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Identification of two novel variants in PRKAG2 gene in Tunisian type 2 diabetic patients with family history of cardiovascular disease
diabetes research and clinical practice 87 (2010) e7–e10
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locate/diabres
Brief report
Identification of two novel variants in PRKAG2 gene in Tunisian type 2 diabetic patients with family history of cardiovascular disease S. Nouira a,b, I. Arfa a, I. Kammoun b, A. Abid c, H. Ouragini a, I. Dorboz a, W. Ghazouani a, S. Ben Fadhel a, M.M. Zorgati a, S. Ben Ammar a, S. Blousa-Chabchoub c, S. Kachboura b, S. Abdelhak a,* a
Molecular Investigation of Genetic Orphan Diseases Research Unit UR04/SP03, Institut Pasteur de Tunis, Tunisia Cardio-net Research Unit UR 09/04, Cardiology Department, Abderahman Mami Hospital, Ariana, Tunisia c National Institute of Nutrition, Tunis, Tunisia b
article info
abstract
Article history:
We report the identification of two novel polymorphisms in the PRKAG2 gene and prelimi-
Received 13 July 2009
nary association study between 50 -UTR and exon 1 polymorphisms with susceptibility to
Accepted 9 November 2009
type 2 diabetes. No association with type 2 diabetes was identified. However, one of these
Published on line 22 December 2009
newly identified polymorphisms (p.Ser20Ile) is likely associated with cardiac disease. # 2009 Published by Elsevier Ireland Ltd.
Keywords: 50 -AMP-activated protein kinase (AMPK) SNP Type 2 diabetes Cardiac failure
1.
Introduction
The 50 -AMP-activated protein kinase (AMPK) is a serinethreonine kinase which plays an important role in different metabolisms such as energetic, lipid and glucose metabolisms. The implication of the AMPK in glucose metabolism has been supported by several studies [1], demonstrating that AMPK increases muscle glucose uptake [2] and ameliorates insulin resistance [3]. Consequently, AMPK is a potentially interesting drug target for the treatment of diabetes.
AMPK is a heterotrimeric protein, consisting of a catalytic subunit (a1 or a2) and two regulatory subunits (b1 or b2 and g1, g2 or g3) [4]. In humans, these subunits are encoded by different genes [4]. The g2 subunit is encoded by the PRKAG2 gene mapped to the chromosome 7q35-q36. A recent study showed that the 26C/T polymorphism in the exon 1 of PRKAG2 gene might be associated with glucose metabolism in Chinese population [5]. To test this hypothesis in Tunisian population, we investigated this polymorphism by direct sequencing in a group of type 2 diabetic patients. In addition to the previously reported polymorphism, we identified two
* Corresponding author at: Institut Pasteur de Tunis, BP 74, 13 Place Pasteur, 1002 Tunis Belve´de`re, Tunisia. Tel.: +216 71 84 91 10; fax: +216 71 79 18 33. E-mail addresses: [email protected] (I. Arfa), [email protected] (S. Abdelhak). 0168-8227/$ – see front matter # 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.diabres.2009.11.009
e8
diabetes research and clinical practice 87 (2010) e7–e10
novel polymorphisms which have been also examined for potential association with type 2 diabetes.
Table 1 – Genotype distribution and allele frequencies of S90G/T and S26C/T polymorphisms. Genotypes
2.
Patients, materials and methods
In this study, we screened 51 unrelated Tunisian individuals (28 type 2 diabetic patients and 23 non diabetic controls). Type 2 diabetes was diagnosed according to the WHO criteria. The mean age of patients is 60.6 years 11 with a mean duration of diabetes 11.7 years 2.1. Approval for the study was obtained from the local ethical committee; all patients gave their informed consent. DNA was extracted from leukocytes using standard methods and polymorphisms screening was performed by direct sequencing of exon 1 of PRKAG2 gene. All data were analyzed using SPSS, x2-test were used for statistical analysis. A value of p < 0.05 was considered statistically significant.
3.
