GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 19, Number 5, 2015 ª Mary Ann Liebert, Inc. Pp. 1–5 DOI: 10.1089/gtmb.2014.0299
ORIGINAL ARTICLE
Simple Method to Genotype the ACTN3 r577x Polymorphism Ines Schadock,1 Augusto Schneider,1 Elton Dias Silva,2 Marcia Rubia Duarte Buchweitz,1 Marcio Nunes Correa,3 Joao Bosco Pesquero,2 Edgar Julian Paredes-Gamero,4 Ronaldo Carvalho Araujo,2 and Carlos Castilho Barros1
The alpha-actinin-3 r577x polymorphism (rs1815739) is one of the most important polymorphisms associated with athletic performance. This single-nucleotide mutation leads to a premature stop codon, resulting in a nonfunctional protein product. The presence of the dominant R allele is associated with full power skeletal muscle contraction. Homozygosity for the X allele is correlated with more efficient energy disposure. Restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) are the standard methods used to genotype this polymorphism, but they are expensive and require special equipments. Here, we present a simple and cost-efficient method to genotype the ACTN3 r577x polymorphism by a single PCR. External primers yield a 690-bp product that indicates the template quality. Internal primers produce a 413-bp product if the R allele is present and a 318-bp product if the X allele is present. Our four-primer genotyping PCR was validated by the standard real-time PCR, generally used to genotype this single-nucleotide polymorphism, demonstrating the accuracy of this method. This protocol is perfect for small- or large-scale cohort genotyping of the ACTN3 r577x polymorphism.
studies of the ACTN3 r577x polymorphism, which has been described as the most important polymorphism to performance in athletes, are necessary. Performance genomics has been used to analyze the sportive talent and injury predisposition in athletes, both of which are important for professional and amateur training protocol development. In the last two decades, numerous reports about the ACTN3 r577x polymorphism estimated the occurrence of this SNP not only in different cohorts of elite athletes but also in population groups beyond professional athletes, such as elderly people, children, and soldiers (Delmonico et al., 2008; Chan et al., 2011; Shang et al., 2012; Ahmetov et al., 2013). The outcomes of these studies were not always consistent and gave rise to speculations regarding the involvement of the r577x polymorphism in the general processes of muscle function, aging, and metabolism (MacArthur and North, 2007; MacArthur et al., 2007; Moran et al., 2007; Holterhoff et al., 2009; Quinlan et al., 2010; Chan et al., 2011). The bottleneck of many studies is the genotyping of an adequately large cohort due to the comparatively expensive techniques used to date. The usual method used to analyze the ACTN3 r577x polymorphism is the restriction fragment length polymorphism (RFLP) technique, for which the DdeI restriction enzyme is used to digest the polymerase chain reaction (PCR)
Introduction
A
lpha-actinin-3 (actn3) is an actin-binding protein expressed almost exclusively in fast-twitch skeletal muscle fibers (North and Beggs, 1996), in which it helps to stabilize the myofibrillar actin filaments in the sarcomere (North and Beggs, 1996). A genetic variation of the actn3 gene leads to the replacement of an arginine (R) with a stop codon (X) at amino acid 577 (r577x polymorphism), resulting in a truncated protein with no biological function (North et al., 1999). This single-nucleotide polymorphism (SNP) is associated with athletic performance and changes in energy metabolism (Quinlan et al., 2010; Ravaglia et al., 2012). The R allele is associated with forceful muscle contractions and better performance in sports, such as sprinting and weightlifting (Yang et al., 2003; Papadimitriou et al., 2008; Santiago et al., 2008; Eynon et al., 2009; Chiu et al., 2011; Erskine et al., 2013; Pimenta et al., 2013; Mikami et al., 2014). The recessive X allele is, as the homozygous X/X genotype, linked to better performance in endurance events (Yang et al., 2003). In addition to the body of literature suggesting a linkage of the ACTN3 r577x polymorphism with athletic talent, a number of reports have reported finding no advantages of any genotype within the cohorts analyzed and study protocols used (Santiago et al., 2010a, 2010b; Ruiz et al., 2011, 2013). Thus, more
1
Laboratory of Nutrigenomics and Metabology, Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil. Department of Biophysics, Federal University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil. Department of Veterinary, Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil. 4 Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil. 2 3
1
2
SCHADOCK ET AL.
