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Genome-wide association studies (GWASs) have identified polymorphic loci associated with coronary artery disease (CAD) risk factors (i.e. serum lipids) in adult populations (42-69 y). We hypothesized that younger populations would show a greater relative genetic component due to fewer confounding variables. We examined the influence of 20 GWAS loci associated with serum lipids and insulin metabolism, in a university student cohort (n = 548; mean age = 24 y), and replicated statistically associated results in a second study cohort of primary school students (n = 810, mean age = 11.5 y). Nineteen loci showed no relationship with studied risk factors in young adults. However, the ancestral allele of the rs646776 (SORT1) locus was strongly associated with increased LDL (C) in young adults [TT: 97.6 ± 1.0 mg/dL (n = 345) versus CT/CC: 87.3 ± 1.0 mg/dL (n = 203); p = 3 × 10(x6)] and children [TT: 94.0 ± 1.3 mg/dL (n = 551) versus CT/CC: 84.7 ± 1.4 mg/dL (n = 259); p = 4 × 10(x6)]. This locus is responsible for 3.6% of population variance in young adults and 2.5% of population variance in children. The effect size of the SORT1 locus is considerably higher in young populations (2.5-4.1%) compared with older subjects (1%).

A 12 kb haplotype upstream of the key signaling protein gene, AKT1, has been associated with insulin resistance and metabolic syndrome (Devaney et al. 2010). The region contains the first exon and promoter sequences of AKT1, but also includes the complete transcript unit for a highly conserved yet uncharacterized zinc finger-containing protein (ZBTB42). One of the component SNPs of the 12 kb haplotype metabolic syndrome haplotype changes a conserved amino acid in the predicted ZBTB42 protein, increasing the potential significance of the ZBTB42 transcript unit for contributing to disease risk. Using RT-PCR of human and mouse cells, we verified that the two exon ZBTB42 was expressed and correctly spliced in human skeletal muscle, and murine C2C12 cells. Production of peptide antibodies showed the expected protein in human (47 kD) and mouse (49 kD) immunoblots, and murine tissue distribution showed strongest expression in muscle and ovary. Immunostaining showed nuclear localization of the ZBTB42 protein in human muscle. Confocal imaging analyses of murine muscle showed ZBTB42 distributed in the nucleoplasm, with particular enrichment in nuclei underlying the neuromuscular junctions. The genetic association data of metabolic syndrome, coupled with the molecular characterization of the ZBTB42 transcript unit and encoded protein presented here, suggests that ZBTB42 may be involved in metabolic syndrome phenotypes.

We performed a meta-analysis of 14 genome-wide association studies of coronary artery disease (CAD) comprising 22,233 individuals with CAD (cases) and 64,762 controls of European descent followed by genotyping of top association signals in 56,682 additional individuals. This analysis identified 13 loci newly associated with CAD at P < 5 × 10⁻⁸ and confirmed the association of 10 of 12 previously reported CAD loci. The 13 new loci showed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6% to 17% increase in the risk of CAD per allele. Notably, only three of the new loci showed significant association with traditional CAD risk factors and the majority lie in gene regions not previously implicated in the pathogenesis of CAD. Finally, five of the new CAD risk loci appear to have pleiotropic effects, showing strong association with various other human diseases or traits.

