The molecular mechanisms by which aging affects stem cell number and function are poorly understood. Murine data have implicated cellular senescence in the loss of muscle stem cells with aging. Here, using human cells and by carrying out experiments within a strictly pre-senescent division count, we demonstrate an impaired capacity for stem cell self-renewal in elderly muscle. We link aging to an increased methylation of the SPRY1 gene, a known regulator of muscle stem cell quiescence. Replenishment of the reserve cell pool was modulated experimentally by demethylation or siRNA knockdown of SPRY1. We propose that suppression of SPRY1 by age-associated methylation in humans inhibits the replenishment of the muscle stem cell pool, contributing to a decreased regenerative response in old age. We further show that aging does not affect muscle stem cell senescence in humans.

Abstract Background: Several driver mutations have been discovered in colorectal cancer (CRC) progressions including KRAS and BRAF that has a practical significant therapeutic and prognostic value. Sequencing technology has advanced and now provides a tool for the discovery of novel driver mutations. In addition, sequencing technologies now provide a tool for whole genome methylation analysis. Here, we performed whole exome sequencing (WES) and whole genome methylation analysis to elucidate the involvement of novel candidate genes the KRAS pathway that may effect colorectal carcinogenesis. Patients and Methods: WES was carried out on genomic DNA extracted from 8 normal-tumor pairs of frozen biopsies from African Americans patients with CRC. WES and Reduced Representation Bisulfite Sequencing (RRBS) were performed. Pyrosequencing and sanger sequencing used for validation of methylation and single nucleotide variants (SNV) respectively. For WES, base call quality recalibration, realignment around indels, SNV calling and variant call recalibration were carried out using Genome Analysis Tool Kit. Variants were then annotated using Annovar. Results: WES uncovered somatic mutations alteration in many genes that are known be mutated in CRC including APC, BRAF, KRAS, Notch1, PIK3C2A, and NDRG4. We discovered a number of Novel SNVs in EID3, RGS3, HNRNPF, and GNAS in tumor samples. One GNAS missense mutation was discovered among KRAS mutated tumors. In addition, the tumor with the GNAS mutation was significantly hypomethylated (P<0.05) in the CpG sites of GNAS promoter as compared with paired normal tissue. The tumor was located in the cecum and identified to be invasive adenocarcinoma with stage IV1b (T4b, N2a, M1). Ingenuity pathway analysis (IPA) showed that GNAS were involved in CRC metastasis signaling via APC, BRAF, GSK3A, KRAS, MLH1, MLH2, MLH3, Notch family, and PIK3C family. Conclusion: This work provides insight into identification of novel somatic mutations in GNAS coupled with promoter hypomethylation at the same locus using WES and RRBS. GNAS SNV resulted in gain of function of G-protein signaling that may play a pivotal role in CRC Identification the biological significance of GNAS in CRC may introduce a new target for colorectal cancer diagnosis and treatment. Citation Format: Hamed Rahi, Sohaila Soltani, Mohammad Daremipouran, Hassan Brim, Eward L. Lee, Alfreda Woods, Wayne Frederick, Adeyinka O. Laiyemo, Joe Devaney, Ron Leavitt, Xueguang Sun, Hassan Ashktorab. Novel mutation and hypomethylation define distinct biological subgroups of altered KRAS colon tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2013-1934

