation and the control group. There was no significant difference between KmNADP+ and KmG6P values in Gd-Med mutated patients with and without hemolytic anemia. Gene expression results of 18 patients without hemolytic anemia were significantly higher than 12 patients with hemolytic anemia. In addition, there was a significant difference between these variables and the control group.
Conclusion: It might be a possible explanation that the substrate binding site of the enzyme in cases with Gd-Med mutation may have undergone post-transcriptional or post-translational modifications, and therefore gene expression might be changed. As a further study, the decrease in gene expressions of patients with hemolytic anemia with Gd-Med mutation can be clarified by evaluating the promoter side of the gene.

Keywords

Enzyme Kinetics, Gene Expression, Glucose-6-phosphate Dehydrogenase, Hemolytic Anemia.

Project Number

TF2013YL2

References

• Ghergurovich JM, Garcia-Canaveras JC, Wang J, Schmidt E, Zhang Z, Teslaa T et al. A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nat Chem Biol. 2020;16:731–9.
• Jacobasch G, Rapoport SM. Hemolytic anemias due to erythrocyte enzyme deficiencies. Mol Aspects Med. 1996;17:143–70.
• Martini G, Toniolo D, Vulliamy T, Luzzatto L, Dono R, Viglietto G et al. Structural analysis of the X-linked gene encoding human glucose 6-phosphate dehydrogenase. EMBO J. 1986;5:1849–55.
• Corcoran CM, Calabro V, Tamagnini G, Town M, Haidar B, Vulliamy TJ et al. Molecular heterogeneity underlying the G6PD mediterranean phenotype. Hum Genet. 1992;88:688-90.
• Özer N, Aksoy Y, Ögüs IH. Kinetic properties of human placental glucose-6-phosphate dehydrogenase. Int J Biochem Cell Biol. 2001;33:221–6.
• Sheikh MH, Hazazi ASJ, Alanazi ANO, Hazazi ISJ, Qaysi AAM, Alali YA et al. Overview on causes and updated management of favism. J Pharm Res Int. 2021;33:249-54.
• Arunachalam AK, Sumithra S, Maddali M, Fouzia NA, Abraham A, George B et al. Molecular characterization of G6PD deficiency: Report of three novel G6PD variants. Indian J Hematol Blood Transfus. 2020;36:349-55.
• Tuli A. Çukurova'da saptanan G6PD varyantlarının mutasyon noktalarının moleküler düzeyde saptanması (Doktora tezi). Adana, Çukurova Üniversitesi, 1994.
• Gomez-Manzo S, Marcial-Quino J, Vanoye-Carlo A, Serrano-Posada H, Ortega-Cuellar D, Gonzalez-Valdez A et al. Glucose-6-phosphate dehydrogenase: Update and analysis of new mutations around the world. Int J Mol Sci. 2016;17:2069.
• Sirdah M, Reading NS, Vankayalapati H, Perkins SL, Shubair ME, Aboud L et al. Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Gaza Strip palestinians. Blood Cells Mol Dis. 2012;49:152–8.
• Aslan KS. Diyarbakır bölgesinde G6PD özellikleri bilinmeyen kişilerde G6PD canton, kaiping ve gaohe mutant allellerinin belirlenmesi (Yüksek lisans tezi). Diyarbakır, Dicle Üniversitesi, 2011.
• Beutler E. G6PD: Population genetics and clinical manifestations. Blood Rev. 1996;10:45-52.
• Schwartz AG, Pashko LL. Dehydroepiandrosterone, glucose-6-phosphate dehydrogenase, and longevity. Ageing Res Rev. 2004;3:171–87.
• Yoshida A. Hemolytic anemia and G6PD deficiency. Science. 1973;179:532–7.
• El-Deen ZME, Hussin NF, Hamid TAA, Migeed ORA, Samy RM. G6PD deficiency and G6PD (mediterranean and silent) polymorphisms in Egyptian infants with neonatal hyperbilirubinemia. Lab Med. 2013;44:228–34.
• Farhud DD, Yazdanpanah L. Glucose-6-phosphate dehydrogenase (G6PD) deficiency. Iran J Public Health. 2008;37:1-18.
• Pamba A, Richardson D, Carter N, Duparc S, Premji Z, Tiono A et al. Clinical spectrum and severity of hemolytic anemia in glucose 6-phosphate dehydrogenase–deficient children receiving dapsone. Blood. 2012;120:4123–33.
• Gomez MS, Marcial QJ, Ortega CD, Serrano PH, Gonzalez VA, Vanoye CA et al. Functional and biochemical analysis of glucose-6-phosphate dehydrogenase (G6PD) variants: Elucidating the molecular basis of G6PD deficiency. Catalysts. 2017;7:1-17.
• Velasco P, Barcia R, Ibarguren I, Sieiro AM, Ramos-Martinez JI. Purification, characterization and kinetic mechanism of glucose-6-phosphate dehydrogenase from mouse liver. Int J Biochem. 1994;26:195–200.
• Demoss RD, Gunsalus IC, Bard RC. Glucose-6-phosphate dehydrogenase. 1952;195:95–105.
• Hopa E. İnsan eritrositlerinden glukoz 6-fosfat dehidrogenaz enziminin saflaştirilmasi, bazi kumarin ve pestisitlerin etkilerinin araştırılması (Doktora Tezi). Balıkesir, Balıkesir Üniversitesi, 2010.
• Wang XT, Au SWN, Lam VMS, Engel PC. Recombinant human glucose-6-phosphate dehydrogenase: evidence for a rapid-equilibrium random-order mechanism. Eur J Biochem. 2002;269:3417–24.
• Cordeiro AT, Thiemann OH, Michels PAM. Inhibition of trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites. Bioorganic Med Chem. 2009;17:2483–9.
