Identification and prioritization of NUAK1 and PPP1CC as positional candidate loci for skeletal muscle strength phenotypes.

Muscle strength is an important determinant in elite sports performance as well as in the activities of daily living. Muscle metabolism also plays a role in the genesis, and therefore prevention, of common pathological conditions and chronic diseases. Even though heritability estimates between 31 and 78% suggest a significant genetic component in muscle strength, only a limited number of genes influencing muscle strength have been identified. This study aimed to identify and prioritize positional candidate genes within a skeletal muscle strength quantitative trait locus on chromosome 12q22-23 for follow-up. A two-staged gene-centered fine-mapping approach using 122 single nucleotide polymorphisms (SNPs) in stage 1 identified a family-based association (n=500) between several tagSNPs located in the ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 (ATP2A2; rs3026468), the NUAK family, SNF1-like kinase, 1 (NUAK1; rs10861553 and rs3741886), and the protein phosphatase 1, catalytic subunit, gamma isoform (PPP1CC; rs1050587 and rs7901769) genes and knee torque production (P values up to 0.00092). In stage 2, family-based association tests on additional putatively functional SNPs (e.g., exonic SNPs, SNPs in transcription factor binding sites or in conserved regions) in an enlarged sample (n=536; 464 individuals overlap with stage 1) did not identify additional associations with muscle strength characteristics. Further in-depth analyses will be necessary to elucidate the exact role of ATP2A2, PPP1CC, and NUAK1 in muscle strength and to find out which functional polymorphisms are at the base of the interindividual strength differences.

Comprehensive fine mapping of chr12q12-14 and follow-up replication identify activin receptor 1B (ACVR1B) as a muscle strength gene.

Muscle strength is important in functional activities of daily living and the prevention of common pathologies. We describe the two-staged fine mapping of a previously identified linkage peak for knee strength on chr12q12-14. First, 209 tagSNPs in/around 74 prioritized genes were genotyped in 500 Caucasian brothers from the Leuven Genes for Muscular Strength study (LGfMS). Combined linkage and family-based association analyses identified activin receptor 1B (ACVR1B) and inhibin ß C (INHBC), part of the transforming growth factor ß pathway regulating myostatin - a negative regulator of muscle mass - signaling, for follow-up. Second, 33 SNPs, selected in these genes based on their likelihood to functionally affect gene expression/function, were genotyped in an extended sample of 536 LGfMS siblings. Strong associations between ACVR1B genotypes and knee muscle strength (P-values up to 0.00002) were present. Of particular interest was the association with rs2854464, located in a putative miR-24-binding site, as miR-24 was implicated in the inhibition of skeletal muscle differentiation. Rs2854464 AA individuals were ∼2% stronger than G-allele carriers. The strength increasing effect of the A-allele was also observed in an independent replication sample (n=266) selected from the Baltimore Longitudinal Study of Aging and a Flemish Policy Research Centre Sport, Physical Activity and Health study. However, no genotype-related difference in ACVR1B mRNA expression in quadriceps muscle was observed. In conclusion, we applied a two-stage fine mapping approach, and are the first to identify and partially replicate genetic variants in the ACVR1B gene that account for genetic variation in human muscle strength.

Protective role of alpha-actinin-3 in the response to an acute eccentric exercise bout.

The ACTN3 gene encodes for the alpha-actinin-3 protein, which has an important structural function in the Z line of the sarcomere in fast muscle fibers. A premature stop codon (R577X) polymorphism in the ACTN3 gene causes a complete loss of the protein in XX homozygotes. This study investigates a possible role for the alpha-actinin-3 protein in protecting the fast fiber from eccentric damage and studies repair mechanisms after a single eccentric exercise bout. Nineteen healthy young men (10 XX, 9 RR) performed 4 series of 20 maximal eccentric knee extensions with both legs. Blood (creatine kinase; CK) and muscle biopsy samples were taken to study differential expression of several anabolic (MyoD1, myogenin, MRF4, Myf5, IGF-1), catabolic (myostatin, MAFbx, and MURF-1), and contraction-induced muscle damage marker genes [cysteine- and glycine-rich protein 3 (CSRP3), CARP, HSP70, and IL-6] as well as a calcineurin signaling pathway marker (RCAN1). Baseline mRNA content of CSRP3 and MyoD1 was 49 + or - 12 and 67 + or - 25% higher in the XX compared with the RR group (P = 0.01-0.045). However, satellite cell number was not different between XX and RR individuals. After eccentric exercise, XX individuals tended to have higher serum CK activity (P = 0.10) and had higher pain scores than RR individuals. However, CSRP3 (P = 0.058) and MyoD1 (P = 0.08) mRNA expression tended to be higher after training in RR individuals compared with XX alpha-actinin-3-deficient subjects. This study suggests a protective role of alpha-actinin-3 protein in muscle damage after eccentric training and an improved stress-sensor signaling, although effects are small.

ACTN3 (R577X) genotype is associated with fiber type distribution.

alpha-Actinin-3 is a Z-disc structural protein found only in type II muscle fibers. The X allele of the R577X polymorphism in the ACTN3 gene results in a premature stop codon and alpha-actinin-3 deficiency in XX homozygotes. Associations between the R577X polymorphism and the muscle-power performance of elite athletes have been described earlier. About 45% of the fiber type proportions are determined by genetic factors. The ACTN3 variant could be one of the contributing genes in the heritability of fiber type distribution through its interaction with calcineurin. The aim of this study was to quantify the association between the polymorphism and muscle fiber type distribution and fast-velocity knee extension strength. Ninety healthy young men (18-29 y) were genotyped for ACTN3 R577X. Knee extensor strength was measured isometrically (45 degrees ) and at different dynamic velocities (100-300 degrees /s) on a programmable dynamometer. Twenty-two XX and twenty-two RR subjects underwent a biopsy of the right vastus lateralis muscle. Fiber type composition was determined by immunohistochemistry. Homozygotes for the R allele show significantly higher relative dynamic quadriceps torques at 300 degrees /s, compared with XX carriers (P < 0.05). Fiber type characteristics differed significantly between the two genotype groups. The percentage surface and number of type IIx fibers were greater in the RR than the XX genotype group (P < 0.05), and alpha-actinin-3 protein content is systematically higher in type IIx compared with type IIa fibers (staining intensity ratio IIx to IIa = 1.17). This study shows that the mechanism, by which the ACTN3 polymorphism has its effect on muscle power, might rely on a control function of fiber type proportions.