Table 1. Sequences for the Allele-Specific (External Primers) and C-R- and T-X-Specific Primers Name hACTN3f hACTN3r hACTN3Tif hACTN3Cir
Sequence
Product size
5¢-CGCCCTTCAACAACTGGCTGGA-3¢ 5¢-GATGAGCCCGAGACAGGCAAGG-3¢ 5¢-CAACACTGCCCGAGGCTGACTG-3¢ 5¢-CATGATGGCACCTCGCTCTCGG-3¢
product of the region around the codon encoding the 577th amino acid (Gineviciene et al., 2011). The genotyping can also be performed by real-time PCR (Paparini et al., 2007) or by sequencing. In the present study, we describe a simple, efficient, and low-cost method using a single PCR with four primers and validate the data by comparing with the commercial real-time PCR method. We believe that the application of our protocol will allow future studies to perform high-throughput identification of the r577x polymorphism in the actn3 gene and to yield more reliable study data. Materials and Methods Sample preparation
Buccal cells were obtained from saliva. Five hundred microliters of saliva was digested with 600 mL of lysis buffer (Cat. 158908, Cell Lysis Solution; Qiagen) and Proteinase K (Cat. 25530-015; Invitrogen) at 55C for 4 h. Protein precipitation was performed by adding 200 mL of Protein Precipitation Solution (Cat. 158912; Qiagen) and further centrifugation at 16,000 g for 10 min. Genomic DNA (gDNA) was precipitated from the supernatant by adding isopropanol (1:1) and centrifuging (16,000 g for 10 min). Before drying, the pellet was washed with 70% ethanol. The gDNA was hydrated with 100 mL of nuclease-free water.
690 bp 690 bp 318 bp 413 bp
with with with with
hACTN3r hACTN3f hACTN3r hACTN3f
volumes of each external primer (hACTN3f and hACTN3r), one volume of the forward internal primer (hACTN3Tif), and two volumes of the reverse internal primer (hACTN3Cir). A volume of 5 mL of the primer mix was added to 10 mL of 2 · GoTaq Green Master Mix (Cat. M7122; Promega) and 5 mL of the DNA sample to yield a reaction volume of 20 mL. The samples were submitted to the following PCR conditions: 95C for 2 min; 35 cycles at 95C for 10 s, 68C for 10 s, and 72C 45 s; and a final step of 72C for 2 min in a thermocycler (Mod. TX25; AmpliTherm Thermal Cycler). PCR products were analyzed in 2% agarose gels with 1:10,000 SYBR Safe DNA gel stain (Invitrogen) and compared to a 100-bp length marker (100-bp DNA Ladder; Invitrogen). Real-time PCR genotyping method
To control the accuracy of the new method, the ACTN3 r577x (rs1815739) polymorphism was identified by the commercially available fluorescence-based TaqMan SNP Genotyping Assay (Assay C_590093_1; Applied Biosystems). The allele-specific set containing probes and flanking
Four-primer PCR protocol
The four primers used are described in Table 1. The final concentrations used were 0.5 mM for the external primers (hACTN3f and hACTN3r) and 0.125 and 0.25 mM for the internal primers (hACTN3Tif and hACTN3Cir, respectively). In practice, primers at 5 mM were mixed in one tube by adding four
FIG. 1. Diagram of polymerase chain reaction (PCR) primers and product lengths. hACTN3f and hACTN3r primers produce a 690-bp (control) product for both the R and X alleles. With the external primers, internal hACTN3Tif only primes synthesis from the X allele (318-bp product) and internal hACTN3Cir only primes synthesis from the R allele (413-bp product).
FIG. 2. ACTN3 r577x polymorphism genotyping. PCR amplification and gel electrophoresis were performed as described previously. Four-primer PCR was performed with two external and two internal primers. The external primers combine to produce the PCR control product (690 bp) in all samples (1–5). The forward internal primer combines with the reverse external primer to produce a 318-bp product, indicating the T mutation, if the X allele is present. The reverse internal primer combines with the forward external primer to produce a 413-bp C-specific product if the R allele is present. The first column contains the ladder (100-bp DNA Ladder; Invitrogen). The last column presents the control PCR product, with water used in place of a gDNA sample. Nonspecific products are much weaker than the control and are of different sizes and therefore do not disturb the diagnosis.
