AGE (2016) 38:7 DOI 10.1007/s11357-015-9869-7
G-395A polymorphism in the promoter region of the KLOTHO gene associates with reduced cognitive impairment among the oldest old Qiukui Hao & Xiang Ding & Langli Gao & Ming Yang & Birong Dong
Received: 23 July 2015 / Accepted: 22 December 2015 # American Aging Association 2016
Abstract This study aimed to examine the possible association between G-395A polymorphism in the promoter region of the KLOTHO gene and cognitive impairment among Chinese nonagenarians and centenarians. This study is a secondary analysis of the Project of Longevity and Aging in Dujiangyan (PLAD) study. Community-dwelling Chinese people aged 90 years or older were included. G-395A (rs1207568) genotyping in the promoter region of the KLOTHO gene was performed using the TaqMan allelic discrimination assay. Cognitive function was assessed with the mini-mental status examination (MMSE). A total of 706 participants (68.0 % female; mean age 93.5 ± 3.6 years) were included. The KLOTHO G-395A polymorphism genotype frequencies for the whole sample were 2.0 % AA, 30.3 % GA, and 67.7 % GG. The GG genotype frequencies for the cognitive impairment and control groups were 70.2 and 62.7 %, respectively. Cognitive impairment prevalence was significantly lower in the GA+AA group than in the GG genotype group (61.4 vs. 69.0 %, p = 0.044). GA+AA genotype subjects had a significantly lower risk of cognitive impairment (odds ratio 0.66; 95 % confidence interval 0.44 to 0.98) than GG genotype individuals after adjusting for age, gender, and other relevant risk factors. KLOTHO G-395A polymorphism associates with reduced cognitive impairment in a sample of Chinese nonagenarians and centenarians. Q. Hao : X. Ding : L. Gao : M. Yang (*) : B. Dong West China Hospital, Sichuan University, Chengdu, Sichuan, China e-mail: [email protected]
Keywords KLOTHO gene . Cognitive impairment . Single nucleotide polymorphism Introduction Aging, which is defined as the age-related decline in physiological functions (Wang and Sun 2009), is inevitable in multicellular organisms. Genetic influences are critical factors in the aging process. KLOTHO is a new anti-aging gene discovered by Kuro-o and colleagues in 1997 (Kuro-o et al. 1997). KLOTHO-deficient mutant mice have shortened life spans and exhibit many phenotypes resembling those found in human aging (e.g., arteriosclerosis, osteoporosis, and hypoactivity) (Kuro-o et al. 1997). Cognitive deterioration is one of the most common consequences of aging in animals and humans (Deary et al. 2005). The prevalence of cognitive impairment increases with advancing age and difficulties in daily living activities and reduces in the quality of life of elderly adults (Ward et al. 2012). The relationship between the KLOTHO gene and cognitive impairment is an interesting line of research. A previous study found that KLOTHO-deficient mice exhibited memory impairment and hippocampal damage earlier than wildtype mice (Nagai et al. 2003). Recent studies showed that the KLOTHO gene, KL-VS, is associated with improved cognition, and increases in klotho expression can decrease premature mortality and network dysfunction (key aspects of Alzheimer’s disease) in human amyloid precursor protein transgenic mice (Dubal et al. 2014, 2015). The human KLOTHO protein shares
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86 % identity with the mouse protein (Nabeshima 2002). The serum level KLOTHO protein also declines with age in humans (Xiao et al. 2004). Dubal and colleagues also found that KLOTHO KL-VS heterozygosity was associated with better executive function and a greater right dorsolateral prefrontal cortex volume in mice (Yokoyama et al. 2015). Based on these findings, we hypothesize that the KLOTHO gene is significantly correlated with cognitive impairment in humans. The KLOTHO gene is located on chromosome 13q12. More than ten mutations or single nucleotide polymorphisms (SNPs) have been found in the human KLOTHO gene, and some of these SNPs are associated with aging-related disease (Wang and Sun 2009). For example, G-395A (rs1207568) is one SNP of the KLOTHO gene located in the promoter region. GG represents the wild-type genotype, whereas GA+AA represents the mutant (A is the mutant allele) for the G-395A polymorphism (Wang et al. 2010). G-395A polymorphism has been