Abstract Background: Whole Exome sequencing (WES) is a tool that is revolutionizing screening for pathogeni single nucleotide variations (SNV) in complex disorders such as cancers. Using matched normal-tumor pairs, it is possible to identify exome-wide germline and somatic variant alterations that may have an effect on disease progression or response to different interventions. The existing analysis pipelines are under continuous enhancements. In analyzing matched pairs, there is a critical assumption that the sequenced data are matched, without any quality check. Identification of germline and rare somatic variants depend on the normal sample being the qualified matched pair. Our aim was to determine if Identity By State (IBS), a genetics concept on measuring relatedness between individuals (matched tumor-normal pairs) can assess somatic landscape between individual using WES data. Materials and Methods: Genomic DNA was extracted from 8 normal-tumor pair tissues from African Americans (males n=3, females n=5) with colorectal cancer (CRC). WES was performed for identification of SNV according to manufacturer recommendations (Illumina HiScan SQ). The low quality reads were discarded and the clean reads were aligned against the human reference genome (HG19) and base quality calibration was completed using SAMTools, and GATK for variant calling and annotation. Pairs with no shared allele, one shared allele, and two shared allele assigned as IBS 0, IBS 1, and IBS 2 respectfully. Alleles in the dataset were coded using A and B and pairwise IBS was computed between all samples. Display of the IBS landscape was done using GenomeRelator. Results: The frequency of IBS from WGS data sets showed that most of the changes were IBS-1 which were heterozygous variants; i.e. AA/BB>AB (somatic) or AB>AA/BB (LOH). Frequency of LOH varied from 4.36% to 73.61%. Homozygous variant (i.e. IBS-0, AA>BB) on the other hand was not common, from 0.00% to 4.17%. Distribution of IBS across genome for matched pairs reveals that most of the IBS were IBS-2, indicating that both alleles between matched normal and tumor were similar. Frequency of IBS-1 and IBS-0 varied amongst the samples. Sample CC1053 had the most allele changes in chr1-22, and Xq, and sample CC1054 showed the second highest allele changes in chr1,3,4,6,8,9,10,11,13, 16, 17,19,20 and 22. Conclusion: Our results showed that the SNV difference between normal-tumor matched pair is relatively small consistent with the assumption of low mutation rate in cancer. IBS approach provides the tumor contents as well as assurance in the selection of samples for sequencing using SNP data. In our samples 88% similarly was found, as shown in IBS landscape across the chromosomes. We concluded that our matched pair samples are appropriately selected and are suitable for analysis of mutation across the samples. Citation Format: Mohammad Daremipouran, Hassan Hassanzadeh Namin, Sohaila Soltani, Joe Devaney, Wayne Frederick, Edward L. Lee, Hassan Brim, Hassan Ashktorab. Paired analysis of matched colon normal and tumor using whole exome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5307. doi:10.1158/1538-7445.AM2013-5307

There is significant and unexplained variability in muscle damage indicators following eccentric exercise, differences that might be due to genetic alterations in muscle structural genes. We determined whether SNP rs11693372 in the promoter of TTN was associated with strength loss and muscle soreness following eccentric exercise. Methods 75 women and 79 men performed 50 eccentric actions of the elbow flexors. Strength was measured pre/post‐exercise, and 4d, 7d and 10d post‐exercise. Soreness was assessed via a visual analog scale at similar time points. Associations were compared between the TTN SNP and strength loss and soreness. Results Analyses revealed significant differences in strength loss and soreness between genotypes for women only. Compared to T allele carriers, women possessing the ancestral CC genotype experienced: 1) less strength loss post‐exercise (CC, N=13, −44.8% ± 4.9; CT, N=31, −63.2% ± 3.2; TT, N=31, −55.9% ± 3.2; p<=0.01) and after 4d (CC, −26.0% ± 7.7; CT, −51.4% ± 4.9; TT, −39.9% ± 4.9; p<0.01); and 2) less muscle soreness at 3d (CC, 40.0mm ± 5.9; CT, 54.1mm ± 3.9; TT, 63.9mm ± 3.9; p=0.01) and 4d (CC: 30.2mm ± 6.8; CT: 50.3mm ± 4.4; TT: 51.4mm ± 4.4; p<0.01) post‐exercise. Conclusions These observations suggest that, for women, possession of the CC alleles mitigated strength loss and the development of muscle soreness following damaging eccentric exercise compared to T allele carriers.