Recently, a genome-wide association study (GWAS) that identified eight single-nucleotide polymorphisms (SNPs) associated with BMI highlighted a possible neuronal influence on the development of obesity. We hypothesized these SNPs would govern the response of BMI and subcutaneous fat to resistance training in young individuals (age = 24 years). We genotyped the eight GWAS-identified SNPs in the article by Willer et al. in a cohort (n = 796) that undertook a 12-week resistance-training program. Females with a copy of the rare allele (C) for rs17782313 (MC4R) had significantly higher BMIs (, Cc/ct: n = 174; 24.70 ± 0.33 kg/m², TT: n = 278; 23.41 ± 0.26 kg/m², P = 0.002), and the SNP explained 1.9% of overall variation in BMI. Males with a copy of the rare allele (T) for rs6548238 (TMEM18) had lower amounts of subcutaneous fat pretraining (CT/TT: n = 65; 156,534 ± 7,415 mm³, CC: n = 136; 177,825 ± 5,139 mm³, P = 0.019) and males with a copy of the rare allele (A) for rs9939609 (FTO) lost a significant amount of subcutaneous fat with exercise (, At/aa: n = 83; -798.35 ± 2,624.30 mm³, TT: n = 47; 9,435.23 ± 3,494.44 mm³, P = 0.021). Females with a copy of the G allele for a missense variant in the SH2B1 (rs7498665) was associated with less change of subcutaneous fat volume with exercise (, Ag/gg: n = 191; 9,813 ± 2,250 mm³ vs. AA: n = 126; 770 ± 2,772 mm³; P = 0.011). These data support the original finding that there is an association between measures of obesity and a variant near the MC4R gene and extends these results to a younger population and implicates FTO, TMEM18, and SH2B1 polymorphisms in subcutaneous fat regulation.

Converging lines of evidence suggest that AKT1 is a major mediator of the responses to insulin,insulin-like growth factor 1 (IGF1), and glucose. AKT1 also plays a key role in the regulation of both muscle cell hypertrophy and atrophy. We hypothesized that AKT1 variants may play a role in the endophenotypes that makeup metabolic syndrome. We studied a 12-kb region including the first exon of the AKT1 gene for association with metabolic syndrome-related phenotypes in four study populations [FAMUSS cohort (n = 574; age 23.7 ± 5.7 years), Strong Heart Study (SHS) (n = 2,134; age 55.5 ± 7.9 years), Dynamics of Health, Aging and Body Composition (Health ABC) (n = 3,075; age 73.6 ± 2.9 years), and Studies of a Targeted Risk Reduction Intervention through Defined Exercise (STRRIDE)(n = 175; age 40–65 years)]. We identified a three SNP haplotype that we call H1, which represents the ancestral alleles eles at the three loci and H2, which represents the derived alleles at the three loci. In young adult European Americans (FAMUSS), H1 was associated with higher fasting glucose levels in females. In middle age Native Americans (SHS), H1 carriers showed higher fasting insulin and HOMA in males, and higher BMI in females. Inolder African-American and European American subjects(Health ABC) H1 carriers showed a higher incidence of metabolic syndrome. Homozygotes for the H1 haplotype showed about twice the risk of metabolic syndrome in both males and females (p < 0.001). In middle-aged European Americans with insulin resistance (STRRIDE) studied by intravenous glucose tolerance test (IVGTT), H1 carriers showed increased insulin resistance due to the Sg component (p = 0.021). The 12-kb haplotype is a risk factor for metabolic syndrome and insulin resistance that needs to be explored in further populations.

