J Pediatr Endocrinol Metab 2017; 30(1): 97–100

Karolina Antosik, Piotr Gnyś, Przemysława Jarosz-Chobot, Małgorzata Myśliwiec, Agnieszka Szadkowska, Maciej Małecki, Wojciech Młynarski and Maciej Borowiec* An analysis of the sequence of the BAD gene among patients with maturity-onset of the young (MODY)

DOI 10.1515/jpem-2016-0239 Keywords: BAD gene; GCK gene; GCK-MODY; monogenic Received June 14, 2016; accepted November 3, 2016; previously diabetes. published online December 9, 2016

Abstract

Background: Monogenic diabetes is a rare disease caused Introduction by single gene mutations. Maturity onset diabetes of the Maturity-onset diabetes of the young (MODY) is a hetero- young (MODY) is one of the major forms of monogenic geneous group of monogenic diabetes forms that result diabetes recognised in the paediatric population. To date, in β-cell dysfunction. MODY is characterised by an auto- 13 genes have been related to MODY development. The somal dominant mode of inheritance and a young age of aim of the study was to analyse the sequence of the BCL2-­ onset, usually occurring prior to 25 years of age [1]. Cur- associated agonist of cell death (BAD) gene in patients rently, defects in 13 genes are known to cause MODY [2–4]. with clinical suspicion of GCK-MODY, but who were nega- The GCK-MODY subtype, responsible for the major- tive for (GCK) gene mutations. ity of MODY cases in the paediatric population, has been Methods: A group of 122 diabetic patients were recruited associated with the presence of heterozygous inactivating from the “Polish Registry for Paediatric and Adolescent mutations in the glucokinase (GCK) gene. The GCK gene Diabetes – nationwide genetic screening for monogenic encodes the enzyme which plays a crucial role in the regu- diabetes” project. The molecular testing was performed lation of secretion from β-cells and hepatic by Sanger sequencing. metabolism. GCK gene expression is regulated at the tran- Results: A total of 10 sequence variants of the BAD gene scriptional and posttranslational levels. The posttrans- were identified in 122 analysed diabetic patients. lational mechanism includes protein-protein interaction Conclusions: Among the analysed patients suspected of specific to liver and pancreatic β-cells [5]. GCK has been MODY, one possible pathogenic variant was identified in shown to interact with proteins such as glucokinase regu- one patient; however, further confirmation is required for latory protein (GKRP) [6] 6-phosphofructo-2-kinase/fruc- a certain identification. tose-2,6-biphosphatase (PFKFB1) [7] and BCL2-associated­ agonist of cell death (BAD) [8, 9]. *Corresponding author: Maciej Borowiec, PhD, Department The protein encoded by the BAD gene is the proapop- of Clinical Genetics, Medical University of Lodz, Pomorska 251, 92-213 Lodz, Poland, Phone: +48 42 272 57 67, totic member of the BCL-2 family of proteins, which share E-mail: [email protected] sequence homologies within conserved regions known as Karolina Antosik: Department of Clinical Genetics, Medical BCL-2 homology domains (BH). The BAD protein itself is University of Lodz, Lodz, Poland one of the BH3-only subset of molecules, which interact Piotr Gnyś: Medeor Plus Hospital, Lodz, Poland with multidomain pro-apoptotic and anti-apoptotic pro- Przemysława Jarosz-Chobot: Department of Pediatrics, teins to regulate apoptosis [10]. and Diabetes, Silesian Medical University of Katowice, Katowice, Poland Although the primary role of the BAD protein is to Małgorzata Myśliwiec: Department of Pediatrics, Oncology, regulate apoptosis, some evidence suggests that it is also Hematology and Endocrinology, Medical University of Gdansk, involved in the glucose metabolism and in glucose-stim- Gdansk, Poland ulated insulin secretion. While the BAD protein has been Agnieszka Szadkowska and Wojciech Młynarski: Department reported to be necessary for maximum GCK activity, it also of Pediatrics, Oncology, Hematology and Diabetology, Medical University of Lodz, Lodz, Poland plays a role in the maintenance of glucose homeostasis Maciej Małecki: Department of Metabolic Diseases, Collegium and in glucose-stimulated insulin secretion by pancreatic Medicum, Jagiellonian University of Krakow, Krakow, Poland β-cells [8, 9].

