GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 19, Number 8, 2015 ª Mary Ann Liebert, Inc. Pp. 1–4 DOI: 10.1089/gtmb.2015.0033
SHORT REPORT
Myostatin Gene Polymorphism in an Elderly Sarcopenic Turkish Population Pinar Tosun Tasar,1 Sevnaz Sahin,1 Emine Karaman,2 Atilla Oz,3 Merve Gulsah Ulusoy,4 Soner Duman,5 Afig Berdeli,3 and Fehmi Akcicek1
Introduction: One of the genetic contributors to sarcopenia predisposition is Myostatin (MSTN), which in humans encodes myostatin, a 376 amino acid growth factor protein that negatively regulates muscle growth. The aim of this study was to investigate MSTN polymorphisms in an elderly sarcopenic population in Turkey and determine how they relate to sarcopenia. Materials and Methods: The study included nursing home residents who were aged ‡65 years. Sarcopenia screening was performed using ‘‘The European Working Group on Sarcopenia in Older People’’ guidelines. Blood sample was taken from each participant and DNA was obtained from the peripheral blood. MSTN polymorphisms were genotyped by polymerase chain reaction and restriction fragment length polymorphism methods. Results: A total of 152 elderly patients were included in the study. The rate of sarcopenia was determined to be 41.4%. The DNA nucleotide sequence of all three MSTN exons was determined for each study participant. Among the 152 patients, only 6 (3.9%) showed an MSTN K153R heterozygous mutation. Among these, three participants were sarcopenic and three were nonsarcopenic. No statistically significant difference in the polymorphism frequency between the sarcopenic and control groups was observed ( p = 0.664). Conclusions: MSTN genotyping revealed that only 3.9% (6/152) of participants had the MSTN K153R heterozygous mutation. Despite the detection of this mutation in the study group, no relationship was found between this mutation and sarcopenia.
age groups: young-old (65–74), middle-old (75–84), and oldold (‡85). The sarcopenia screening was conducted as specified in the 2010 report by the European Union Geriatric Association, ‘‘The European Working Group on Sarcopenia in Older People.’’ The criteria for sarcopenia diagnosis include a decrease in muscle mass and decline in muscle strength and/or physical performance (Cruz-Jentoft et al., 2010). In this study, patients’ physical performance was determined based on the 6-m walking speed and muscle strength as measured with a handgrip device (Takei physical fitness test). The fat-free mass (FFM) was measured using a bioimpedence analysis device (Body Composition Analyzer SC-330). A walking speed of more than 0.8 m/s and muscle strength of 20 kg for women and 30 kg for men were considered normal. The FFM of the study group was compared with healthy community-dwelling young adults aged 18–45 years with no illnesses and not taking medications, who had consented to participate in the study. The body surface area (BSA) was calculated using the DuBois formula (BSA = kg0.425 · m0.725 · 0.007184). The FFM was divided by the BSA to determine the muscle mass. Values below the
Introduction
S
arcopenia is an age-dependent loss of muscle mass and function (Rosenberg, 1997). Currently, there is no consensus for the definitive diagnosis of this disorder, but diagnosis criteria for sarcopenia can include a decrease in muscle mass and a decline in muscle strength and/or physical performance (Cruz-Jentoft et al., 2010; Cesari et al., 2012). Myostatin (MSTN) is one of the genes that can contribute to a genetic predisposition to develop sarcopenia (Huygens et al., 2004). In humans, MSTN encodes the growth factor myostatin, which negatively regulates muscle growth (McPherron et al., 1997). To date, no research has been conducted on the effect of MSTN polymorphisms on sarcopenia in Turkey. The purpose of this study was to determine if a relationship exists between MSTN polymorphism and sarcopenia in an elderly population in Turkey. Materials and Methods
The study group comprised 152 elderly nursing home residents aged ‡65 years. The patients were divided into three 1
Division of Geriatric Medicine, Department of Internal Medicine, Faculty of Medicine, Ege University, Izmir, Turkey. Department of Internal Medicine, Faculty of Nursing, Ege University, Izmir, Turkey. Division of Molecular Medicine Laboratory, Department of Pediatrics, Faculty of Medicine, Ege University, Izmir, Turkey. Departments of 4Biostatistics and 5Internal Medicine, Faculty of Medicine, Ege University, Izmir, Turkey.