reported to be associated with age-related diseases. For example, the A allele increased the risk of essential hypertension (Wang et al. 2010) and cardioembolic stroke (Kim et al. 2006), whereas the G allele increased the risk of osteoarthritis (Tsezou et al. 2008; Zhang et al. 2007). The associations of the KLOTHO gene allele with bone mineral density (Kawano et al. 2002; Yamada et al. 2005), coronary artery disease (Imamura et al. 2006; Rhee et al. 2006), and cognitive impairment (Shimokata et al. 2006) remain controversial. However, the relationship between G-395A polymorphism and cognitive impairment has only been studied in humans aged 60–79 years old. Furthermore, cognitive function differences assessed using the mini-mental status examination (MMSE) have not been significant (Shimokata et al. 2006). Therefore, the relationship between G-395A polymorphism and cognitive impairment remains unclear in very old populations (80+ years old). In 2005, our team performed a cross-sectional study of 870 adults aged 90 years and older. Most participants had lived in their homelands for their entire lifetime and had never been exposed to immigrants (Yue et al. 2010; Zhou et al. 2012). Thus, the participants in this study were stable and represented the Chinese population well. The prevalence of cognitive impairment in this population is approximately 66 %. This high rate allowed us to examine the relationship between the KLOTHO G-395A SNP and cognitive impairment. Thus, we detected this relationship in this specific cohort.
Material and methods Study population Data were collected from the Project of Longevity and Aging in Dujiangyan (PLAD), a cross-sectional study conducted in the small southwestern Chinese town of Dujiangyan in April 2005. The methods of the PLAD study have been previously reported (Yue et al. 2010; Zhou et al. 2012). Briefly, a total of 1115 community members aged 90 years and older were screened via face-to-face interviews. Of these community members, 870 agreed to participate in the study. Trained medical staff then collected anthropometric measurements and blood samples from all participants in their homes or community centers. Written informed consent was obtained from all subjects or their legal proxies. The Research Ethics Committee of Sichuan University approved the study protocol. Subjects without blood samples, MMSE scores (129 cases), or the results of relevant clinical covariates (35 cases) were excluded from the current analyses, which resulted in a study sample of 706 participants (226 males, 480 females). KLOTHO genotype analysis Genomic DNA was isolated from whole blood samples using commercial DNA isolation kits from QIAGEN (Chatsworth, CA, USA) based on standard procedures. The KLOTHO promoter region G-395A (rs1207568) was genotyped using the TaqMan allelic discrimination assay, as previously described by Wang and colleagues (Wang et al. 2010). The following primers and probes (Takara, Dalian, China) were used: & & & &
Forward primer 5′-TAGGGCCCGGCAGGAT-3′ Reverse primer 5′-CCTGGAGCGGCTTCGTC-3′ Probe A 5′-(FAM) CCCCAAGTCGGGAAA AGTTGGTC (TAMRA)-3′ Probe G 5′-(HEX) CCCCCAAGTCGGGGA AAGTTGGTC (TAMRA)-3′
The PCR reaction was performed in 20 μl reaction volumes containing 10 μl of Premix Ex Taq, 1.5 μl of each primer, 0.5 μl of probe A, 1 μl of probe G, 1 μl of genomic DNA, and 4.25 μl of sterile, double-distilled water. The reaction conditions were an initial denaturation step at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. This
AGE (2016) 38:7
reaction was performed and analyzed on a Thermal Cycler Dice Real Time System (Takara, Dalian, China). We also randomly selected 10 % of the samples for forward and reverse sequencing to confirm promoter region KLOTHO G-395A genotype. The results were identical to those of the TaqMan allelic discrimination assay. Evaluation of cognitive function Cognitive function was assessed with the 30-item MMSE. This widely used test evaluates orientation, attention, calculation, language, and recall. Most items of the MMSE rely on visual and auditory abilities (Holtsberg et al. 1995), which may be a challenge in very old populations. Specifically, 100 participants (28 men and 72 women) in our very old populations did not complete the MMSE test due to visual or hearing impairment. To address this problem, we excluded