Results
The investigation of the 26C/T polymorphism, identified as associated with type 2 diabetes in Chinese population, showed no significant statistical difference between Tunisian type 2 diabetic subjects compared to nondiabetic controls (x2 = 0.06, p = 0.96) (Table 1). When screening the 26C/T polymorphism, we identified two novel substitutions: 90G/T in the 50 -UTR region and 59 G/ T (p.Ser20Ile) in exon 1 of PRKAG2 gene (NM_016203.3). We have tested the hypothesis that these polymorphisms might be associated with glucose metabolism.
Type 2 diabetic patients (N = 28)
Normal controls (N = 23)
90G/T polymorphism GT and TT GG f(G/T)
3 (10) 25 (90) 0.93/0.07
1(4) 22 (96) 0.98/0.02
26C/T polymorphism CC CT and TT f(C/T)
25(89) 3(11) 0.97/0.03
20(87) 3(13) 0.97/0.03
Key: Data are in N (%), X2 = 0.94, p-value = 0.62 for 90G/T and X2 = 0.06, p = 0.96 for 26C/T.
The genotype distribution and allele frequencies for the 90G/T variant did not significantly differ between the two groups (x2 = 0.8, p = 0.67) (Table 1). We observed that this polymorphism is located in a highly conserved region between mammalians suggesting that it might disrupt a functionally important regulatory sequence (Fig. 1a). To test this hypothesis, we used a program for predicting transcription factor binding sites (http://www.gene-regulation.com). The result shows that the 90G/T polymorphism leads to the alteration of a transcription factor binding site for YY1 in the presence of thymine. Among the two studied groups, the polymorphism p.Ser20Ile was detected only in one diabetic patient at heterozygous state, and absent from the whole investigated pool of patients and controls (a total of 102 alleles). This suggests that this polymorphism is a rare variant.
Fig. 1 – (a) Alignments of the genomic sequence of PRKAG2 for six mammalian species around the variant in the 50 -UTR (http://www.ensembl.org genomic sequence alignment). (b) Multiple alignment of amino acids sequence of vertebrate PRKAG2 gene around serine residue in position 20 (http://genome.ucsc.edu).
diabetes research and clinical practice 87 (2010) e7–e10
Fig. 2 – Pedigree of type 2 diabetic case (individual shown by an arrow in whom the p.Ser20Ile has been identified). Individuals IV-5, IV-6, IV-7 and IV-8 underwent cardiological examination to check for their status. No particular cardiac disease was identified. The status of the other individuals was identified by interviewing family members.
e9
The p.Ser20Ile identified only in one diabetic patient. Clinical and familial history of this patient showed that he has no particular cardiac complications but a family history of cardiac disease (Fig. 2). Other family members accepted to undergo clinical and genetic investigation (Fig. 2). No clear correlation between this variant and the cardiac disease was identified. As this variation leads to an amino acid change at the protein level, this mutation could be either a very rare deleterious variant or could be a pathogenic, incompletely penetrant, variant associated to cardiac disease. SIFT and Pmut analysis of the sequence variant p.Ser20Ile predicts this change to be deleterious to the function of the protein. Analysis of conservation of the protein sequence across species shows that the residue p.Ser20Ile is conserved across vertebrate species (Fig. 1b). This preliminary study needs further investigation and confirmation in other populations to elucidate the relationship between the newly reported polymorphism p.Ser20Ile and glucose metabolism as well as cardiac complication, as several mutations in AMPK have been shown to underlie numerous metabolic cardiac diseases [9,10,13].
Conflicts of interest There are no conflicts of interest.
Multiple alignments show that the substitution p.Ser20Ile affects amino acids which are highly conserved among species (Fig. 1b). To predict the putative effect of the substitution on protein function, we used two in silico methods: SIFT ‘‘Sorting Tolerent From Intolerant’’ that predicts the functional importance of an amino acid based on the alignment of highly similar orthologous and/or paralogous protein sequences [6]; and Pmut that predicts the functional effect of substitutions [7]. The Ser20Ile is predicted to be ‘‘not tolerant’’ by SIFT and ‘‘pathological’’ by Pmut.