ACTN3 R577X POLYMORPHISM GENOTYPING BY PCR
primers was used along with a premade PCR master mix containing AmpliTaq DNA polymerase Gold (Applied Biosystems) in a reaction volume of 20 mL. PCR conditions consisted of a 10-min heat activation step (95C), followed by 50 cycles of 15 s at 95C and 1 min at 60C. Amplification was performed in a real-time ABI 7500 PCR machine (Applied Biosystems). Results Primer design and testing
The r577x polymorphism in the actn3 gene (rs1815739) is a nonsense C-T transition that converts the codon for arginine into a premature stop codon. In the four-primer PCR described here, the external primers, hACTN3f and hACTN3r, produce an amplicon of 690 bp in length in all genotypes and are used as an internal PCR control, demonstrating the template quality (Figs. 1 and 2). The internal primers are specific for each allele, yielding a 413-bp product in the presence of the R allele and a 318-bp product in the presence of the X allele (Figs. 1 and 2). The internal primers run in opposing directions, forming PCR
3
products with the external primers previously described, allowing the products resolution by size on the same gel. All primers anneal at the same temperature. At lower temperatures, the internal primers always anneal to the template making it impossible to distinguish between different genotypes. However, at 68C, the internal primers anneal only to their specific alleles due to a base mismatch with the unspecific allele in the penultimate base at the 3¢ end of these primers (Ye et al., 2001). Therefore, in a simple PCR protocol using these four primers, it is possible to identify all genotypes. PCR accuracy confirmation
The result obtained with the four-primer PCR method was confirmed using the fluorescence-based TaqMan SNP Genotyping Assay. The reactions were performed with the same DNA samples and in the same order. All results confirmed the utility of the new method, showing no discrepancies between the two methods (Fig. 3A, B). We also tested this PCR on 100 samples, and the results were always easy to analyze (data not shown).
FIG. 3. Method validation. Diagram showing the result of the fluorescence-based TaqMan SNP genotyping Assay C_590093_1 (Applied Biosystems). (A) Plotting area on the two-color amplicon analysis; (B) plate profile containing samples 1–5, the three controls (R/R, R/X, and X/X), the negative controls (NC), and the results. All results matched with the simple PCR protocol shown in Figure 2.
4 Discussion
Since the discovery of r577x polymorphism in the actn3 gene, hundreds of studies have been performed to elucidate the correlation of this mutation with energy disposure, athletic performance, and metabolic diseases. Each year, several articles studying this polymorphism have been published. Despite the importance of this mutation in these areas, several questions require more data to be uncovered. In this panorama, studies of population in different parts of the world are necessary and can be facilitated when a simple and low-cost genotyping method is available. The ACTN3 r577x polymorphism is one of the most promising genetic polymorphisms discussed to predict athletic talent. A series of studies reported an underrepresentation of the X/X genotype and/or the higher occurrence of the R allele of this gene in cohorts of professional power athletes worldwide (Yang et al., 2003; Niemi and Majamaa, 2005; Druzhevskaya et al., 2008; Papadimitriou et al., 2008; Roth et al., 2008; Santiago et al., 2008; Eynon et al., 2009; Massidda et al., 2009; Ahmetov et al., 2011; Chiu et al., 2011; Cieszczyk et al., 2011; Eynon et al., 2012; Pimenta et al., 2013; Mikami et al., 2014). On the other hand, it seems that the X/X genotype might be beneficial for endurance athletes (Niemi and Majamaa, 2005; Eynon et al., 2009; Eynon et al., 2012). Furthermore, the ACTN3 r577x polymorphism has been associated with energy metabolism (MacArthur et al., 2007; Moran et al., 2007; Quinlan et al., 2010) in humans and mice, inevitably raising questions about the linkage of this gene with diseases, such as type 2 diabetes and metabolic syndrome. Nearly all articles that support or do not support current opinions conclude about their findings with caution because the studies suffer from the same bottleneck—the number of study participants included in their statistical analyses is too low. The commonly used methods (RFLP, real-time PCR, or sequencing) are time- and cost-intensive and sometimes limit large-scale analysis. To overcome these problems, here, we present a simple method to identify the ACTN3 r577x polymorphism in a single PCR. At the same time, this approach allows for the amplification of all three PCR products in the same reaction mixture. Another advantage of our multiplex PCR is that both external and internal primers provide amplicons of different sizes, allowing for visualization of all three products in only one agarose gel run. Nonspecific gel bands appear to be very weak and run a proper distance away from the specific ones; thus, these bands are negligible. In comparison, the specific gel bands are rich and easily identifiable. The amplification efficiency is the main problem in multiplex PCR, which functions like a competition assay between the products. If one product has a higher amplification efficiency compared to others, it tends to consume faster the substrates and thereby hinders the production of other amplicons. To solve this problem, we had to reduce the concentration of internal primers, especially for the forward internal primer (hACTN3Tif), which had to be four times more diluted than external primers and two times than the reverse internal primer, according to the described protocol in the Materials and Methods section. The different concentrations helped to correct the different amplification efficiency of the products (Supplementary Figs. S1 and S2; Supple-
SCHADOCK ET AL.