2 3
1
2
20th percentile of the control group were accepted as low muscle mass (Yu et al., 2014). This study was approved by the Ege University Ethics Committee. Procedure
A 1 mL blood sample was taken from each study participant and DNA was isolated from 200 mL of the sample blood for gene analysis. The total genomic DNA was extracted from peripheral leukocytes using an automatic DNA isolation kit (GmbH; Qiagen). The concentration of the eluted DNA was measured at 260 nm and the purity was assessed by the 260/280 nm ratio detected by a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.). DNA samples with a 260/ 280 nm absorbance of >1.8 were accepted as pure. The DNA concentration of the final 200 mL of eluate was adjusted to 50 ng/mL. Electrophoresis in a 1% agarose gel was performed using 2 mL (100 ng) of DNA. Each polymerase chain reaction (PCR) consisted of 1 mL (100 ng) genomic DNA, 2.5 mL enhancer buffer (20 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2), 0.5 mL dNTP mix (0.2 mM), 1 mL each of forward and reverse primers (10 pmol/mL; Invitrogen), 1.0 U PlatinumTaq DNA Polymerase (Invitrogen), and deionized water to yield a 25 mL total volume. A gradient program was run on a VERITI Gradient Thermal Cycler PE (Applied Biosystems). Positive PCR products were enzymatically purified using Exo-SAP Amersham, which is a mixture of exonucleases and shrimp alkaline phosphatase. The chain elongation termination method with both sense and antisense primers (bidirectional sequencing) was used to sequence the PCR products (BigDye chemistry cycle sequencing). Amplicons were assessed by agarose gel electrophoresis and positive amplicons were purified using the BigDye XTerminator Purification Kit (Applied Biosystems). Purified products were analyzed by capillary gel electrophoresis using an automatic DNA analysis system (ABI3130xl; Applied Biosystems). The resulting nucleotide sequences were compared with reference sequences (NM_005259) in the NCBI electronic database, and mutations or polymorphisms were determined according to sequence NP_005250. The distribution of the identified mutations, allele frequency, and correlation with phenotypic characteristics were determined. Statistical analysis
The sarcopenic and nonsarcopenic subjects in the study group were compared. Data were analyzed using a commercially available statistics program (SPSS version 18; IBM), while the statistical power was determined using G*Power (version 3.1.9.2). Numerical variables were expressed as mean – standard deviation and categorical variables were analyzed using the chi-square test. p-Values of £ 0.05 were considered statistically significant. Power analysis revealed that to detect an effect size of 3% with a statistical power of 0.80 and a significance level (a) of 0.05, a sample size of at least 137 patients was required.
TOSUN TASAR ET AL.
Table 1. Sarcopenia Patient Demographic Data, Clinical Characteristics, Anthropometric Measurements, and Muscle Performance Average age (years) Age group Young-old (65–74) Middle-old (75–84) Old-old (‡85) Gender Women Men Average 6-m walking speed (m/s) Walking speed Normal (‡0.8 m/s) Low (
SHORT REPORT
Myostatin Gene Polymorphism in an Elderly Sarcopenic Turkish Population Pinar Tosun Tasar,1 Sevnaz Sahin,1 Emine Karaman,2 Atilla Oz,3 Merve Gulsah Ulusoy,4 Soner Duman,5 Afig Berdeli,3 and Fehmi Akcicek1
Introduction: One of the genetic contributors to sarcopenia predisposition is Myostatin (MSTN), which in humans encodes myostatin, a 376 amino acid growth factor protein that negatively regulates muscle growth. The aim of this study was to investigate MSTN polymorphisms in an elderly sarcopenic population in Turkey and determine how they relate to sarcopenia. Materials and Methods: The study included nursing home residents who were aged ‡65 years. Sarcopenia screening was performed using ‘‘The European Working Group on Sarcopenia in Older People’’ guidelines. Blood sample was taken from each participant and DNA was obtained from the peripheral blood. MSTN polymorphisms were genotyped by polymerase chain reaction and restriction fragment length polymorphism methods. Results: A total of 152 elderly patients were included in the study. The rate of sarcopenia was determined to be 41.4%. The DNA nucleotide sequence of all three MSTN exons was determined for each study participant. Among the 152 patients, only 6 (3.9%) showed an MSTN K153R heterozygous mutation. Among these, three participants were sarcopenic and three were nonsarcopenic. No statistically significant difference in the polymorphism frequency between the sarcopenic and control groups was observed ( p = 0.664). Conclusions: MSTN genotyping revealed that only 3.9% (6/152) of participants had the MSTN K153R heterozygous mutation. Despite the detection of this mutation in the study group, no relationship was found between this mutation and sarcopenia.
