The effects of the CREBRF gene variant rs373863828 on skeletal muscle mass and strength in people of Polynesian ancestry

Abstract

Introduction: The primary function of human skeletal muscle is to facilitate mechanical movement, however, equally important to this its role in metabolic health. Although environmental factors are the main factors determining lean muscle mass and strength, emerging evidence suggests genetic variants are associated with skeletal muscle phenotypes. In this context, people of Polynesian ancestry are often perceived as huge and tall individuals, and questions have raised whether this large figure and increased body size could be due to genetics and not only the surrounding factors in their environment. Recently, the cyclic adenosine monophosphate-response element-binding protein 3 regulatory factor (CREBRF) gene (rs373863828: G > A; p. Arg457Gln) have shown to play a significant associate with larger body composition in the form of body mass index. However, it was currently debated whether this increase in BMI was specifically the lean mass, fat mass, and bone mass. Recently, proposed research has now suggested that the rs373863828-A allele is associated with increased lean mass in both infant and adults in the Samoan population. Aim: To determine whether rs373863828 genotype is associated with upper and lower limb skeletal muscle mass and strength in men of Polynesian ancestry, and additionally examine the potential role of the CREBRF gene and rs373863828-A allele in rodent muscle fibretype and C2C12 myoblast differentiation into myotubes. Methods: DXA scans of 164 GG and 80 AG/AA genotyped men of Polynesian ancestry were analysed for skeletal muscle mass, by measuring cross-sectional region of upper and lower limb lean mass, while using the DXA software region of interest setting (enCORE v18 software application, GE Healthcare Medicine). In a subgroup (36 GG and 22 AA/AG genotypes), we performed handgrip strength test, isometric and isotonic knee flexion and extension contractions, and lastly vertical jump tests to measure the upper and lower limb muscle strength. To determine the effects of rs373863828-A allele on muscle fibretype, we used immunofluorescent staining to fibretype the gastrocnemius, soleus, and the tibialis anterior of 12 weeks old male wild type (WT) and CREBRF knock-in (KI) mice. Analysis for fibretype count, surface area and cross-sectional area of each muscle tissue was done using automated image analysis implemented in the FiJi (NIH, Bethesda, MD, USA) environment a macro/plugin called MuscleJ. Finally, we measured the expression level of the CREBRF gene and other related genes in C2C12 myoblasts and differentiated myotubes, to determine if CREBRF gene is affected by muscle differentiation. Results: We demonstrated that the DXA scans of 244 young men of Polynesian ancestry, the rs373863828-A allele does not associate with whole-body composition, regional sections and also cross-sectional regions analysis of the arm and leg (P> 0.05). However, there was an overall trend for slight increase lean mass, particularly in the lower limb from the cross-sectional analysis region of the calf (P= 0.07), and concordantly type IIB fibre percentage area of the tibialis anterior muscle showed a trend for a slight increase from our mice experiment. More so, from our muscle strength analysis, the rs373863828-A allele was significantly associated with only one of our muscle strength measure, particularly the peak isometric knee flexion torque (P= 0.018). Lastly, we also demonstrated that CREBRF gene expression was markedly higher in C2C12 myotubes than in myoblasts (P< 0.006). Conclusion: We concluded that the rs373863828-A allele did not associate with skeletal muscle mass and muscle fibretype, however, it associated with lower limb strength in our small sample size experiments. Furthermore, CREBRF gene being highly expressed in C2C12 differentiated myotubes may suggests a potential role in muscle differentiation. However, further research is warranted using larger sample size, interventional exercise studies, and also C2C12 myotube CREBRF knockdown gene experiments would be ideal for future directions for this study.