Objective: Glycated hemoglobin (HbA₁(c)), used to monitor and diagnose diabetes, is influenced by average glycemia over a 2- to 3-month period. Genetic factors affecting expression, turnover, and abnormal glycation of hemoglobin could also be associated with increased levels of HbA₁(c). We aimed to identify such genetic factors and investigate the extent to which they influence diabetes classification based on HbA₁(c) levels., Research Design and Methods: We studied associations with HbA₁(c) in up to 46,368 nondiabetic adults of European descent from 23 genome-wide association studies (GWAS) and 8 cohorts with de novo genotyped single nucleotide polymorphisms (SNPs). We combined studies using inverse-variance meta-analysis and tested mediation by glycemia using conditional analyses. We estimated the global effect of HbA₁(c) loci using a multilocus risk score, and used net reclassification to estimate genetic effects on diabetes screening., Results: Ten loci reached genome-wide significant association with HbA(1c), including six new loci near FN3K (lead SNP/P value, rs1046896/P = 1.6 × 10⁻²⁶), HFE (rs1800562/P = 2.6 × 10⁻²⁰), TMPRSS6 (rs855791/P = 2.7 × 10⁻¹⁴), ANK1 (rs4737009/P = 6.1 × 10⁻¹²), SPTA1 (rs2779116/P = 2.8 × 10⁻⁹) and ATP11A/TUBGCP3 (rs7998202/P = 5.2 × 10⁻⁹), and four known HbA₁(c) loci: HK1 (rs16926246/P = 3.1 × 10⁻⁵⁴), MTNR1B (rs1387153/P = 4.0 × 10⁻¹¹), GCK (rs1799884/P = 1.5 × 10⁻²⁰) and G6PC2/ABCB11 (rs552976/P = 8.2 × 10⁻¹⁸). We show that associations with HbA₁(c) are partly a function of hyperglycemia associated with 3 of the 10 loci (GCK, G6PC2 and MTNR1B). The seven nonglycemic loci accounted for a 0.19 (% HbA₁(c)) difference between the extreme 10% tails of the risk score, and would reclassify ∼2% of a general white population screened for diabetes with HbA₁(c)., Conclusions: GWAS identified 10 genetic loci reproducibly associated with HbA₁(c). Six are novel and seven map to loci where rarer variants cause hereditary anemias and iron storage disorders. Common variants at these loci likely influence HbA₁(c) levels via erythrocyte biology, and confer a small but detectable reclassification of diabetes diagnosis by HbA₁(c).

A small 12-kb haplotype upstream of the AKT1 gene has been found to be associated with insulin resistance phenotypes. We sought to define the functional consequences of the three component polymorphic loci (rs1130214, rs10141867, rs33925946) on AKT1 and the upstream ZBTB42 gene. 5' RACE analysis of AKT1 transcripts in human skeletal muscle biopsies showed the predominant promoter to be 2.5 kb upstream of exon 2, and distinct from those promoters previously reported in rat. We then studied the effect of each of the three haplotype polymorphisms in transcriptional reporter assays in muscle, bone, and fat cell culture models, and found that each modulated enhancer and repressor activity are in a cell-specific and differentiation-specific manner. Our results in promoter assays are consistent with the human phenotype data; we found an anabolic effect on muscle and bone with increased mRNA expression of AKT1, and catabolic effect on fat with decreased expression. To test the hypothesis that rs10141867 affects transcription levels of the novel zinc finger protein ZBTB42 in vivo, we developed the allele-specific expression assay using Taqman technology to test for allelic differences within heterozygotes. The allele containing the derived polymorphism (haplotype H2) showed a 1.75-fold increase in expression in human skeletal muscle. Our data show a particularly complex effect of the component polymorphisms of a single haplotype on cells and tissues, suggesting that the coordination of different tissue-specific effects may have driven selection for the H2 haplotype. In light of the recent abundance of SNP association studies, our approach can serve as a method for exploring the biological function of polymorphisms that show significant genotype/phenotype associations.

Baseline muscle size and muscle adaptation to exercise are traits with high variability across individuals. Recent research has implicated several chemokines and their receptors in the pathogenesis of many conditions that are influenced by inflammatory processes, including muscle damage and repair. One specific chemokine, chemokine (C-C motif) ligand 2 (CCL2), is expressed by macrophages and muscle satellite cells, increases expression dramatically following muscle damage, and increases expression further with repeated bouts of exercise, suggesting that CCL2 plays a key role in muscle adaptation. The present study hypothesizes that genetic variations in CCL2 and its receptor (CCR2) may help explain muscle trait variability. College-aged subjects [n = 874, Functional Single-Nucleotide Polymorphisms Associated With Muscle Size and Strength (FAMUSS) cohort] underwent a 12-wk supervised strength-training program for the upper arm muscles. Muscle size (via MR imaging) and elbow flexion strength (1 repetition maximum and isometric) measurements were taken before and after training. The study participants were then genotyped for 11 genetic variants in CCL2 and five variants in CCR2. Variants in the CCL2 and CCR2 genes show strong associations with several pretraining muscle strength traits, indicating that inflammatory genes in skeletal muscle contribute to the polygenic system that determines muscle phenotypes. These associations extend across both sexes, and several of these genetic variants have been shown to influence gene regulation.

