Chin J Integr Med
•1•
ORIGINAL ARTICLE Association between Genetic Variants and Characteristic Symptoms of Type 2 Diabetes: A Matched Case-Control Study DOU Hao-ying (窦昊颖)1, WANG Yuan-yuan (王媛媛)1, YANG Nan (杨 楠)1, HENG Ming-li (衡明莉)2, ZHOU Xuan (周 萱)2, BU Huai-en (步怀恩)2, XU Fang (徐 芳)2, ZHAO Tie-niu (赵铁牛)2, HUANG He (黄 鹤)3, and WANG Hong-wu (王泓午)2 Objective: To examine the association of genetic variants with characteristic symptoms of type 2 ABSTRACT Objective: Methods:: A matched case-control study was performed to investigate the association diabetes mellitus (T2DM). Methods between common variants in four genes (CDKAL1, GLIS3, GRK5, and TCF7L2) and symptoms of T2DM. Symptoms were examined with questionnaire for 710 subjects. Genomic DNA was extracted from peripheral blood mononuclear cell by salting-out procedure. Genotyping was carried out by direct sequencing of the unpurified Result:: Most of the T2DM patients pressented characteristic symptoms, polymerase chain reaction products. Result such as feeling weak in limbs (P =0.0057), hand tremor (P =0.0208), bradymasesis (P =0.0234), and polyuria (P =0.0051). Some of the T2DM patients shared characteristic symptoms, such as desire for cold drinks (P =0.0304), polyphagia (P =0.0051), and furred tongue (P =0.028). The impaired glucose regulation (IGR) cases took only one characteristic symptom of frequent micturition (P =0.0422). GLIS3 rs7034200 and GRK5 rs10886471 were significantly associated with increased T2DM risk (GLIS3 rs7034200 under dominant model: P =0.0307; GRK5 rs10886471 under recessive model: P =0.0092). However, only the rs10886471 polymorphism in GRK5 showed a significant effect on both differentiated symptoms and T2DM risk. The C-allele was involved in both dampness-heat encumbering the Pi (Spleen) syndrome (P =0.047) and qi-yin deficiency syndrome (P =0.002) via increased GRK5 Conclusion:: Both T2DM and IGR exhibited its corresponding characteristic symptoms. The variants of expression. Conclusion GRK5 were involved with both qi-yin deficiency syndrome and dampness-heat encumbering Pi syndrome. KEYWORDS type 2 diabetes mellitus, impaired glucose regulation, Chinese medicine, syndromes, genetic variants
Type 2 diabetes mellitus (T2DM) has become a globally major health problem and is projected to affect 300 million people by the year 2025. (1) It is regarded as a metabolic disorder disease resulting from gene-environment interactions. Till now, genome-wide association studies (GWASs) have identified more than 40 genetic variants which are associated with T2DM.(2) Among these, CDKAL1 and TCF7L2, are highly replicated biological candidate genes. One of the two genes, the TCF7L2 gene is the most frequently replicated adult-onset T2DM susceptibility gene in Europeans, West Africans, and Chinese. (3,4) In this context, based on a metaanalysis, we recently have found that in Chinese Han population, the rs7903146C/T polymorphism of TCF7L2 gene is associated with T2DM risk, while the polymorphisms of rs12255372G/T and rs290487T/C are not.(5) The CDKAL1 gene has been reported to reduce glucose-induced first-phase insulin exocytosis by facilitating adenosine triphosphate (ATP) generation, glucose-induced ATP sensitive K+ hannels (KATP) responsiveness and subsequent Ca2+
channel activity,(6) hence conferring an increased risk for T2DM.(7,8) Some studies have confirmed that the CDKAL1 rs7754840 is one of the most commonly susceptibility genes in Asian populations, including Chinese, Japanese, and Korean.(9,10) Beside the above two genes, another two variants (GLIS3 and GRK5) have also been reported to be involved in T2DM by fine mapping and multiple GWASs. The GLIS3,
©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2015 Supported by the Project of Science and Technology Development of Tianjin Municipality, China (No. 033113211), the Key Cultivated Academic Construction Project of State Administrative Bureau for Prophylactic Medicine of Traditional Chinese Medicine, China (No. 2012) 1. Department of Nursing, Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 2. Department of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 3. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin (300072), China Correspondence to: Prof. WANG Hong-wu, Tel: 86-2259596240, E-mail: [email protected] DOI: 10.1007/s11655-015-2290-3
Chin J Integr Med
•2•
involved with pancreatic beta cell development and insulin gene expression, has been proved its association with fasting glucose levels.(11,12) Coding product of this gene, a Kruppel-like zinc finger transcription factor, was proposed to be a key player in the regulation of pancreatic beta cell development and insulin gene expression.(13) As a newly found susceptibility gene locus, the GLIS3 rs7034200 exhibited much more influence on T2DM in Chinese Hans than in Europeans, and its contribution to T2DM risk was mainly mediated through impaired beta cell function.(14) As for GRK5, recent studies demonstrated that its association with T2DM seems to be specific to East Asians.(15) The allele of rs10886471 was also associated with higher GRK5 mRNA expression levels, higher fasting insulin, but not with fasting glucose, suggesting that it might impair insulin sensitivity by increasing inflammatory response and consequently contributes to T2DM risk in Chinese Hans or East Asians.(15,16)
stagnation" were three major syndromes.