these subjects when the data were analyzed. The cutoff point of MMSE is highly variable and ranges from 17/30 to 29/30 in Asian people (Rosli et al. 2015). Because the majority of participants were illiterate in our study, the subjects were categorized into two groups based on these scores: cognitive impairment (scores of 0–18) and free of cognitive impairment (scores of 19–30). This cutoff point has been shown to be 80 to 90 % sensitive and 80 to 100 % specific for cognitive impairment diagnosis in the Chinese population (Cui et al. 2011; Katzman et al. 1988; Tombaugh and McIntyre 1992; Zhu et al. 2006). MMSE administrators were trained by experienced geriatricians to ensure high-quality assessment and methodological reliability. The following contents were included: (1) reviewing MMSE procedures and grading system, as outlined in a short booklet and video; (2) observing tutors administering the MMSE to standardized patients; and (3) receiving supervision while administering the MMSE to standardized patients. Evaluation of potential confounders The following information were included: age, gender, educational levels (illiteracy, primary school, or secondary school and advanced), cigarette smoking status (smoking or not), alcohol drinking status (drinking or not), and exercise habits (yes or no). Height and weight were measured using a wall-mounted stadiometer and
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digital floor scale to the nearest 0.1 cm and 0.1 kg, respectively. The body mass index (BMI) was calculated by dividing weight in kilograms by the height in meters squared (kg/m2). The waist circumference (WC) was measured to the nearest 0.1 cm using a steel measuring tape. This measurement was performed in a standing position on naked skin at the end of light exhalation. Diabetes was diagnosed according to the American Diabetes Association criteria (Mellitus 2003) or if the subjects had diabetes history and were currently using antidiabetic agents. Hypertension was diagnosed according to the JNC VII criteria (Chobanian et al. 2003) and characterized by a systolic BP (SBP) of ≥140 mmHg and/or a diastolic BP (DBP) of ≥90 mmHg. Hypertension was also diagnosed if subjects had confirmed clinical diagnoses of hypertension according to formal medical records and were currently taking antihypertension medications. A history of stroke or coronary artery disease (CAD), which may be linked to cognitive impairment, was also included in the analyses. Laboratory analyses included the total cholesterol (TC), triglycerides (TG), highdensity lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and serum uric acid (SUA) levels. Statistical analysis All statistical analyses were performed using the SPSS for Windows software package, version 11.5 (SPSS Inc., Chicago, IL, USA). Two-tailed p values of
G-395A polymorphism in the promoter region of the KLOTHO gene associates with reduced cognitive impairment among the oldest old Qiukui Hao & Xiang Ding & Langli Gao & Ming Yang & Birong Dong
Received: 23 July 2015 / Accepted: 22 December 2015 # American Aging Association 2016
Abstract This study aimed to examine the possible association between G-395A polymorphism in the promoter region of the KLOTHO gene and cognitive impairment among Chinese nonagenarians and centenarians. This study is a secondary analysis of the Project of Longevity and Aging in Dujiangyan (PLAD) study. Community-dwelling Chinese people aged 90 years or older were included. G-395A (rs1207568) genotyping in the promoter region of the KLOTHO gene was performed using the TaqMan allelic discrimination assay. Cognitive function was assessed with the mini-mental status examination (MMSE). A total of 706 participants (68.0 % female; mean age 93.5 ± 3.6 years) were included. The KLOTHO G-395A polymorphism genotype frequencies for the whole sample were 2.0 % AA, 30.3 % GA, and 67.7 % GG. The GG genotype frequencies for the cognitive impairment and control groups were 70.2 and 62.7 %, respectively. Cognitive impairment prevalence was significantly lower in the GA+AA group than in the GG genotype group (61.4 vs. 69.0 %, p = 0.044). GA+AA genotype subjects had a significantly lower risk of cognitive impairment (odds ratio 0.66; 95 % confidence interval 0.44 to 0.98) than GG genotype individuals after adjusting for age, gender, and other relevant risk factors. KLOTHO G-395A polymorphism associates with reduced cognitive impairment in a sample of Chinese nonagenarians and centenarians. Q. Hao : X. Ding : L. Gao : M. Yang (*) : B. Dong West China Hospital, Sichuan University, Chengdu, Sichuan, China e-mail: [email protected]