PURPOSE: Much heterogeneity exists in markers of muscle damage following intense eccentric exercise. While the mechanisms for such differences are not completely understood, genetic differences may explain some of this observed variation. We screened numerous single nucleotide polymorphisms (SNPs) to test for associations with indicators of muscle damage. METHODS: 75 women and 79 men performed 50 maximal eccentric contractions of the elbow flexors of their non-dominant arm. 3 maximal isometric contractions were performed pre/post-exercise as well as 4d, 7d and 10d post-exercise to assess strength loss. Serum creatine kinase (CK) activity was measured at the same time points. Soreness was reported via a visual analog scale at 13h, 3d, and 4d post-exercise. RESULTS: We found significant associations of a SNP in Slc30a8 (rs13266634) with strength loss, CK activity, and soreness post-exercise. Distribution of alleles was 57% ancestral CC, 37% CT, and 6% TT. Analysis revealed significant differences between genotypes for men only. TT carriers experienced less strength loss post-exercise (N=10; -29.8% ± 6.3) as compared to CT (N=26;-47.9% ± 3.5) and CC carriers (N=36;-51.9% ± 2.8) (p=0.0452). Less strength loss was also experienced for TT carriers at 4d post-exercise (TT: -13.5% ± 9.1; CT: -30.6% ± 5.7; CC: -40.4% ± 4.8; p=0.0392) and 7d post-exercise (TT:-2.9% ± 9.1; CT:-18.1% ± 5.9; CC:-30.5% ± 4.8; p=0.0453) as compared to CT and CC. Men possessing the TT alleles also had lower levels of serum CK activity 4d post-exercise (TT: 1024 ± 3835 U/L; CT: 9540 ± 2463; CC: 12153 ± 2124; p <0.001) and lower soreness at 3d post-exercise (TT: 27.2mm ± 7.0; CT: 49.3mm ± 4.3; CC: 57.9mm ± 3.7; p= 0.0203) and 4d (TT:15.6mm ± 6.8; CT: 35.3mm ± 4.2; CC: 44.9mm ± 3.6; p=0.0279). CONCLUSIONS: For men, possession of the Slc30a8 TT alleles for SNP rs13266634 mitigated the effects of damaging eccentric exercise as compared to men possessing the CT or ancestral CC alleles. SLC30A8 is a zinc transporter gene, and the rs13266634 SNP has been found to be related to diabetes and impaired pro-insulin to insulin conversion. These results may implicate alterations in zinc-induced metabolism as factors related to exertional muscle damage. Supported in part by Medinova Inc.

We previously reported that a single nucleotide polymorphism (SNP) of the AKT1 gene in males was associated with body composition, which could have important implications for understanding the genetic underpinnings of diseases such as obesity and osteoporosis. Males homozygous for AKT1 G205T rare allele (TT) had larger bones and lower subcutaneous fat compared to males with the heterozygous or the wildtype alleles. However, these data were calculated from only one anatomical site, the upper arm. PURPOSE: We wanted to extend these findings using whole body assessment of bone and fat to determine if the AKT1 SNP was associated with total body composition in males and females. We hypothesized that individuals who possessed the rare allele of AKT1 G205T would have less total body fat tissue and greater bone density than individuals who possessed the wild type alleles of G205T. METHODS: Anthropometric measures of height, weight, arm circumference, and skinfolds were recorded. Body composition was performed using dual energy x-ray absorptiometry (DEXA) that provided bone mineral density, fat free mass, percent tissue fat (%TF), and fat mass for total body and body regions (e.g. arms, legs). Genotyping was performed using a TaqMan allele discrimination assay that employed the 5' nuclease activity of Taq polymerase to detect a fluorescent reporter signal generated during PCR reactions. An ANOVA was used to detect significant differences between the genotypes and the dependent measures. RESULTS: Of the 127 subjects tested to date, 56.7% (n=72) were wild type (GG), 32.3% (n=41) were heterozygous (GT), and 11.0% (n=14) were homozygous for the rare allele (TT). Individuals who possessed the rare allele of AKT1 (GT and TT) demonstrated a trend for lower %TF in the arm (23.6 ± 1.5% (mean±SE)) compared to individuals with the wildtype alleles (GG =25.8 ± 1.3%) (p=.09). No significant differences were found between genotypes and dependent measures of total body composition or for any other body region. CONCLUSIONS: These data indicate the AKT1 genotype in males and females is not associated with whole body composition measured by DEXA, but support previous work showing an association with body composition of the arm. This research was Supported by an ACSM Foundation Research Grant from the American College of Sports Medicine Foundation.