GWASs have confirmed more than 40 genetic variants associated with T2DM, (2) however, the pathogenic mechanisms of the most genes still remained open. Moreover, the GWASs have not shed light on the association between genetic variants and symptoms. As Chinese medicine (CM) is concerned, the doctors pay considerable attention to symptoms in contrast with gene variants. Over two thousand years, T2DM had a name for diabetes-related symptoms "Xiaoke", which meant losing weight and thirst.(17,18) By long time observation, doctors of CM considered that symptoms and signs were related with the diagnosis of T2DM just because human body and its functioning are holistic.(19) According to the symptoms and signs of T2DM, such as tiredness, desire for cold drinks, polyphagia (increased hunger), polyuria, emaciation, and color of urine, T2DM is classically divided into five syndromes. These syndromes include "yin deficiency heat exuberance syndrome", "dampness-heat encumbering Pi (Spleen) syndrome", "qi-yin deficiency syndrome", "yin-yang deficiency syndrome", and "blood stasis in the vessels and venation syndrome". This classification standard of T2DM is recommended by the guiding principle of new drug clinical research for CM.(20) According to this principle, each syndrome has a group of characteristic symptoms, and the "dampness-heat encumbering the Pi syndrome" is a basic type.(21) However, Tong, et al(22) indicated that "Gan (Liver) and Wei (Stomach) heat stagnation", "Wei and Chang (Intestine) excess heat", and "qi
Study design
Although there has been a great deal of dramatically controversy in the chief syndrome, only few studies concentrate on biological interpretation of characteristic signs and differentiating syndromes. To address these situations, we carefully evaluated the association between genetic variants and characteristic symptoms of T2DM by a matched casecontrol study. This study is based on the following two hypotheses. First, we hypothesize that T2DM should show characteristic symptoms since it is a metabolic disorder disease resulting from gene-environment interactions. Second, we suppose that differential expression of various molecules are involved with differential syndromes. The inherent gene pattern and metabolic profiles of exposure might work together to help doctors tailor therapies.
METHODS The 1:1 matched case-control study was performed to investigate the association between common variants in four genes and T2DM. That was to say, one case was paired up with one control subject on same gender, during the same period of survey, and in the same area. What's more, the age differences between the two groups were less than or equal to 3. Confounding factors were controlled effectively including age and gender in this method.
Study Participants The included individuals recruited from two villages which located in Jinghai country of Tianjin, China from 2009 to 2011. The sample sizes based on 1:1 matched case-control study were calculated as follows: P1=(OR×P0)/(1 + P0 + OR×P0) P=OR/(1 + OR) m= M=
[Zα/2+Zβ P(1–P)] (P–0.5)2 m P0(1–P1)+P1(1–P0)
In these formulae, α= 0.05 (bilateral), Power (1–β)=0.9. Then, it could be concluded that Z α = 1.960, Z β =1.282. P 0 and P 1 denoted the estimated exposure rates of T2DM in the control and case groups, respectively. OR represented the relative risk of T2DM which was caused by each
Chin J Integr Med
gene variants. All the values above could be acquired via consulting literature.(5,14,15,23) In addition, m and M stood for inconsistent and requisite pair numbers. All the participants were adults with at least 5-year, residence in Tianjin. The subjects, who were diagnosed as T2DM according to the American Diabetes Association (ADA) guidelines, were included in the study. (24) The definition was for a single raised glucose reading with symptoms, otherwise raised values on two occasions, of either: fasting plasma glucose (FPG) 7.0 mmol/L (126 mg/dL) or with a glucose tolerance test, 2 h after the oral dose a plasma glucose 11.1 mmol/L (199 mg/dL). Patients with impaired glucose regulation (IGR) were also eligible for inclusion: people whose FPG were between 6.1–6.9 mmol/L (110–125 mg/dL), or whose 2-h plasma glucose after a 75-g oral glucose challenge were 7.8–11.1 mmol/L (140–199 mg/dL). Among them, patients whose FPG were between 6.1–6.9 mmol/L (110–125 mg/dL), and 2-h plasma glucose after a 75-g oral glucose challenge were < 7.8 mmol/L (140 mg/dL) were considered as impaired fasting glucose (IFG). On the contrary, patients whose FPG were < 7.0 mmol/L (126 mg/dL), and 2-h plasma glucose after a 75-g oral glucose challenge were 7.8–11.1 mmol/L (140–199 mg/dL) were considered as impaired glucose tolerance (IGT). The inclusion criteria for control subjects were fasting glucose 5.6 mmol/L (100 mg/dL) and 2-h plasma glucose after a 75-g oral glucose challenge was T gene
Discrimination of type 2 diabetes mellitus corresponding
polymorphism in newly diagnosed type 2 diabetes patients.