Objectives: We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD)., Background: Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with noncarriers., Methods: The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports., Results: A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups., Conclusions: The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study., (Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Previous studies have reported associations of polymorphisms in the IGF1 gene with phenotypes of body composition (BC). The purpose of this study was to identify phenotypes of BC and physical function that were associated with the IGF1 promoter polymorphism (rs35767, -C1245T). Subjects from the Health, Aging, and Body Composition Study, white males and females (n = 925/836) and black males and females (533/705) aged 70-79 years were genotyped for the polymorphism. Phenotypes of muscle size and function, bone mineral density, and BC were analyzed for associations with this polymorphism. To validate and compare these findings, a cohort of young (mean age = 24.6, SD = 5.9) white men and women (n = 173/296) with similar phenotypic measurements were genotyped. An association with BC was identified in elderly females when significant covariates (physical activity, age, smoking status, body mass index) were included. White women with C/C genotype had 3% more trunk fat and 2% more total fat than those with C/T (P < 0.05). Black women with C/C genotype had 3% less total lean mass and 3% less muscle mass than their T/T counterparts (P < 0.05). Associations were identified with muscle strength in white women (P < 0.01) that were in agreement with the C/C genotype having lower muscle function. Thus, in an elderly population but not a young population, a polymorphism in the IGF1 gene may be predictive of differences in body composition, primarily in black females.

Novel eccentric (lengthening contraction) exercise typically results in muscle damage, which manifests as prolonged muscle dysfunction, delayed onset muscle soreness, and leakage of muscle proteins into circulation. There is a large degree of variability in the damage response of individuals to eccentric exercise, with higher responders at risk for potentially fatal rhabdomyolysis. We hypothesized that single nucleotide polymorphisms (SNPs) in chemokine ligand 2 (CCL2) and its receptor chemokine receptor 2 (CCR2) associate with the high degrees of variability in the muscle damage response. We based this hypothesis on CCL2's roles in macrophage and satellite cell signaling in injured muscle. DNA was obtained from 157 untrained men and women following maximal eccentric exercise. Strength loss, soreness, serum creatine kinase (CK), and myoglobin levels before and during recovery from a single exercise bout were tested for association with 16 SNPs in CCL2 and CCR2. The rare alleles for rs768539 and rs3918358 (CCR2) were significantly (P<0.05) associated with lower preexercise strength in men, whereas CCL2 SNPs (rs13900, rs1024611, and rs1860189) and CCR2 (rs1799865) were associated with altered preexercise CK levels in women. During recovery, the rs3917878 genotype (CCL2) was associated with attenuated strength recovery in men and an elevated CK response in women. CCR2 variants were associated with slower strength recovery in women (rs3918358) and elevated soreness (rs1799865) across all subjects. In summary, we found that SNPs in CCL2 and CCR2 are associated with exercise-induced muscle damage and that the presence of certain variants may result in an exaggerated damage response to strenuous exercise.

Smoking is a leading global cause of disease and mortality. We established the Oxford-GlaxoSmithKline study (Ox-GSK) to perform a genome-wide meta-analysis of SNP association with smoking-related behavioral traits. Our final data set included 41,150 individuals drawn from 20 disease, population and control cohorts. Our analysis confirmed an effect on smoking quantity at a locus on 15q25 (P = 9.45 x 10(-19)) that includes CHRNA5, CHRNA3 and CHRNB4, three genes encoding neuronal nicotinic acetylcholine receptor subunits. We used data from the 1000 Genomes project to investigate the region using imputation, which allowed for analysis of virtually all common SNPs in the region and offered a fivefold increase in marker density over HapMap2 (ref. 2) as an imputation reference panel. Our fine-mapping approach identified a SNP showing the highest significance, rs55853698, located within the promoter region of CHRNA5. Conditional analysis also identified a secondary locus (rs6495308) in CHRNA3.