age groups: young-old (65–74), middle-old (75–84), and oldold (‡85). The sarcopenia screening was conducted as specified in the 2010 report by the European Union Geriatric Association, ‘‘The European Working Group on Sarcopenia in Older People.’’ The criteria for sarcopenia diagnosis include a decrease in muscle mass and decline in muscle strength and/or physical performance (Cruz-Jentoft et al., 2010). In this study, patients’ physical performance was determined based on the 6-m walking speed and muscle strength as measured with a handgrip device (Takei physical fitness test). The fat-free mass (FFM) was measured using a bioimpedence analysis device (Body Composition Analyzer SC-330). A walking speed of more than 0.8 m/s and muscle strength of 20 kg for women and 30 kg for men were considered normal. The FFM of the study group was compared with healthy community-dwelling young adults aged 18–45 years with no illnesses and not taking medications, who had consented to participate in the study. The body surface area (BSA) was calculated using the DuBois formula (BSA = kg0.425 · m0.725 · 0.007184). The FFM was divided by the BSA to determine the muscle mass. Values below the
Introduction
S
arcopenia is an age-dependent loss of muscle mass and function (Rosenberg, 1997). Currently, there is no consensus for the definitive diagnosis of this disorder, but diagnosis criteria for sarcopenia can include a decrease in muscle mass and a decline in muscle strength and/or physical performance (Cruz-Jentoft et al., 2010; Cesari et al., 2012). Myostatin (MSTN) is one of the genes that can contribute to a genetic predisposition to develop sarcopenia (Huygens et al., 2004). In humans, MSTN encodes the growth factor myostatin, which negatively regulates muscle growth (McPherron et al., 1997). To date, no research has been conducted on the effect of MSTN polymorphisms on sarcopenia in Turkey. The purpose of this study was to determine if a relationship exists between MSTN polymorphism and sarcopenia in an elderly population in Turkey. Materials and Methods
The study group comprised 152 elderly nursing home residents aged ‡65 years. The patients were divided into three 1
Division of Geriatric Medicine, Department of Internal Medicine, Faculty of Medicine, Ege University, Izmir, Turkey. Department of Internal Medicine, Faculty of Nursing, Ege University, Izmir, Turkey. Division of Molecular Medicine Laboratory, Department of Pediatrics, Faculty of Medicine, Ege University, Izmir, Turkey. Departments of 4Biostatistics and 5Internal Medicine, Faculty of Medicine, Ege University, Izmir, Turkey.
2 3
1
2
20th percentile of the control group were accepted as low muscle mass (Yu et al., 2014). This study was approved by the Ege University Ethics Committee. Procedure
A 1 mL blood sample was taken from each study participant and DNA was isolated from 200 mL of the sample blood for gene analysis. The total genomic DNA was extracted from peripheral leukocytes using an automatic DNA isolation kit (GmbH; Qiagen). The concentration of the eluted DNA was measured at 260 nm and the purity was assessed by the 260/280 nm ratio detected by a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.). DNA samples with a 260/ 280 nm absorbance of >1.8 were accepted as pure. The DNA concentration of the final 200 mL of eluate was adjusted to 50 ng/mL. Electrophoresis in a 1% agarose gel was performed using 2 mL (100 ng) of DNA. Each polymerase chain reaction (PCR) consisted of 1 mL (100 ng) genomic DNA, 2.5 mL enhancer buffer (20 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2), 0.5 mL dNTP mix (0.2 mM), 1 mL each of forward and reverse primers (10 pmol/mL; Invitrogen), 1.0 U PlatinumTaq DNA Polymerase (Invitrogen), and deionized water to yield a 25 mL total volume. A gradient program was run on a VERITI Gradient Thermal Cycler PE (Applied Biosystems). Positive PCR products were enzymatically purified using Exo-SAP Amersham, which is a mixture of exonucleases and shrimp alkaline phosphatase. The chain elongation termination method with both sense and antisense primers (bidirectional sequencing) was used to sequence the PCR products (BigDye chemistry cycle sequencing). Amplicons were assessed by agarose gel electrophoresis and positive amplicons were purified using the BigDye XTerminator Purification Kit (Applied Biosystems). Purified products were analyzed by capillary gel electrophoresis using an automatic DNA analysis system (ABI3130xl; Applied Biosystems). The resulting nucleotide sequences were compared with reference sequences (NM_005259) in the NCBI electronic database, and mutations or polymorphisms were determined according to sequence NP_005250. The distribution of the identified mutations, allele frequency, and correlation with phenotypic characteristics were determined. Statistical analysis
The sarcopenic and nonsarcopenic subjects in the study group were compared. Data were analyzed using a commercially available statistics program (SPSS version 18; IBM), while the statistical power was determined using G*Power (version 3.1.9.2). Numerical variables were expressed as mean – standard deviation and categorical variables were analyzed using the chi-square test. p-Values of £ 0.05 were considered statistically significant. Power analysis revealed that to detect an effect size of 3% with a statistical power of 0.80 and a significance level (a) of 0.05, a sample size of at least 137 patients was required.
TOSUN TASAR ET AL.
Table 1. Sarcopenia Patient Demographic Data, Clinical Characteristics, Anthropometric Measurements, and Muscle Performance Average age (years) Age group Young-old (65–74) Middle-old (75–84) Old-old (‡85) Gender Women Men Average 6-m walking speed (m/s) Walking speed Normal (‡0.8 m/s) Low (