to different traditional Chinese medicine syndromes based
J Tradit Chin Med (Chin) 2012;53:1397-1400.
on plasma fatty acid profiles and chemometric methods. J
22. Tong XL, Dong L, Chen L, Zhen Z. Treatment of diabetes using traditional Chinese medicine: past, present and future. Am J Chin Med 2012;40:877-886.
Ethnopharmacol 2012;143:463-468. 35. He YX, Li X, Wu YY, Wang YM, Luo GA. Gene expression of CDKM2A, CDKN2B, IGF2BP2 and CDKAL1 in diabetic
23. Dehwah MAS, Wang M, Huang QY. CDKAL1 and type
nephropathy patients with CM syndrome differentiations.
2 diabetes: a global meta-analysis. Genet Mol Res
World Sci Tech/Modern Tradit Chin Med Mater Med (Chin)
2010;9:1109-1120. 24. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2013;36:S67-S74. 25. Ji L, Li H, Guo X, Li Y, Hu R, Zhu Z. Impact of baseline BMI on glycemic control and weight change with metformin monotherapy in Chinese type 2 diabetes patients: phase Ⅳ
2009;11:356-360. 36. Guo NT, Zhu XH, Lin M. Relation between Chinese medicine syndrome and red blood cell gene expression in type 2 diabetes patients. J Chin Med (Chin) 2004;19:215-216. (Received September 28, 2014) Edited by YUAN Lin
•1•
ORIGINAL ARTICLE Association between Genetic Variants and Characteristic Symptoms of Type 2 Diabetes: A Matched Case-Control Study DOU Hao-ying (窦昊颖)1, WANG Yuan-yuan (王媛媛)1, YANG Nan (杨 楠)1, HENG Ming-li (衡明莉)2, ZHOU Xuan (周 萱)2, BU Huai-en (步怀恩)2, XU Fang (徐 芳)2, ZHAO Tie-niu (赵铁牛)2, HUANG He (黄 鹤)3, and WANG Hong-wu (王泓午)2 Objective: To examine the association of genetic variants with characteristic symptoms of type 2 ABSTRACT Objective: Methods:: A matched case-control study was performed to investigate the association diabetes mellitus (T2DM). Methods between common variants in four genes (CDKAL1, GLIS3, GRK5, and TCF7L2) and symptoms of T2DM. Symptoms were examined with questionnaire for 710 subjects. Genomic DNA was extracted from peripheral blood mononuclear cell by salting-out procedure. Genotyping was carried out by direct sequencing of the unpurified Result:: Most of the T2DM patients pressented characteristic symptoms, polymerase chain reaction products. Result such as feeling weak in limbs (P =0.0057), hand tremor (P =0.0208), bradymasesis (P =0.0234), and polyuria (P =0.0051). Some of the T2DM patients shared characteristic symptoms, such as desire for cold drinks (P =0.0304), polyphagia (P =0.0051), and furred tongue (P =0.028). The impaired glucose regulation (IGR) cases took only one characteristic symptom of frequent micturition (P =0.0422). GLIS3 rs7034200 and GRK5 rs10886471 were significantly associated with increased T2DM risk (GLIS3 rs7034200 under dominant model: P =0.0307; GRK5 rs10886471 under recessive model: P =0.0092). However, only the rs10886471 polymorphism in GRK5 showed a significant effect on both differentiated symptoms and T2DM risk. The C-allele was involved in both dampness-heat encumbering the Pi (Spleen) syndrome (P =0.047) and qi-yin deficiency syndrome (P =0.002) via increased GRK5 Conclusion:: Both T2DM and IGR exhibited its corresponding characteristic symptoms. The variants of expression. Conclusion GRK5 were involved with both qi-yin deficiency syndrome and dampness-heat encumbering Pi syndrome. KEYWORDS type 2 diabetes mellitus, impaired glucose regulation, Chinese medicine, syndromes, genetic variants
Type 2 diabetes mellitus (T2DM) has become a globally major health problem and is projected to affect 300 million people by the year 2025. (1) It is regarded as a metabolic disorder disease resulting from gene-environment interactions. Till now, genome-wide association studies (GWASs) have identified more than 40 genetic variants which are associated with T2DM.(2) Among these, CDKAL1 and TCF7L2, are highly replicated biological candidate genes. One of the two genes, the TCF7L2 gene is the most frequently replicated adult-onset T2DM susceptibility gene in Europeans, West Africans, and Chinese. (3,4) In this context, based on a metaanalysis, we recently have found that in Chinese Han population, the rs7903146C/T polymorphism of TCF7L2 gene is associated with T2DM risk, while the polymorphisms of rs12255372G/T and rs290487T/C are not.(5) The CDKAL1 gene has been reported to reduce glucose-induced first-phase insulin exocytosis by facilitating adenosine triphosphate (ATP) generation, glucose-induced ATP sensitive K+ hannels (KATP) responsiveness and subsequent Ca2+