Keywords KLOTHO gene . Cognitive impairment . Single nucleotide polymorphism Introduction Aging, which is defined as the age-related decline in physiological functions (Wang and Sun 2009), is inevitable in multicellular organisms. Genetic influences are critical factors in the aging process. KLOTHO is a new anti-aging gene discovered by Kuro-o and colleagues in 1997 (Kuro-o et al. 1997). KLOTHO-deficient mutant mice have shortened life spans and exhibit many phenotypes resembling those found in human aging (e.g., arteriosclerosis, osteoporosis, and hypoactivity) (Kuro-o et al. 1997). Cognitive deterioration is one of the most common consequences of aging in animals and humans (Deary et al. 2005). The prevalence of cognitive impairment increases with advancing age and difficulties in daily living activities and reduces in the quality of life of elderly adults (Ward et al. 2012). The relationship between the KLOTHO gene and cognitive impairment is an interesting line of research. A previous study found that KLOTHO-deficient mice exhibited memory impairment and hippocampal damage earlier than wildtype mice (Nagai et al. 2003). Recent studies showed that the KLOTHO gene, KL-VS, is associated with improved cognition, and increases in klotho expression can decrease premature mortality and network dysfunction (key aspects of Alzheimer’s disease) in human amyloid precursor protein transgenic mice (Dubal et al. 2014, 2015). The human KLOTHO protein shares
7
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Page 2 of 8
86 % identity with the mouse protein (Nabeshima 2002). The serum level KLOTHO protein also declines with age in humans (Xiao et al. 2004). Dubal and colleagues also found that KLOTHO KL-VS heterozygosity was associated with better executive function and a greater right dorsolateral prefrontal cortex volume in mice (Yokoyama et al. 2015). Based on these findings, we hypothesize that the KLOTHO gene is significantly correlated with cognitive impairment in humans. The KLOTHO gene is located on chromosome 13q12. More than ten mutations or single nucleotide polymorphisms (SNPs) have been found in the human KLOTHO gene, and some of these SNPs are associated with aging-related disease (Wang and Sun 2009). For example, G-395A (rs1207568) is one SNP of the KLOTHO gene located in the promoter region. GG represents the wild-type genotype, whereas GA+AA represents the mutant (A is the mutant allele) for the G-395A polymorphism (Wang et al. 2010). G-395A polymorphism has been reported to be associated with age-related diseases. For example, the A allele increased the risk of essential hypertension (Wang et al. 2010) and cardioembolic stroke (Kim et al. 2006), whereas the G allele increased the risk of osteoarthritis (Tsezou et al. 2008; Zhang et al. 2007). The associations of the KLOTHO gene allele with bone mineral density (Kawano et al. 2002; Yamada et al. 2005), coronary artery disease (Imamura et al. 2006; Rhee et al. 2006), and cognitive impairment (Shimokata et al. 2006) remain controversial. However, the relationship between G-395A polymorphism and cognitive impairment has only been studied in humans aged 60–79 years old. Furthermore, cognitive function differences assessed using the mini-mental status examination (MMSE) have not been significant (Shimokata et al. 2006). Therefore, the relationship between G-395A polymorphism and cognitive impairment remains unclear in very old populations (80+ years old). In 2005, our team performed a cross-sectional study of 870 adults aged 90 years and older. Most participants had lived in their homelands for their entire lifetime and had never been exposed to immigrants (Yue et al. 2010; Zhou et al. 2012). Thus, the participants in this study were stable and represented the Chinese population well. The prevalence of cognitive impairment in this population is approximately 66 %. This high rate allowed us to examine the relationship between the KLOTHO G-395A SNP and cognitive impairment. Thus, we detected this relationship in this specific cohort.