mentary Data are available online at www.liebertpub.com/ gtmb). We also tested the protocol with two other commercial enzymes. Naturally, in these cases, we had to optimize conditions for each specific PCR kit. We found that it is most important to keep the high annealing temperature of our protocol and the final concentration of each primer. To confirm the accuracy of our method, we ran the same samples in parallel by real-time PCR using the commercially available PCR set to identify the ACTN3 r577x polymorphism. The real-time PCR results confirmed the outcome of our PCR. Therefore, we suggest that our PCR method be used in large- or small-scale screening to identify the ACTN3 r577x polymorphism. Author Disclosure Statement
No competing financial interests exist. References
Ahmetov, II, Druzhevskaya AM, Lyubaeva EV, et al. (2011) The dependence of preferred competitive racing distance on muscle fibre type composition and ACTN3 genotype in speed skaters. Exp Physiol 96:1302–1310. Ahmetov, II, Gavrilov DN, Astratenkova IV, et al. (2013) The association of ACE, ACTN3 and PPARA gene variants with strength phenotypes in middle school-age children. J Physiol Sci 63:79–85. Chan S, Seto JT, Houweling PJ, et al. (2011) Properties of extensor digitorum longus muscle and skinned fibers from adult and aged male and female Actn3 knockout mice. Muscle Nerve 43:37–48. Chiu LL, Wu YF, Tang MT, et al. (2011) ACTN3 genotype and swimming performance in Taiwan. Int J Sports Med 32:476–480. Cieszczyk P, Eider J, Ostanek M, et al. (2011) Association of the ACTN3 R577X polymorphism in polish power-orientated athletes. J Hum Kinet 28:55–61. Delmonico MJ, Zmuda JM, Taylor BC, et al. (2008) Association of the ACTN3 genotype and physical functioning with age in older adults. J Gerontol A Biol Sci 63:1227–1234. Druzhevskaya AM, Ahmetov, II, Astratenkova IV, Rogozkin VA (2008) Association of the ACTN3 R577X polymorphism with power athlete status in Russians. Eur J Appl Physiol 103:631–634. Erskine RM, Williams AG, Jones DA, et al. (2013) The individual and combined influence of ACE and ACTN3 genotypes on muscle phenotypes before and after strength training. Scand J Med Sci Sports; doi: 10.1111/sms.12055. Eynon N, Duarte JA, Oliveira J, et al. (2009) ACTN3 R577X polymorphism and Israeli top-level athletes. Int J Sports Med 30:695–698. Eynon N, Ruiz JR, Femia P, et al. (2012) The ACTN3 R577X polymorphism across three groups of elite male European athletes. PLoS One 7:e43132. Gineviciene V, Pranculis A, Jakaitiene A, et al. (2011) Genetic variation of the human ACE and ACTN3 genes and their association with functional muscle properties in Lithuanian elite athletes. Medicina (Kaunas) 47:284–290. Holterhoff CK, Saunders RH, Brito EE, Wagner DS (2009) Sequence and expression of the zebrafish alpha-actinin gene family reveals conservation and diversification among vertebrates. Dev Dyn 238:2936–2947. MacArthur DG, North KN (2007) ACTN3: A genetic influence on muscle function and athletic performance. Exerc Sports Sci Rev 35:30–34.