channel activity,(6) hence conferring an increased risk for T2DM.(7,8) Some studies have confirmed that the CDKAL1 rs7754840 is one of the most commonly susceptibility genes in Asian populations, including Chinese, Japanese, and Korean.(9,10) Beside the above two genes, another two variants (GLIS3 and GRK5) have also been reported to be involved in T2DM by fine mapping and multiple GWASs. The GLIS3,
©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2015 Supported by the Project of Science and Technology Development of Tianjin Municipality, China (No. 033113211), the Key Cultivated Academic Construction Project of State Administrative Bureau for Prophylactic Medicine of Traditional Chinese Medicine, China (No. 2012) 1. Department of Nursing, Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 2. Department of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 3. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin (300072), China Correspondence to: Prof. WANG Hong-wu, Tel: 86-2259596240, E-mail: [email protected] DOI: 10.1007/s11655-015-2290-3
Chin J Integr Med
•2•
involved with pancreatic beta cell development and insulin gene expression, has been proved its association with fasting glucose levels.(11,12) Coding product of this gene, a Kruppel-like zinc finger transcription factor, was proposed to be a key player in the regulation of pancreatic beta cell development and insulin gene expression.(13) As a newly found susceptibility gene locus, the GLIS3 rs7034200 exhibited much more influence on T2DM in Chinese Hans than in Europeans, and its contribution to T2DM risk was mainly mediated through impaired beta cell function.(14) As for GRK5, recent studies demonstrated that its association with T2DM seems to be specific to East Asians.(15) The allele of rs10886471 was also associated with higher GRK5 mRNA expression levels, higher fasting insulin, but not with fasting glucose, suggesting that it might impair insulin sensitivity by increasing inflammatory response and consequently contributes to T2DM risk in Chinese Hans or East Asians.(15,16)
stagnation" were three major syndromes.
GWASs have confirmed more than 40 genetic variants associated with T2DM, (2) however, the pathogenic mechanisms of the most genes still remained open. Moreover, the GWASs have not shed light on the association between genetic variants and symptoms. As Chinese medicine (CM) is concerned, the doctors pay considerable attention to symptoms in contrast with gene variants. Over two thousand years, T2DM had a name for diabetes-related symptoms "Xiaoke", which meant losing weight and thirst.(17,18) By long time observation, doctors of CM considered that symptoms and signs were related with the diagnosis of T2DM just because human body and its functioning are holistic.(19) According to the symptoms and signs of T2DM, such as tiredness, desire for cold drinks, polyphagia (increased hunger), polyuria, emaciation, and color of urine, T2DM is classically divided into five syndromes. These syndromes include "yin deficiency heat exuberance syndrome", "dampness-heat encumbering Pi (Spleen) syndrome", "qi-yin deficiency syndrome", "yin-yang deficiency syndrome", and "blood stasis in the vessels and venation syndrome". This classification standard of T2DM is recommended by the guiding principle of new drug clinical research for CM.(20) According to this principle, each syndrome has a group of characteristic symptoms, and the "dampness-heat encumbering the Pi syndrome" is a basic type.(21) However, Tong, et al(22) indicated that "Gan (Liver) and Wei (Stomach) heat stagnation", "Wei and Chang (Intestine) excess heat", and "qi
Study design
Although there has been a great deal of dramatically controversy in the chief syndrome, only few studies concentrate on biological interpretation of characteristic signs and differentiating syndromes. To address these situations, we carefully evaluated the association between genetic variants and characteristic symptoms of T2DM by a matched casecontrol study. This study is based on the following two hypotheses. First, we hypothesize that T2DM should show characteristic symptoms since it is a metabolic disorder disease resulting from gene-environment interactions. Second, we suppose that differential expression of various molecules are involved with differential syndromes. The inherent gene pattern and metabolic profiles of exposure might work together to help doctors tailor therapies.