Acknowledgements We wish to thank the patients and their family members. This study was supported by the Tunisian Ministry of Higher Education, Scientific and Technological Research, the Tunisian Ministry of Health, the NEPAD/NABnet T2D project and EMRO-COMSTECH RAB&GH grant.
references
4.
Discussion
AMPK plays an important role in the pathway of glucose metabolism. The activation of this kinase may lower blood glucose levels [8]. It has been demonstrated that different functional variations in human PRKAG2 gene were associated with glycogen content in heart, liver, and skeletal muscle [9,10]. In this report, we have studied the association between glucose metabolism and 3 polymorphisms in the PRKAG2 gene: the 26C/T and two newly identified variations the 90G/T and p.Ser20Ile. Our results suggest that all these polymorphisms are likely not associated with the susceptibility to T2D in the studied sample. This is not consistent with previous studies [5]. The newly identified polymorphism in the 50 -UTR of PRKAG2 gene, 90G/T disrupted a binding site of a transcription factor YY1 that has been shown to regulate a variety of promoters [11] in relation to glucose metabolism. It is known that YY1 in the liver may modulate expression of a number of genes in relation to glucose metabolism [12]. Preliminary investigation showed no association between the 90G/T and the diabetics patients.
[1] G.A. Rutter, G. Da Silva Xavier, I. Leclerc, Roles of 50 -AMPactivated protein kinase (AMPK) in mammalian glucose homoeostasis, Biochem. J. 375 (2003) 1–16. [2] R. Bergeron, R.R. Russell 3rd, L.H. Young, J.M. Ren, M. Marcucci, A. Lee, et al., Effect of AMPK activation on muscle glucose metabolism in conscious rats, Am. J. Physiol. 276 (1999) E938–944. [3] N.B. Ruderman, J.M. Cacicedo, S. Itani, N. Yagihashi, A.K. Saha, J.M. Ye, et al., Malonyl-CoA and AMP-activated protein kinase (AMPK): possible links between insulin resistance in muscle and early endothelial cell damage in diabetes, Biochem. Soc. Trans. 31 (2003) 202–206. [4] W.W. Winder, Energy-sensing and signaling by AMPactivated protein kinase in skeletal muscle, J. Appl. Physiol. 91 (2001) 1017–1028. [5] M. Xu, X. Li, J.G. Wang, P. Du, J. Hong, W. Gu, et al., Glucose and lipid metabolism in relation to novel polymorphisms in the 50 -AMP-activated protein kinase gamma2 gene in Chinese, Mol. Genet. Metab. 86 (2005) 372–378. [6] P.C. Ng, S. Henikoff, Predicting deleterious amino acid substitutions, Genome Res. 11 (2001) 863–874. [7] C. Ferrer-Costa, M. Orozco, X. de la Cruz, Use of bioinformatics tools for the annotation of disease-
e10
diabetes research and clinical practice 87 (2010) e7–e10
associated mutations in animal models, Proteins 61 (2005) 878–887. [8] D. Carling, AMPK, Curr. Biol. 14 (2004) R220. [9] M.H. Gollob, M.S. Green, A.S. Tang, T. Gollob, A. Karibe, A.S. Ali Hassan, et al., Identification of a gene responsible for familial Wolff–Parkinson–White syndrome, N. Engl. J. Med. 344 (2001) 1823–1831. [10] M. Arad, B.J. Maron, J.M. Gorham, W.H. Johnson Jr., J.P. Saul, A.R. Perez-Atayde, et al., Glycogen storage diseases presenting as hypertrophic cardiomyopathy, N. Engl. J. Med. 352 (2005) 362–372.
[11] M.J. Thomas, E. Seto, Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key? Gene 236 (1999) 197–208. [12] C.Q. He, N.Z. Ding, W. Fan, YY1 repressing peroxisome proliferator-activated receptor delta promoter, Mol. Cell Biochem. 308 (2008) 247–252. [13] M. Arad, D.W. Benson, A.R. Perez-Atayde, W.J. McKenna, E.A. Sparks, R.J. Kanter, et al., Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy, J. Clin. Invest. 109 (2002) 357–362.