ACTN3 R577X POLYMORPHISM GENOTYPING BY PCR
MacArthur DG, Seto JT, Raftery JM, et al. (2007) Loss of ACTN3 gene function alters mouse muscle metabolism and shows evidence of positive selection in humans. Nat Genet 39:1261–1265. Massidda M, Vona G, Calo CM (2009) Association between the ACTN3 R577X polymorphism and artistic gymnastic performance in Italy. Genet Test Mol Biomarkers 13:377– 380. Mikami E, Fuku N, Murakami H, et al. (2014) ACTN3 R577X genotype is associated with sprinting in elite Japanese athletes. Int J Sports Med 35:172–177. Moran CN, Yang N, Bailey ME, et al. (2007) Association analysis of the ACTN3 R577X polymorphism and complex quantitative body composition and performance phenotypes in adolescent Greeks. Eur J Hum Genet 15:88–93. Niemi AK, Majamaa K (2005) Mitochondrial DNA and ACTN3 genotypes in Finnish elite endurance and sprint athletes. Eur J Human Genet 13:965–969. North KN, Beggs AH (1996) Deficiency of a skeletal muscle isoform of alpha-actinin (alpha-actinin-3) in merosin-positive congenital muscular dystrophy. Neuromuscul Disord 6:229– 235. North KN, Yang N, Wattanasirichaigoon D, et al. (1999) A common nonsense mutation results in alpha-actinin-3 deficiency in the general population. Nat Genet 21:353–354. Papadimitriou ID, Papadopoulos C, Kouvatsi A, Triantaphyllidis C (2008) The ACTN3 gene in elite Greek track and field athletes. Int J Sports Med 29:352–355. Paparini A, Ripani M, Giordano GD, et al. (2007) ACTN3 genotyping by real-time PCR in the Italian population and athletes. Med Sci Sports Exerc 39:810–815. Pimenta EM, Coelho DB, Veneroso CE, et al. (2013) Effect of ACTN3 gene on strength and endurance in soccer players. J Strength Cond Res 27:3286–3292. Quinlan KG, Seto JT, Turner N, et al. (2010) Alpha-actinin-3 deficiency results in reduced glycogen phosphorylase activity and altered calcium handling in skeletal muscle. Hum Mol Genet 19:1335–1346. Ravaglia S, De Filippi P, Pichiecchio A, et al. (2012) Can genes influencing muscle function affect the therapeutic response to enzyme replacement therapy (ERT) in late-onset type II glycogenosis? Mol Genet Metab 107:104–110.
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Roth SM, Walsh S, Liu D, et al. (2008) The ACTN3 R577X nonsense allele is under-represented in elite-level strength athletes. Eur J Hum Genet 16:391–394. Ruiz JR, Fernandez del Valle M, Verde Z, et al. (2011) ACTN3 R577X polymorphism does not influence explosive leg muscle power in elite volleyball players. Scandinavian J Med Sci Sports 21:e34–e41. Ruiz JR, Santiago C, Yvert T, et al. (2013) ACTN3 genotype in Spanish elite swimmers: no ‘‘heterozygous advantage’’. Scand J Med Sci Sports 23:e162–e167. Santiago C, Gonzalez-Freire M, Serratosa L, et al. (2008) ACTN3 genotype in professional soccer players. Br J Sports Med 42:71–73. Santiago C, Rodriguez-Romo G, Gomez-Gallego F, et al. (2010a) Is there an association between ACTN3 R577X polymorphism and muscle power phenotypes in young, nonathletic adults? Scand J Med Sci Sports 20:771–778. Santiago C, Ruiz JR, Muniesa CA, et al. (2010b) Does the polygenic profile determine the potential for becoming a world-class athlete? Insights from the sport of rowing. Scand J Med Sci Sports 20:e188–e194. Shang X, Zhang F, Zhang L, Huang C (2012) ACTN3 R577X polymorphism and performance phenotypes in young Chinese male soldiers. J Sports Sci 30:255–260. Yang N, MacArthur DG, Gulbin JP, et al. (2003) ACTN3 genotype is associated with human elite athletic performance. Am J Hum Genet 73:627–631. Ye S, Dhillon S, Ke X, et al. (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:E88-8.
Address correspondence to: Carlos Castilho Barros, PhD Universidade Federal de Pelotas—UFPel Laborato´rio de Nutrifisiogenoˆmica e Metabologia R. Gomes Carneiro no 01 Sala 239 Pelotas Rio Grande do Sul CEP 96010-610 Brazil E-mail: [email protected]
ORIGINAL ARTICLE
Simple Method to Genotype the ACTN3 r577x Polymorphism Ines Schadock,1 Augusto Schneider,1 Elton Dias Silva,2 Marcia Rubia Duarte Buchweitz,1 Marcio Nunes Correa,3 Joao Bosco Pesquero,2 Edgar Julian Paredes-Gamero,4 Ronaldo Carvalho Araujo,2 and Carlos Castilho Barros1
The alpha-actinin-3 r577x polymorphism (rs1815739) is one of the most important polymorphisms associated with athletic performance. This single-nucleotide mutation leads to a premature stop codon, resulting in a nonfunctional protein product. The presence of the dominant R allele is associated with full power skeletal muscle contraction. Homozygosity for the X allele is correlated with more efficient energy disposure. Restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) are the standard methods used to genotype this polymorphism, but they are expensive and require special equipments. Here, we present a simple and cost-efficient method to genotype the ACTN3 r577x polymorphism by a single PCR. External primers yield a 690-bp product that indicates the template quality. Internal primers produce a 413-bp product if the R allele is present and a 318-bp product if the X allele is present. Our four-primer genotyping PCR was validated by the standard real-time PCR, generally used to genotype this single-nucleotide polymorphism, demonstrating the accuracy of this method. This protocol is perfect for small- or large-scale cohort genotyping of the ACTN3 r577x polymorphism.