METHODS The 1:1 matched case-control study was performed to investigate the association between common variants in four genes and T2DM. That was to say, one case was paired up with one control subject on same gender, during the same period of survey, and in the same area. What's more, the age differences between the two groups were less than or equal to 3. Confounding factors were controlled effectively including age and gender in this method.
Study Participants The included individuals recruited from two villages which located in Jinghai country of Tianjin, China from 2009 to 2011. The sample sizes based on 1:1 matched case-control study were calculated as follows: P1=(OR×P0)/(1 + P0 + OR×P0) P=OR/(1 + OR) m= M=
[Zα/2+Zβ P(1–P)] (P–0.5)2 m P0(1–P1)+P1(1–P0)
In these formulae, α= 0.05 (bilateral), Power (1–β)=0.9. Then, it could be concluded that Z α = 1.960, Z β =1.282. P 0 and P 1 denoted the estimated exposure rates of T2DM in the control and case groups, respectively. OR represented the relative risk of T2DM which was caused by each
Chin J Integr Med
gene variants. All the values above could be acquired via consulting literature.(5,14,15,23) In addition, m and M stood for inconsistent and requisite pair numbers. All the participants were adults with at least 5-year, residence in Tianjin. The subjects, who were diagnosed as T2DM according to the American Diabetes Association (ADA) guidelines, were included in the study. (24) The definition was for a single raised glucose reading with symptoms, otherwise raised values on two occasions, of either: fasting plasma glucose (FPG) 7.0 mmol/L (126 mg/dL) or with a glucose tolerance test, 2 h after the oral dose a plasma glucose 11.1 mmol/L (199 mg/dL). Patients with impaired glucose regulation (IGR) were also eligible for inclusion: people whose FPG were between 6.1–6.9 mmol/L (110–125 mg/dL), or whose 2-h plasma glucose after a 75-g oral glucose challenge were 7.8–11.1 mmol/L (140–199 mg/dL). Among them, patients whose FPG were between 6.1–6.9 mmol/L (110–125 mg/dL), and 2-h plasma glucose after a 75-g oral glucose challenge were < 7.8 mmol/L (140 mg/dL) were considered as impaired fasting glucose (IFG). On the contrary, patients whose FPG were < 7.0 mmol/L (126 mg/dL), and 2-h plasma glucose after a 75-g oral glucose challenge were 7.8–11.1 mmol/L (140–199 mg/dL) were considered as impaired glucose tolerance (IGT). The inclusion criteria for control subjects were fasting glucose 5.6 mmol/L (100 mg/dL) and 2-h plasma glucose after a 75-g oral glucose challenge was T gene
Discrimination of type 2 diabetes mellitus corresponding
polymorphism in newly diagnosed type 2 diabetes patients.
to different traditional Chinese medicine syndromes based
J Tradit Chin Med (Chin) 2012;53:1397-1400.
on plasma fatty acid profiles and chemometric methods. J
22. Tong XL, Dong L, Chen L, Zhen Z. Treatment of diabetes using traditional Chinese medicine: past, present and future. Am J Chin Med 2012;40:877-886.
Ethnopharmacol 2012;143:463-468. 35. He YX, Li X, Wu YY, Wang YM, Luo GA. Gene expression of CDKM2A, CDKN2B, IGF2BP2 and CDKAL1 in diabetic
23. Dehwah MAS, Wang M, Huang QY. CDKAL1 and type
nephropathy patients with CM syndrome differentiations.
2 diabetes: a global meta-analysis. Genet Mol Res
World Sci Tech/Modern Tradit Chin Med Mater Med (Chin)
2010;9:1109-1120. 24. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2013;36:S67-S74. 25. Ji L, Li H, Guo X, Li Y, Hu R, Zhu Z. Impact of baseline BMI on glycemic control and weight change with metformin monotherapy in Chinese type 2 diabetes patients: phase Ⅳ
2009;11:356-360. 36. Guo NT, Zhu XH, Lin M. Relation between Chinese medicine syndrome and red blood cell gene expression in type 2 diabetes patients. J Chin Med (Chin) 2004;19:215-216. (Received September 28, 2014) Edited by YUAN Lin