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The aim of this study was to analyse the sequence of DNA sequencing was performed using a BigDye Terminator v3.1 the BAD gene in patients with clinical phenotype of GCK- Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencher software v4.1.4 (GeneCodes, Ann Arbor, MI, USA) was MODY, but who were negative for GCK gene mutations. used for the comparative analysis of examined sequences (BAD gene RefSeq: NM_004322.3).

Materials and methods Bioinformatic tools The project was approved by the Bioethics Committee of the Medical University of Lodz (RNN/172/11/KE). The UCSC Genome Browser and the Human Mutation Database were The study group comprised 122 patients with clinical suspicion used to assess the impact of identified mutations/polymorphisms in of GCK-MODY diabetes. Patient recruitment was performed as a the BAD gene on the functionality of the BAD protein. part of the “Polish Registry for Pediatric and Adolescent ­Diabetes – Three bioinformatic tools incorporating analyses of the inter- ­nationwide genetic screening for monogenic diabetes” project. The species conservation of amino acid residues were used to determine clinical criteria used for selecting the study group were as those the effect of point mutations: SIFT, PolyPhen-2 and PROVEAN. Using described by Borowiec et al. [11]. In 122 selected patients, GCK gene these programs it can be determined whether an amino acid substitu- mutations were previously excluded. Detailed clinical characteristics tion is neutral or deleterious for the particular amino acid sequence. of the patients are shown in Tables 1 and 2.

Molecular methods Results

Mutations in the BAD gene were identified by direct sequencing Among the 122 patients, 10 sequence nucleotide changes of DNA extracted from peripheral blood leukocytes. The DNA was in the BAD gene sequence were identified (Figure 1). extracted using the AxyPrep™ Blood Genomic DNA Miniprep Kit (Axygen, Union City, CA, USA) according to the manufacturer’s There was no change in 25 patients. Seventy-nine patients instructions. Prior to sequencing, PCR was performed using four were the carriers of one nucleotide changes in the BAD specifically designed pairs of oligonucleotide primers spanning the sequence. More than one nucleotide changes were identi- coding, 5′ and 3′ untranslated regions and intron-exon boundaries. fied in 18 patients. These are located throughout the entire gene in the Table 1: Clinical characteristics of patients. intron, exon, 5′ and 3′ UTR sequences. Seven changes are classified as single nucleotide Features Values polymorphisms (SNP), which have known frequencies in Patients, n 122 the Caucasian population. Five are located in the coding Sex, male/female 59/63 sequence, one in the intronic sequence and one in both Age of onset of diabetes, years 11.54 ± 4.50 the intronic and 3′ UTR sequence of the BAD gene alterna- Age of genetic testing, years 13.51 ± 5.87 tive splice form. Three nucleotide changes are not depos- Ketoacidosis at diagnosis 9/109 (8.26%) ited in public databases: two in the intronic sequence and Polydipsia/polyuria at diagnosis 59/112 (52.68%) Birthweight, g 3342 ± 651 one in both the intronic and 5′ UTR sequence of the BAD BMI at examination, kg/m2 20.59 ± 4.28 sequence alternative split form.