studies of the ACTN3 r577x polymorphism, which has been described as the most important polymorphism to performance in athletes, are necessary. Performance genomics has been used to analyze the sportive talent and injury predisposition in athletes, both of which are important for professional and amateur training protocol development. In the last two decades, numerous reports about the ACTN3 r577x polymorphism estimated the occurrence of this SNP not only in different cohorts of elite athletes but also in population groups beyond professional athletes, such as elderly people, children, and soldiers (Delmonico et al., 2008; Chan et al., 2011; Shang et al., 2012; Ahmetov et al., 2013). The outcomes of these studies were not always consistent and gave rise to speculations regarding the involvement of the r577x polymorphism in the general processes of muscle function, aging, and metabolism (MacArthur and North, 2007; MacArthur et al., 2007; Moran et al., 2007; Holterhoff et al., 2009; Quinlan et al., 2010; Chan et al., 2011). The bottleneck of many studies is the genotyping of an adequately large cohort due to the comparatively expensive techniques used to date. The usual method used to analyze the ACTN3 r577x polymorphism is the restriction fragment length polymorphism (RFLP) technique, for which the DdeI restriction enzyme is used to digest the polymerase chain reaction (PCR)
Introduction
A
lpha-actinin-3 (actn3) is an actin-binding protein expressed almost exclusively in fast-twitch skeletal muscle fibers (North and Beggs, 1996), in which it helps to stabilize the myofibrillar actin filaments in the sarcomere (North and Beggs, 1996). A genetic variation of the actn3 gene leads to the replacement of an arginine (R) with a stop codon (X) at amino acid 577 (r577x polymorphism), resulting in a truncated protein with no biological function (North et al., 1999). This single-nucleotide polymorphism (SNP) is associated with athletic performance and changes in energy metabolism (Quinlan et al., 2010; Ravaglia et al., 2012). The R allele is associated with forceful muscle contractions and better performance in sports, such as sprinting and weightlifting (Yang et al., 2003; Papadimitriou et al., 2008; Santiago et al., 2008; Eynon et al., 2009; Chiu et al., 2011; Erskine et al., 2013; Pimenta et al., 2013; Mikami et al., 2014). The recessive X allele is, as the homozygous X/X genotype, linked to better performance in endurance events (Yang et al., 2003). In addition to the body of literature suggesting a linkage of the ACTN3 r577x polymorphism with athletic talent, a number of reports have reported finding no advantages of any genotype within the cohorts analyzed and study protocols used (Santiago et al., 2010a, 2010b; Ruiz et al., 2011, 2013). Thus, more
1
Laboratory of Nutrigenomics and Metabology, Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil. Department of Biophysics, Federal University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil. Department of Veterinary, Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil. 4 Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil. 2 3
1
2
SCHADOCK ET AL.
Table 1. Sequences for the Allele-Specific (External Primers) and C-R- and T-X-Specific Primers Name hACTN3f hACTN3r hACTN3Tif hACTN3Cir
Sequence
Product size
5¢-CGCCCTTCAACAACTGGCTGGA-3¢ 5¢-GATGAGCCCGAGACAGGCAAGG-3¢ 5¢-CAACACTGCCCGAGGCTGACTG-3¢ 5¢-CATGATGGCACCTCGCTCTCGG-3¢
product of the region around the codon encoding the 577th amino acid (Gineviciene et al., 2011). The genotyping can also be performed by real-time PCR (Paparini et al., 2007) or by sequencing. In the present study, we describe a simple, efficient, and low-cost method using a single PCR with four primers and validate the data by comparing with the commercial real-time PCR method. We believe that the application of our protocol will allow future studies to perform high-throughput identification of the r577x polymorphism in the actn3 gene and to yield more reliable study data. Materials and Methods Sample preparation
Buccal cells were obtained from saliva. Five hundred microliters of saliva was digested with 600 mL of lysis buffer (Cat. 158908, Cell Lysis Solution; Qiagen) and Proteinase K (Cat. 25530-015; Invitrogen) at 55C for 4 h. Protein precipitation was performed by adding 200 mL of Protein Precipitation Solution (Cat. 158912; Qiagen) and further centrifugation at 16,000 g for 10 min. Genomic DNA (gDNA) was precipitated from the supernatant by adding isopropanol (1:1) and centrifuging (16,000 g for 10 min). Before drying, the pellet was washed with 70% ethanol. The gDNA was hydrated with 100 mL of nuclease-free water.