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locate/diabres
Brief report
Identification of two novel variants in PRKAG2 gene in Tunisian type 2 diabetic patients with family history of cardiovascular disease S. Nouira a,b, I. Arfa a, I. Kammoun b, A. Abid c, H. Ouragini a, I. Dorboz a, W. Ghazouani a, S. Ben Fadhel a, M.M. Zorgati a, S. Ben Ammar a, S. Blousa-Chabchoub c, S. Kachboura b, S. Abdelhak a,* a
Molecular Investigation of Genetic Orphan Diseases Research Unit UR04/SP03, Institut Pasteur de Tunis, Tunisia Cardio-net Research Unit UR 09/04, Cardiology Department, Abderahman Mami Hospital, Ariana, Tunisia c National Institute of Nutrition, Tunis, Tunisia b
article info
abstract
Article history:
We report the identification of two novel polymorphisms in the PRKAG2 gene and prelimi-
Received 13 July 2009
nary association study between 50 -UTR and exon 1 polymorphisms with susceptibility to
Accepted 9 November 2009
type 2 diabetes. No association with type 2 diabetes was identified. However, one of these
Published on line 22 December 2009
newly identified polymorphisms (p.Ser20Ile) is likely associated with cardiac disease. # 2009 Published by Elsevier Ireland Ltd.
Keywords: 50 -AMP-activated protein kinase (AMPK) SNP Type 2 diabetes Cardiac failure
1.
Introduction
The 50 -AMP-activated protein kinase (AMPK) is a serinethreonine kinase which plays an important role in different metabolisms such as energetic, lipid and glucose metabolisms. The implication of the AMPK in glucose metabolism has been supported by several studies [1], demonstrating that AMPK increases muscle glucose uptake [2] and ameliorates insulin resistance [3]. Consequently, AMPK is a potentially interesting drug target for the treatment of diabetes.
AMPK is a heterotrimeric protein, consisting of a catalytic subunit (a1 or a2) and two regulatory subunits (b1 or b2 and g1, g2 or g3) [4]. In humans, these subunits are encoded by different genes [4]. The g2 subunit is encoded by the PRKAG2 gene mapped to the chromosome 7q35-q36. A recent study showed that the 26C/T polymorphism in the exon 1 of PRKAG2 gene might be associated with glucose metabolism in Chinese population [5]. To test this hypothesis in Tunisian population, we investigated this polymorphism by direct sequencing in a group of type 2 diabetic patients. In addition to the previously reported polymorphism, we identified two
* Corresponding author at: Institut Pasteur de Tunis, BP 74, 13 Place Pasteur, 1002 Tunis Belve´de`re, Tunisia. Tel.: +216 71 84 91 10; fax: +216 71 79 18 33. E-mail addresses: [email protected] (I. Arfa), [email protected] (S. Abdelhak). 0168-8227/$ – see front matter # 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.diabres.2009.11.009
e8
diabetes research and clinical practice 87 (2010) e7–e10
novel polymorphisms which have been also examined for potential association with type 2 diabetes.
Table 1 – Genotype distribution and allele frequencies of S90G/T and S26C/T polymorphisms. Genotypes
2.
Patients, materials and methods
In this study, we screened 51 unrelated Tunisian individuals (28 type 2 diabetic patients and 23 non diabetic controls). Type 2 diabetes was diagnosed according to the WHO criteria. The mean age of patients is 60.6 years 11 with a mean duration of diabetes 11.7 years 2.1. Approval for the study was obtained from the local ethical committee; all patients gave their informed consent. DNA was extracted from leukocytes using standard methods and polymorphisms screening was performed by direct sequencing of exon 1 of PRKAG2 gene. All data were analyzed using SPSS, x2-test were used for statistical analysis. A value of p < 0.05 was considered statistically significant.
3.