BACKGROUND: Limited investigation suggests that the ciliary nerotrophic factor G1357A (CNTF GA) polymorphism alters muscle function among middle-aged and older adults. PURPOSE: To examine associations between the CNTF GA polymorphism and muscle strength response to a unilateral, upper-arm resistance training (RT) program in healthy, young adults. METHODS: Subjects were 810 (mean ± SEM, 23.8 ± 0.4yr) men (42%) and women (58%). Of these, 75% were CNTF GG (248 men and 361 women), 22% CNTF GA (78 men and 97 women) and 3% CNTF AA (11 men and 15 women). Subjects performed a supervised 12wk (2d-wk) RT program of the non-dominant arm (trained, T) with the dominant arm (untrained, UT) as comparison. One-repetition maximum (1RM) measured peak dynamic elbow-flexor muscle strength in T and UT pre- and post-RT. Multiple variable and repeated measure ANO VA tested if muscle strength differed among CNTF genotypes and gender with body mass index, ethnicity, and age as covariates pre-, post-, and pre- to post-RT. Muscle strength phenotypes did not differ among CNTF GA and AA genotypes so these were combined and statistical analyses repeated. Results are reported comparing CNTF GG versus GA/AA genotypes. RESULTS: 1RM did not differ by CNTF genotype in T and UT in the total sample pre-, post-, or pre- to post-RT (P<0.05). However, gender interacted significantly with CNTF genotype to alter 1RM response to RT. Among men 1RM was not different between CNTF genotypes in T and UT pre- and post-RT (P<0.05). Similarly, the 1RM increase in T did not differ between CNTF genotypes pre-to post-RT (p>0.05).In contrast, men with the CNTF A allele (i.e., GA/AA) gained more absolute (1.4 ± 0.2 vs 0.7 ± 0.1kg), relative (12.2 ± 1.7vs 6.1 ± 0.9%), and allometric (0.2 ± 0.0 vs 0.1 ± 0. lkg-kg-0.67) 1RM in UT than CNTF GG homozygotes pre- to post- RT (p<0.05). Among women 1RM in T and UT was not different between CNTF genotypes pre-, post-, and pre- to post-RT (P<0.05). CONCLUSION: Men with the A allele of the CNTF GA polymorphism gained greater absolute, relative, and allometric dynamic muscle strength in UT than men with the CNTF GG genotype. The CNTF GA polymorphism did not alter muscle strength response to RT in T among men or women. Our findings suggest the CNTF GA polymorphism is involved with neurologic adaptations to RT among men.

PURPOSE: Genetic variation in insulin-like growth factor 2 (IGF2), a peptide hormone within the IGF system, is associated with adiposity in adult males. Genetic variants have not been identified to explain exercise adherence, though 50% of the variability in adulthood physical activity levels is due to genetic factors. Thus, we examined if the IGF2 G3579C polymorphism (IGF2 GC) and adiposity associated with adherence to a resistance training (RT) program. METHODS: Subjects were 649 (mean ± SEM, 24.5 ± 0.2 yr) mostly white (78%), men (41%) and women (59%) with a body mass index (BMI) of 24.7±0.7 kg/m2 and had not performed RT in the previous yr. Subj ects enrolled in a 12 wk (2 d/wk) supervised unilateral upper-arm RT program. Exercise adherence was categorized into two groups: completers (n=597, completed 24 RT sessions) and drop outs (DO) (n=52, seceded prior to completing 24 RT sessions with a mean of 9.0 ± 0.9 sessions completed). Adiposity was categorized by BMI as normal weight (n=400, NW=21.9 ± 0.1 kg/m2) and overweight (n=249, OW=29.2 ±0.3 kg/m2). Chi-Square (χ2) tested for differences in exercise adherence among IGF2 GC genotype and adiposity groups. RESULTS: More IGF2 CC homozygotes (n=1 93) did not complete the RT program compared to carriers of the IGF2 G allele (n=456, GC/GG), 13.0 vs 5.9% DO, respectively (p<0.01). In addition, more NW subjects with the IGF2 CC genotype (n=1 10, 22.0 ± 0.1 kg/m2) did not complete the RT program than NW carriers of the IGF2G allele (n=283, 21.9 ±0.1 kg/m2), 10.0 vs 4.0% DO (p<0.05). Exercise adherence was not associated with IGF2 GC genotype in the OW subjects (p>0.05). CONCLUSIONS: NW adults with the IGF2 G allele appear to be more likely to adhere to an RT program than those with the IGF2 CC genotype. However, no association between the IGF2 GC genotype and exercise adherence was found in OW subjects. Further work is needed to determine the pathways through which IGF2 GC genotype and adiposity may associate with exercise adherence.