Material and methods Study population Data were collected from the Project of Longevity and Aging in Dujiangyan (PLAD), a cross-sectional study conducted in the small southwestern Chinese town of Dujiangyan in April 2005. The methods of the PLAD study have been previously reported (Yue et al. 2010; Zhou et al. 2012). Briefly, a total of 1115 community members aged 90 years and older were screened via face-to-face interviews. Of these community members, 870 agreed to participate in the study. Trained medical staff then collected anthropometric measurements and blood samples from all participants in their homes or community centers. Written informed consent was obtained from all subjects or their legal proxies. The Research Ethics Committee of Sichuan University approved the study protocol. Subjects without blood samples, MMSE scores (129 cases), or the results of relevant clinical covariates (35 cases) were excluded from the current analyses, which resulted in a study sample of 706 participants (226 males, 480 females). KLOTHO genotype analysis Genomic DNA was isolated from whole blood samples using commercial DNA isolation kits from QIAGEN (Chatsworth, CA, USA) based on standard procedures. The KLOTHO promoter region G-395A (rs1207568) was genotyped using the TaqMan allelic discrimination assay, as previously described by Wang and colleagues (Wang et al. 2010). The following primers and probes (Takara, Dalian, China) were used: & & & &
Forward primer 5′-TAGGGCCCGGCAGGAT-3′ Reverse primer 5′-CCTGGAGCGGCTTCGTC-3′ Probe A 5′-(FAM) CCCCAAGTCGGGAAA AGTTGGTC (TAMRA)-3′ Probe G 5′-(HEX) CCCCCAAGTCGGGGA AAGTTGGTC (TAMRA)-3′
The PCR reaction was performed in 20 μl reaction volumes containing 10 μl of Premix Ex Taq, 1.5 μl of each primer, 0.5 μl of probe A, 1 μl of probe G, 1 μl of genomic DNA, and 4.25 μl of sterile, double-distilled water. The reaction conditions were an initial denaturation step at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. This
AGE (2016) 38:7
reaction was performed and analyzed on a Thermal Cycler Dice Real Time System (Takara, Dalian, China). We also randomly selected 10 % of the samples for forward and reverse sequencing to confirm promoter region KLOTHO G-395A genotype. The results were identical to those of the TaqMan allelic discrimination assay. Evaluation of cognitive function Cognitive function was assessed with the 30-item MMSE. This widely used test evaluates orientation, attention, calculation, language, and recall. Most items of the MMSE rely on visual and auditory abilities (Holtsberg et al. 1995), which may be a challenge in very old populations. Specifically, 100 participants (28 men and 72 women) in our very old populations did not complete the MMSE test due to visual or hearing impairment. To address this problem, we excluded these subjects when the data were analyzed. The cutoff point of MMSE is highly variable and ranges from 17/30 to 29/30 in Asian people (Rosli et al. 2015). Because the majority of participants were illiterate in our study, the subjects were categorized into two groups based on these scores: cognitive impairment (scores of 0–18) and free of cognitive impairment (scores of 19–30). This cutoff point has been shown to be 80 to 90 % sensitive and 80 to 100 % specific for cognitive impairment diagnosis in the Chinese population (Cui et al. 2011; Katzman et al. 1988; Tombaugh and McIntyre 1992; Zhu et al. 2006). MMSE administrators were trained by experienced geriatricians to ensure high-quality assessment and methodological reliability. The following contents were included: (1) reviewing MMSE procedures and grading system, as outlined in a short booklet and video; (2) observing tutors administering the MMSE to standardized patients; and (3) receiving supervision while administering the MMSE to standardized patients. Evaluation of potential confounders The following information were included: age, gender, educational levels (illiteracy, primary school, or secondary school and advanced), cigarette smoking status (smoking or not), alcohol drinking status (drinking or not), and exercise habits (yes or no). Height and weight were measured using a wall-mounted stadiometer and
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digital floor scale to the nearest 0.1 cm and 0.1 kg, respectively. The body mass index (BMI) was calculated by dividing weight in kilograms by the height in meters squared (kg/m2). The waist circumference (WC) was measured to the nearest 0.1 cm using a steel measuring tape. This measurement was performed in a standing position on naked skin at the end of light exhalation. Diabetes was diagnosed according to the American Diabetes Association criteria (Mellitus 2003) or if the subjects had diabetes history and were currently using antidiabetic agents. Hypertension was diagnosed according to the JNC VII criteria (Chobanian et al. 2003) and characterized by a systolic BP (SBP) of ≥140 mmHg and/or a diastolic BP (DBP) of ≥90 mmHg. Hypertension was also diagnosed if subjects had confirmed clinical diagnoses of hypertension according to formal medical records and were currently taking antihypertension medications. A history of stroke or coronary artery disease (CAD), which may be linked to cognitive impairment, was also included in the analyses. Laboratory analyses included the total cholesterol (TC), triglycerides (TG), highdensity lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and serum uric acid (SUA) levels. Statistical analysis All statistical analyses were performed using the SPSS for Windows software package, version 11.5 (SPSS Inc., Chicago, IL, USA). Two-tailed p values of