Results
The investigation of the 26C/T polymorphism, identified as associated with type 2 diabetes in Chinese population, showed no significant statistical difference between Tunisian type 2 diabetic subjects compared to nondiabetic controls (x2 = 0.06, p = 0.96) (Table 1). When screening the 26C/T polymorphism, we identified two novel substitutions: 90G/T in the 50 -UTR region and 59 G/ T (p.Ser20Ile) in exon 1 of PRKAG2 gene (NM_016203.3). We have tested the hypothesis that these polymorphisms might be associated with glucose metabolism.
Type 2 diabetic patients (N = 28)
Normal controls (N = 23)
90G/T polymorphism GT and TT GG f(G/T)
3 (10) 25 (90) 0.93/0.07
1(4) 22 (96) 0.98/0.02
26C/T polymorphism CC CT and TT f(C/T)
25(89) 3(11) 0.97/0.03
20(87) 3(13) 0.97/0.03
Key: Data are in N (%), X2 = 0.94, p-value = 0.62 for 90G/T and X2 = 0.06, p = 0.96 for 26C/T.
The genotype distribution and allele frequencies for the 90G/T variant did not significantly differ between the two groups (x2 = 0.8, p = 0.67) (Table 1). We observed that this polymorphism is located in a highly conserved region between mammalians suggesting that it might disrupt a functionally important regulatory sequence (Fig. 1a). To test this hypothesis, we used a program for predicting transcription factor binding sites (http://www.gene-regulation.com). The result shows that the 90G/T polymorphism leads to the alteration of a transcription factor binding site for YY1 in the presence of thymine. Among the two studied groups, the polymorphism p.Ser20Ile was detected only in one diabetic patient at heterozygous state, and absent from the whole investigated pool of patients and controls (a total of 102 alleles). This suggests that this polymorphism is a rare variant.
Fig. 1 – (a) Alignments of the genomic sequence of PRKAG2 for six mammalian species around the variant in the 50 -UTR (http://www.ensembl.org genomic sequence alignment). (b) Multiple alignment of amino acids sequence of vertebrate PRKAG2 gene around serine residue in position 20 (http://genome.ucsc.edu).
diabetes research and clinical practice 87 (2010) e7–e10
Fig. 2 – Pedigree of type 2 diabetic case (individual shown by an arrow in whom the p.Ser20Ile has been identified). Individuals IV-5, IV-6, IV-7 and IV-8 underwent cardiological examination to check for their status. No particular cardiac disease was identified. The status of the other individuals was identified by interviewing family members.
e9
The p.Ser20Ile identified only in one diabetic patient. Clinical and familial history of this patient showed that he has no particular cardiac complications but a family history of cardiac disease (Fig. 2). Other family members accepted to undergo clinical and genetic investigation (Fig. 2). No clear correlation between this variant and the cardiac disease was identified. As this variation leads to an amino acid change at the protein level, this mutation could be either a very rare deleterious variant or could be a pathogenic, incompletely penetrant, variant associated to cardiac disease. SIFT and Pmut analysis of the sequence variant p.Ser20Ile predicts this change to be deleterious to the function of the protein. Analysis of conservation of the protein sequence across species shows that the residue p.Ser20Ile is conserved across vertebrate species (Fig. 1b). This preliminary study needs further investigation and confirmation in other populations to elucidate the relationship between the newly reported polymorphism p.Ser20Ile and glucose metabolism as well as cardiac complication, as several mutations in AMPK have been shown to underlie numerous metabolic cardiac diseases [9,10,13].
Conflicts of interest There are no conflicts of interest.
Multiple alignments show that the substitution p.Ser20Ile affects amino acids which are highly conserved among species (Fig. 1b). To predict the putative effect of the substitution on protein function, we used two in silico methods: SIFT ‘‘Sorting Tolerent From Intolerant’’ that predicts the functional importance of an amino acid based on the alignment of highly similar orthologous and/or paralogous protein sequences [6]; and Pmut that predicts the functional effect of substitutions [7]. The Ser20Ile is predicted to be ‘‘not tolerant’’ by SIFT and ‘‘pathological’’ by Pmut.