690 bp 690 bp 318 bp 413 bp
with with with with
hACTN3r hACTN3f hACTN3r hACTN3f
volumes of each external primer (hACTN3f and hACTN3r), one volume of the forward internal primer (hACTN3Tif), and two volumes of the reverse internal primer (hACTN3Cir). A volume of 5 mL of the primer mix was added to 10 mL of 2 · GoTaq Green Master Mix (Cat. M7122; Promega) and 5 mL of the DNA sample to yield a reaction volume of 20 mL. The samples were submitted to the following PCR conditions: 95C for 2 min; 35 cycles at 95C for 10 s, 68C for 10 s, and 72C 45 s; and a final step of 72C for 2 min in a thermocycler (Mod. TX25; AmpliTherm Thermal Cycler). PCR products were analyzed in 2% agarose gels with 1:10,000 SYBR Safe DNA gel stain (Invitrogen) and compared to a 100-bp length marker (100-bp DNA Ladder; Invitrogen). Real-time PCR genotyping method
To control the accuracy of the new method, the ACTN3 r577x (rs1815739) polymorphism was identified by the commercially available fluorescence-based TaqMan SNP Genotyping Assay (Assay C_590093_1; Applied Biosystems). The allele-specific set containing probes and flanking
Four-primer PCR protocol
The four primers used are described in Table 1. The final concentrations used were 0.5 mM for the external primers (hACTN3f and hACTN3r) and 0.125 and 0.25 mM for the internal primers (hACTN3Tif and hACTN3Cir, respectively). In practice, primers at 5 mM were mixed in one tube by adding four
FIG. 1. Diagram of polymerase chain reaction (PCR) primers and product lengths. hACTN3f and hACTN3r primers produce a 690-bp (control) product for both the R and X alleles. With the external primers, internal hACTN3Tif only primes synthesis from the X allele (318-bp product) and internal hACTN3Cir only primes synthesis from the R allele (413-bp product).
FIG. 2. ACTN3 r577x polymorphism genotyping. PCR amplification and gel electrophoresis were performed as described previously. Four-primer PCR was performed with two external and two internal primers. The external primers combine to produce the PCR control product (690 bp) in all samples (1–5). The forward internal primer combines with the reverse external primer to produce a 318-bp product, indicating the T mutation, if the X allele is present. The reverse internal primer combines with the forward external primer to produce a 413-bp C-specific product if the R allele is present. The first column contains the ladder (100-bp DNA Ladder; Invitrogen). The last column presents the control PCR product, with water used in place of a gDNA sample. Nonspecific products are much weaker than the control and are of different sizes and therefore do not disturb the diagnosis.
ACTN3 R577X POLYMORPHISM GENOTYPING BY PCR
primers was used along with a premade PCR master mix containing AmpliTaq DNA polymerase Gold (Applied Biosystems) in a reaction volume of 20 mL. PCR conditions consisted of a 10-min heat activation step (95C), followed by 50 cycles of 15 s at 95C and 1 min at 60C. Amplification was performed in a real-time ABI 7500 PCR machine (Applied Biosystems). Results Primer design and testing
The r577x polymorphism in the actn3 gene (rs1815739) is a nonsense C-T transition that converts the codon for arginine into a premature stop codon. In the four-primer PCR described here, the external primers, hACTN3f and hACTN3r, produce an amplicon of 690 bp in length in all genotypes and are used as an internal PCR control, demonstrating the template quality (Figs. 1 and 2). The internal primers are specific for each allele, yielding a 413-bp product in the presence of the R allele and a 318-bp product in the presence of the X allele (Figs. 1 and 2). The internal primers run in opposing directions, forming PCR
3
products with the external primers previously described, allowing the products resolution by size on the same gel. All primers anneal at the same temperature. At lower temperatures, the internal primers always anneal to the template making it impossible to distinguish between different genotypes. However, at 68C, the internal primers anneal only to their specific alleles due to a base mismatch with the unspecific allele in the penultimate base at the 3¢ end of these primers (Ye et al., 2001). Therefore, in a simple PCR protocol using these four primers, it is possible to identify all genotypes. PCR accuracy confirmation
The result obtained with the four-primer PCR method was confirmed using the fluorescence-based TaqMan SNP Genotyping Assay. The reactions were performed with the same DNA samples and in the same order. All results confirmed the utility of the new method, showing no discrepancies between the two methods (Fig. 3A, B). We also tested this PCR on 100 samples, and the results were always easy to analyze (data not shown).