• Corrons JLV, Pujades A. Heterogeneity of “Mediterranean type” glucose-6-phosphate dehydrogenase (g6pd) deficiency in spain and description of two new variants associated with favism. Hum Genet. 1982;60:216–21.
• Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 2000;21:1037–53.
• Bourzac KM, LaVine LJ, Rice MS. Analysis of DAPI and SYBR Green I as alternatives to ethidium bromide for nucleic acid staining in agarose gel electrophoresis. J Chem Educ. 2003;80:1292–6.
• Karpati M, Gazit E, Goldman B, Frisch A, Colombo R, Peleg L. Specific mutations in the HEXA gene among Iraqi jewish tay-sachs disease carriers: Dating of Founder Ancestor. Neurogenetics. 2004;5:35–40.
• Nakhaee A, Salimi S, Zadehvakili A, Dabiri S, Noora M, Rezaei M et al. The prevalence of mediterranean mutation of glucose-6-phosphate dehydrogenase (G6PD) in Zahedan. Zahedan Journal of Research in Medical Sciences. 2012;14:39–43.
• Noori-Daloii MR, Najafi L, Mohammad Ganji S, Hajebrahimi Z, Sanati MH. Molecular identification of mutations in G6PD gene in patients with favism in Iran. J Physiol Biochem. 2004;60:273–7.
• Fakruddin M, Chowdhury A. Pyrosequencing-an alternative to traditional sanger sequencing. Am J Biochem Biotechnol. 2012;8:14–20.
• Hong GF. A method for sequencing single-stranded cloned DNA in both directions. Biosci Rep. 1981;1:243–52.
• Von Dornum M, Ruvolo M. Phylogenetic relationships of the new world Monkeys (Primates, platyrrhini) based on nuclear G6PD DNA sequences. Mol Phylogenet Evol. 1999;11:459–76.
• MacLean D, Jones JDG, Studholme DJ. Application of “next-generation” sequencing technologies to microbial genetics. Nat Rev Microbiol. 2009;7:287–96.
• Wagle A, Jivraj S, Garlock GL, Stapleton SR. Insulin regulation of glucose-6-phosphate dehydrogenase gene expression is rapamycin-sensitive and requires phosphatidylinositol 3-kinase. J Biol Chem. 1998;273:14968–74.
• Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3:71–85.
• Sanna B. G6PD aktivitesi referans değerleri altında olan bireylerde gen ekspresyonlarının karşılaştırılması (Yüksek Lisans Tezi). Adana, Çukurova Üniversitesi, 2015.
• Beutler E. G6PD: Population genetics and clinical manifestations. Blood Rev. 1996;10:45–52.
• Akisin YA, Arslan Z, Ceylaner S, Akar N. Glucose-6-phosphate dehydrogenase gene Ala365Thr mutation in an Iraqi family with confusing clinical differences. Turkish J Biochem. 2021;46:729 31.
• Gürbüz N, Aksu TA, Noorden CJF. Biochemical and cytochemical evaluation of heterozygote individuals with glucose-6-phosphate dehydrogenase deficiency. Acta histochem. 2005;107:261-7.
• Sukumar S, Mukherjee MB, Colah RB, Mohanty D. Molecular basis of G6PD deficiency in India. Blood Cells Mol Dis. 2004;33:141–5.
• Silva HS, Lima AN, Andrade GB, Gaia KC, Costa LC, Campos AK et al. Molecular genotyping of G6PD mutations and duffy blood group in afro-descendant communities from Brazilian Amazon. Genet Mol Biol. 2018;41:758–65.
• Chen Y, Zhang SF. Diagnostic value and clinical significance of G6PD activity for the mediterranean anemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2015;23:1108-11.
• Gerli GC, Beretta L, Bianchi M, Agostoni A, Gualandri V, Orsini GB. Erythrocyte superoxide dismutase, catalase and glutathione peroxidase in glucose‐6‐phosphate dehydrogenase deficiency. Scand J Haematol. 1982;29:135–40.
• Testa U, Meloni T, Lania A, Battistuzzi G, Cutillo S, Luzzatto L. Genetic heterogeneity of glucose 6-phosphate dehydrogenase deficiency in Sardinia. Hum Genet. 1980;56:99–105.
• Sirdah M, Reading NS, Vankayalapati H, Prchal JT. A computational study of structural differences of binding of NADP+ and G6P substrates to G6PD Mediterranean. Blood Cells Mol Dis. 2021;89:1–7.
• Batetta B, Pulisci D, Bonatesta RR, Sanna F, Piras S, Mulas MF et al. G6PD activity and gene expression in leukemic cells from G6PD deficient subjects. Cancer Lett. 1999;140:53-8.
• Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Jarvela I. Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia. Gut. 2003;52:647–52.
• Xu DD, Wen FQ, Rong LV, Zhang M, Chen YS, Chen XW. Gene promoter methylation in glucose-6-phosphate dehydrogenase deficiency. Zhongguo Dang Dai Er Ke Za Zhi. 2016;12:405-9.
• Wang J, Xiao QZ, Chen YM, Yi S, Liu D, Liu YH et al. DNA hypermethylation and X chromosome inactivation are major determinants of phenotypic variation in women heterozygous for G6PD mutations. Blood Cells Mol Dis. 2014;53:241-5.
• Mini MV, Scalera L, Martini G. High levels of transcription driven by a 400 bp segment of the human G6PD promoter. Biochem Biophys Res Commun. 1990;170:1203-9.
• Menounos PG, Garinis GA, George P. Glucose-6-phosphate dehydrogenase deficiency does not result from mutations in the promoter region of the G6PD gene. J Clin Lab Anal. 2003;17:90-2.