PURPOSE The Angiotensin Converting Enzyme Insertion allele (ACE I) is favorably associated with endurance performance, whereas the ACE Deletion allele (ACE D) with muscle strength and power-oriented performance. The purpose of this study was to examine if the ACE I/D polymorphism was related to the muscle strength and size responses to a unilateral, upper arm resistance training (RT) program among apparently healthy, young adults. We hypothesized the ACE D allele would be associated with greater increases in muscle strength and size than the ACE I allele. METHODS Subjects were 620 (mean ± SEM, 24.5±1.7 yr) mostly white (80%), mean (42%) and women (58%) who had not performed RT in the previous yr. Subjects completed 12 wk (2 d/wk) of supervised upper-arm RT. Peak elbow flexor muscle strength (kg) was assessed by isometric testing and 1 repetition maximum (1RM). Biceps muscle cross sectional area (cm2) was measured by magnetic resonance imaging. Multivariate ANOVA tested if muscle strength and size differed between ACE I/D pre-RT, post-RT, and pre- to post-RT. Because ACE II (n=146) and ID (n=288) did not differ in their RT response, these genotypes were combined and data are reported for ACE II/ID (n=434) versus DD (n=186). RESULTS Pre-RT absolute elbow flexor peak isometric (45.0±0.7 vs 45.4±1.1 kg) and 1RM (9.1±0.1 vs 9.2±0.2 kg) strength and biceps size (17.9±0.2 vs 17.2±0.4 cm2) did not differ between ACE II/ID and DD, respectively. Similarly, post-RT absolute elbow flexor peak isometric (53.6±0.8 vs 52.2±1.3 kg) and 1RM (13.1±0.1 vs 13.2±0.2 kg) strength and biceps size (21.2±0.3 vs 20.6±0.4 cm2) were not different between ACE II/ID and DD (p > 0.05). Absolute (8.6±0.4 vs 6.8±0.6 kg), relative (% of initial values) (22.2±1.0 vs 16.8±1.6 %) and allometric (0.5±0.0 vs 0.4±0.0 kg*kg−0.67) peak elbow flexor isometric strength increases pre- to post-RT were greater for ACE II/ID than DD, respectively (p < 0.01). In contrast, absolute (4.0±0.1 vs 4.0±0.1 kg), relative (51.0±1.5 vs 51.2±2.3 %) and allometric (0.2±0.0 vs 0.2±0.0 kg*kg−0.67) peak elbow flexor strength gains as measured by 1RM; and absolute (3.3±0.1 vs 3.4±0.1 cm2) and relative (18.6±0.5 vs 19.9±0.8 %) muscle size gains were not different between ACE II/ID and DD pre- to post-RT (p > 0.05). CONCLUSION Contrary to our hypothesis, ACE DD gained less peak elbow flexor isometric strength than ACE II/ID genotypes pre- to post-RT, despite similar gains in 1RM and muscle size. Reasons for these differences between the muscle strength response to RT and ACE I/D genotypes are not clear and merit further investigation. Supported by NIH-NINDS R01 NS40606-02

There is an association between physical activity and increased bone mass. This is demonstrated by the observation that dominant limbs have greater bone mass than non-dominant limbs, and that athletes loading their dominant limbs preferentially while exercising (e.g., tennis players) develop greater bilateral disparity. PURPOSE To determine if elbow flexion resistance training causes cortical bone growth in the humerus, and to determine if a growth difference exists between the proximal and distal humeral regions. METHODS 20 subjects (10F, 10M) (24.5±1.5 y, 72.3±3.7 kg, 166.4±3.8 cm, mean ± SEM) participated in a 12wk non-dominant biceps and triceps strength training program employing progressive periodized training consisting of 4wks of 12 reps (65–75% 1RM), 5wks of 8 reps (75–82% 1RM) and 3wks of 6 reps (83–90% 1RM). Exercises included a preacher bench curl, overhead triceps extension, concentration curl, triceps extension, and standing curl. Subjects were studied pre- and post-training using MRI. A series of axial slices (15 slices, 16mm slice thickness, 0mm interscan spacing, 256 × 192 matrix, 22 × 22cm FOV) were collected to study a 24cm length of the upper arm. Volumes were calculated in both non-dominant (trained) and dominant (untrained) arms by determining the number of pixels in an assigned region of interest (ROI) within each successive slice, calculating the area (cm2) of each ROI, multiplying by the slice thickness (1.6cm), and summing the successive slice volumes. Pre-training volumes subtracted from post-training volumes yielded training-induced change in volume. RESULTS Training increased cortical bone volume in the distal shaft of the non-dominant humerus (9.6cm) by 7.0±2.1%, with no gain in the same region of dominant humerus (N = 20). Cortical bone volume in the proximal shaft of the non-dominant humerus increased by 1.1±0.7% (p = 0.0375, paired t-test vs distal humerus), with no gain in the same region of dominant humerus (10 subjects completed to date). CONCLUSIONS Twelve weeks strength training of the non-dominant upper arm is sufficient to cause significant adaptation of the distal humeral cortex; however, less adaptive growth occurs at the proximal end of the humerus. Supported by: NIH-IDS RO1 NS40606-02