FIG. 3. Method validation. Diagram showing the result of the fluorescence-based TaqMan SNP genotyping Assay C_590093_1 (Applied Biosystems). (A) Plotting area on the two-color amplicon analysis; (B) plate profile containing samples 1–5, the three controls (R/R, R/X, and X/X), the negative controls (NC), and the results. All results matched with the simple PCR protocol shown in Figure 2.
4 Discussion
Since the discovery of r577x polymorphism in the actn3 gene, hundreds of studies have been performed to elucidate the correlation of this mutation with energy disposure, athletic performance, and metabolic diseases. Each year, several articles studying this polymorphism have been published. Despite the importance of this mutation in these areas, several questions require more data to be uncovered. In this panorama, studies of population in different parts of the world are necessary and can be facilitated when a simple and low-cost genotyping method is available. The ACTN3 r577x polymorphism is one of the most promising genetic polymorphisms discussed to predict athletic talent. A series of studies reported an underrepresentation of the X/X genotype and/or the higher occurrence of the R allele of this gene in cohorts of professional power athletes worldwide (Yang et al., 2003; Niemi and Majamaa, 2005; Druzhevskaya et al., 2008; Papadimitriou et al., 2008; Roth et al., 2008; Santiago et al., 2008; Eynon et al., 2009; Massidda et al., 2009; Ahmetov et al., 2011; Chiu et al., 2011; Cieszczyk et al., 2011; Eynon et al., 2012; Pimenta et al., 2013; Mikami et al., 2014). On the other hand, it seems that the X/X genotype might be beneficial for endurance athletes (Niemi and Majamaa, 2005; Eynon et al., 2009; Eynon et al., 2012). Furthermore, the ACTN3 r577x polymorphism has been associated with energy metabolism (MacArthur et al., 2007; Moran et al., 2007; Quinlan et al., 2010) in humans and mice, inevitably raising questions about the linkage of this gene with diseases, such as type 2 diabetes and metabolic syndrome. Nearly all articles that support or do not support current opinions conclude about their findings with caution because the studies suffer from the same bottleneck—the number of study participants included in their statistical analyses is too low. The commonly used methods (RFLP, real-time PCR, or sequencing) are time- and cost-intensive and sometimes limit large-scale analysis. To overcome these problems, here, we present a simple method to identify the ACTN3 r577x polymorphism in a single PCR. At the same time, this approach allows for the amplification of all three PCR products in the same reaction mixture. Another advantage of our multiplex PCR is that both external and internal primers provide amplicons of different sizes, allowing for visualization of all three products in only one agarose gel run. Nonspecific gel bands appear to be very weak and run a proper distance away from the specific ones; thus, these bands are negligible. In comparison, the specific gel bands are rich and easily identifiable. The amplification efficiency is the main problem in multiplex PCR, which functions like a competition assay between the products. If one product has a higher amplification efficiency compared to others, it tends to consume faster the substrates and thereby hinders the production of other amplicons. To solve this problem, we had to reduce the concentration of internal primers, especially for the forward internal primer (hACTN3Tif), which had to be four times more diluted than external primers and two times than the reverse internal primer, according to the described protocol in the Materials and Methods section. The different concentrations helped to correct the different amplification efficiency of the products (Supplementary Figs. S1 and S2; Supple-
SCHADOCK ET AL.
mentary Data are available online at www.liebertpub.com/ gtmb). We also tested the protocol with two other commercial enzymes. Naturally, in these cases, we had to optimize conditions for each specific PCR kit. We found that it is most important to keep the high annealing temperature of our protocol and the final concentration of each primer. To confirm the accuracy of our method, we ran the same samples in parallel by real-time PCR using the commercially available PCR set to identify the ACTN3 r577x polymorphism. The real-time PCR results confirmed the outcome of our PCR. Therefore, we suggest that our PCR method be used in large- or small-scale screening to identify the ACTN3 r577x polymorphism. Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Carlos Castilho Barros, PhD Universidade Federal de Pelotas—UFPel Laborato´rio de Nutrifisiogenoˆmica e Metabologia R. Gomes Carneiro no 01 Sala 239 Pelotas Rio Grande do Sul CEP 96010-610 Brazil E-mail: [email protected]