Last HbA1c concentration, % 6.59 ± 1.50 Among the five variants located in the coding C-peptide level, pmol/L 502 ± 256 sequence, four are synonymous substitutions. Only Data are presented as numbers, percentage values or mean ± stand- one identified variant (c.462G>C) results in an amino ard deviation (SD). acid change: p.Trp154Cys (rs139093260). The carrier of the identified variant (p.Trp154Cys) displayed a clini- Table 2: The kind of treatment. cal course and positive family history of diabetes, which strongly suggested the presence of GCK-MODY diabetes. Treatments n = 122 He was diagnosed with diabetes by chance at 10 years Insulin 58 (47.54%) of age, presenting with polydipsia and polyuria but Oral hypoglycaemic agents 15 (12.29%) no symptoms of ketoacidosis. Neither insulin nor oral Insulin + oral hypoglycaemic agents 5 (4.10%) hypoglycaemic agents were required. No islet antibod- Lack of treatment 38 (31.15%) ies were found. The level of HbA1c at the time of genetic Lack of data 6 (4.92%) testing was 5%. The patient also had additional clinical Data are presented as percentage values. features such as allergy to furry animals and adenoid. In

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Figure 1: The result of BAD gene sequencing in the alternative transcript forms. Red color indicates heterozygous nucleotide substitution in the coding regions.

some other carriers of the four synonymous variants, dia- dephosphorylated state [13], in its phosphorylated state, it betes coexisting­ with additional clinical features such as is found in the mitochondrial complex with GCK. hypercholesterolaemia, obesity, food allergy, Hashimoto By using Bad−/− and Bad3SA (a non-phosphorytable disease or conductive hearing loss. form) murine models, an association has been found Bioinformatic analysis in PolyPhen-2 predicts the con- between the presence of dephosphorylated BAD and sequence of p.Trp154Cys for the BAD protein as “probably GCK loss-of-function, resulting in glucose intolerance, damaging”, SIFT regards the variant as “damaging” and impaired glucose-stimulated insulin secretion by β-cells PROVEAN as “deleterious”. and loss of hepatic glucose sensing [8, 9]. The defects observed in the Bad−/− and Bad3SA murine models are similar to those seen in mice with a liver-specific deletion Discussion of GCK and in people with GCK-MODY diabetes, in whom GCK mutations have been found [14]. This is the first study to analyse the sequence of the BAD Some of the GCK mutations did not affect the func- gene in patients with a suspicion of monogenic diabetes tionality of the protein but still caused mild diabetes. (GCK-MODY). The identification of a multiprotein complex Three GCK mutants, R36W, A53S and V367M, have kinetic in the liver mitochondria consisting of BAD, protein kinase and thermal stability properties similar to those of wild- A (PKA), protein phosphatase 1 (PP1), Wiskott Aldrich type GCK and have been thought to cause GCK-MODY by family member (WAVE-1) and GCK revealed the presence irregularities in protein-protein interaction [15]. There- of a link between BAD and glucose metabolism [8]. As fore, it appears that not only mutations in the GCK gene glucose stimulates insulin secretion, a much more signifi- can interrupt protein-protein interaction with BAD, but cant discovery was that BAD resides in the same complex also changes in the BAD gene. in the liver as in pancreatic β-cells [9]. To summarise, the BAD gene was sequenced in The dual role of BAD is known to be regulated patients with clinical suspicion of GCK-MODY but who through the phosphorylation and dephosphorylation of were known to be negative for GCK gene mutations. As three evolutionary-conserved­ serine residues (S112, S136, a result, one example of a base pair substitution causing S155 – murine numeration) within the BH3-only domain nonsynonymous replacement of an amino acid was found. [12]. While the BAD protein activates pro-apoptotic mol- Therefore, it is possible that a change in the amino acid ecules and causes multiple stages of apoptosis in its sequence of the BAD gene, which regulates the activity

Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-09-26 100 Antosik et al.: An analysis of the sequence of the BAD gene among patients with MODY of the GCK enzyme, may have an impact on the glucose 2. Siddiqui K, Musambil M, Nazir N. Maturity onset diabetes of the metabolism in this patient. Furthermore, three differ- young (MODY)-History, first case reports and recent advances. Gene 2015;555:66–71. ent bioinformatic tools found the p.Trp154Cys variant to 3. Bowman P, Flanagan SE, Edghill EL, Damhuis A, Shepherd MH, have “probably damaging”, ”damaging” or “deleterious” et al. Heterozygous ABCC8 mutations are a cause of MODY. effects, confirming its possible pathogenic influence on ­Diabetologia 2012;55:123–7. the structure and function of the BAD protein. 4. Bonnefond A, Philippe J, Durand E, Dechaume A, Huyvaert M, One limitation of the study was the lack of a DNA et al. Whole-Exome sequencing and high throughput geno­ sample from the diabetic mother and diabetic grand- typing identified KCNJ11 as the thirteenth MODY gene. PLoS One 2012;7:1–8. mother of the patient with this variant. This sample would 5. Osbak KK, Colclough K, Saint-Martin C, Beer NL, Bellanne- have been valuable in confirming the segregation of the Chantelot C, et al. Update on mutations in glucokinase (GCK), observed change in the amino acid sequence in diabetic which cause maturity-onset diabetes of the young, permanent family members. neonatal diabetes, and hyperinsulinemic . Hum To conclude, this is the first study to analyse the Mutat 2009;30:1512–6. 6. Agius L. Glucokinase and molecular aspects of liver sequence of the BAD gene among patients suspected of metabolism. Biochem J 2008;414:1–18. possessing monogenic diabetes. We have identified one 7. Baltrusch S, Lenzen S, Okar DA, Lange AJ, Tiedge M. Char- interesting gene variant which may have functional impli- acterization of glucokinase-binding protein epitopes by a cations, but its pathogenicity still needs to be confirmed. phage-displayed peptide library – identification of 6-phosphof- ructo-2-kinase/-2,6-bisphosphatase as a novel interac- Acknowledgments: This study was supported by funds tion partner. J Biol Chem 2001;276:43915–23. 8. Danial NN, Gramm CF, Scorrano L, Zhang CY, Krauss S, et al. from the National Science Centre, project no. 2011/01/N/ BAD and glucokinase reside in a mitochondrial complex that NZ5/02758. integrates and apoptosis. Nature 2003;424:952–6. Author contributions: All the authors have accepted 9. Danial NN, Walensky LD, Zhang CY, Choi CS, Fisher JK, et al. responsibility for the entire content of the submitted man- Dual role of proapoptotic BAD in insulin secretion and uscript and approved submission. survival. Nat Med 2008;14:144–53. 10. Danial NN. BCL-2 family proteins: Critical checkpoints of apop- Research funding: None declared. totic cell death. Clin Cancer Res 2007;13:7254–63. Employment or leadership: None declared. 11. Borowiec M, Antosik K, Fendler W, Deja G, Jarosz-Chobot P, et al. Honorarium: None declared. Novel glucokinase mutations in patients with monogenic diabe- Competing interests: The funding organization(s) played tes – clinical outline of GCK-MD and potential for founder effect no role in the study design; in the collection, analysis, and in Slavic population. Clin Genet 2012;81:278–83. interpretation of data; in the writing of the report; or in the 12. Danial NN. BAD: undertaker by night, candyman by day. ­Oncogene 2008;27:S53–70. decision to submit the report for publication. 13. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, et al. Proapoptotic BAX and BAK: A requisite gateway to mito- chondrial dysfunction and death. Science 2001;292:727–30. 14. Postic C, Shiota M, Niswender KD, Jetton TL, Chen YJ, et al. Dual References roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre 1. Tattersall RB, Fajans SS. A difference between the inheritance recombinase. J Biol Chem 1999;274:305–15. of classical juvenile-onset and maturity-onset type diabetes of 15. Philipson LH, Roe MW. When BAD is good for beta cells. Cell young people. Diabetes 1975;24:44–53. Metab 2008;7:280–1.

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