Acknowledgements We wish to thank the patients and their family members. This study was supported by the Tunisian Ministry of Higher Education, Scientific and Technological Research, the Tunisian Ministry of Health, the NEPAD/NABnet T2D project and EMRO-COMSTECH RAB&GH grant.
references
4.
Discussion
AMPK plays an important role in the pathway of glucose metabolism. The activation of this kinase may lower blood glucose levels [8]. It has been demonstrated that different functional variations in human PRKAG2 gene were associated with glycogen content in heart, liver, and skeletal muscle [9,10]. In this report, we have studied the association between glucose metabolism and 3 polymorphisms in the PRKAG2 gene: the 26C/T and two newly identified variations the 90G/T and p.Ser20Ile. Our results suggest that all these polymorphisms are likely not associated with the susceptibility to T2D in the studied sample. This is not consistent with previous studies [5]. The newly identified polymorphism in the 50 -UTR of PRKAG2 gene, 90G/T disrupted a binding site of a transcription factor YY1 that has been shown to regulate a variety of promoters [11] in relation to glucose metabolism. It is known that YY1 in the liver may modulate expression of a number of genes in relation to glucose metabolism [12]. Preliminary investigation showed no association between the 90G/T and the diabetics patients.
[1] G.A. Rutter, G. Da Silva Xavier, I. Leclerc, Roles of 50 -AMPactivated protein kinase (AMPK) in mammalian glucose homoeostasis, Biochem. J. 375 (2003) 1–16. [2] R. Bergeron, R.R. Russell 3rd, L.H. Young, J.M. Ren, M. Marcucci, A. Lee, et al., Effect of AMPK activation on muscle glucose metabolism in conscious rats, Am. J. Physiol. 276 (1999) E938–944. [3] N.B. Ruderman, J.M. Cacicedo, S. Itani, N. Yagihashi, A.K. Saha, J.M. Ye, et al., Malonyl-CoA and AMP-activated protein kinase (AMPK): possible links between insulin resistance in muscle and early endothelial cell damage in diabetes, Biochem. Soc. Trans. 31 (2003) 202–206. [4] W.W. Winder, Energy-sensing and signaling by AMPactivated protein kinase in skeletal muscle, J. Appl. Physiol. 91 (2001) 1017–1028. [5] M. Xu, X. Li, J.G. Wang, P. Du, J. Hong, W. Gu, et al., Glucose and lipid metabolism in relation to novel polymorphisms in the 50 -AMP-activated protein kinase gamma2 gene in Chinese, Mol. Genet. Metab. 86 (2005) 372–378. [6] P.C. Ng, S. Henikoff, Predicting deleterious amino acid substitutions, Genome Res. 11 (2001) 863–874. [7] C. Ferrer-Costa, M. Orozco, X. de la Cruz, Use of bioinformatics tools for the annotation of disease-
e10
diabetes research and clinical practice 87 (2010) e7–e10
associated mutations in animal models, Proteins 61 (2005) 878–887. [8] D. Carling, AMPK, Curr. Biol. 14 (2004) R220. [9] M.H. Gollob, M.S. Green, A.S. Tang, T. Gollob, A. Karibe, A.S. Ali Hassan, et al., Identification of a gene responsible for familial Wolff–Parkinson–White syndrome, N. Engl. J. Med. 344 (2001) 1823–1831. [10] M. Arad, B.J. Maron, J.M. Gorham, W.H. Johnson Jr., J.P. Saul, A.R. Perez-Atayde, et al., Glycogen storage diseases presenting as hypertrophic cardiomyopathy, N. Engl. J. Med. 352 (2005) 362–372.
[11] M.J. Thomas, E. Seto, Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key? Gene 236 (1999) 197–208. [12] C.Q. He, N.Z. Ding, W. Fan, YY1 repressing peroxisome proliferator-activated receptor delta promoter, Mol. Cell Biochem. 308 (2008) 247–252. [13] M. Arad, D.W. Benson, A.R. Perez-Atayde, W.J. McKenna, E.A. Sparks, R.J. Kanter, et al., Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy, J. Clin. Invest. 109 (2002) 357–362.