Unlabelled: Participation in regular aerobic exercise has been shown to increase arterial size and that exercise-induced vascular remodeling may be regional rather than systemic. However, these issues have been minimally investigated concerning resistance training., Purposes: To determine whether 1) resistance training of the nondominant arm elicits an increase in diameter of the brachial artery and 2) unilateral training induces arterial remodeling in the contralateral arm., Methods: Twenty-four previously untrained participants, consisting of 18 females (aged 22.3 +/- 5.1 yr) and 6 males (aged 21.7 +/- 1.8 yr), participated in unilateral strength training of the biceps and triceps for 12 wk using their nondominant arm. Isotonic (one-repetition maximum, 1RM) and isometric (ISO) strength of the biceps were assessed before and after training on both arms. Brachial artery diameter and biceps muscle cross-sectional area (CSA) of both arms were also measured before and after training using magnetic resonance imaging (MRI)., Results: Brachial artery diameter increased 5.47% (P < 0.05) in the nondominant trained arm with no change observed in the dominant untrained arm. Biceps CSA increased 18.3% (P < 0.05) in the trained arm with no change (P > 0.05) in the untrained limb. Nondominant 1RM and ISO strength increased by 35.1% and 16.8%, respectively (P < 0.05 for both), although there were no significant changes (P > 0.05) in the contralateral arm. A modest correlation was found between the increases in CSA and in brachial artery diameter (r2 = 0.19, P = 0.039)., Conclusions: These results indicate that upper arm vascular remodeling, manifesting as increased brachial artery diameter, can result from resistance training and that these changes are localized to the trained limb and associated with increases in CSA.

The present study examined associations between the ciliary neurotrophic factor (CNTF) 1357 G --> A polymorphism and the muscle strength response to a unilateral, upper arm resistance-training (RT) program among healthy, young adults. Subjects were 754 Caucasian men (40%) and women (60%) who were genotyped and performed a training program of the nondominant (trained) arm with the dominant (untrained) arm as a comparison. Peak elbow flexor strength was measured with one repetition maximum, isometric strength with maximum voluntary contraction, and bicep cross-sectional area with MRI in the trained and untrained arms before and after training. Women with the CNTF GG genotype gained more absolute isometric strength, as measured by MVC (6.5 +/- 0.3 vs. 5.2 +/- 0.5 kg), than carriers of the CNTF A1357 allele in the trained arm pre- to posttraining (P < 0.05). No significant associations were seen in men. Women with the CNTF GG genotype gained more absolute dynamic (1.0 +/- 0.1 vs. 0.6 +/- 0.1 kg) and allometric (0.022 +/- 0.0 vs. 0.015 +/- 0.0 kg/kg(-0.67)) strength, as measured by 1 RM, than carriers of the CNTF A1357 allele in the untrained arm pre- to posttraining (P < 0.05). No significant associations were seen in men. No significant associations, as measured by cross-sectional area, were seen in men or women. The CNTF 1357 G --> A polymorphism explains only a small portion of the variability in the muscle strength response to training in women.

The purpose of this study was to assess the association of age with muscle mass and strength in a group of young adults before and after 12 weeks of progressive resistance training. Eight hundred twenty-six young males and females (age 24.34 +/- 5.69 yr, range 18-39 yr) completed a strictly supervised 12-week unilateral resistance training program of the nondominant arm. Isometric (maximal voluntary contraction [MVC]) and dynamic strength (1 repetition maximum [1RM]) of the elbow flexors and cross-sectional area (CSA) of the biceps-brachii using magnetic resonance imaging (MRI) scans were measured before and after training. Pearson correlation coefficients were calculated for size and strength variables and age. In addition, the cohort was divided into groups according to decade of life and differences assessed by analysis of variance. Age correlated significantly and positively with all pretraining measures of muscle size and strength (CSA: r = 0.191, p < 0.001; MVC: r = 0.109, p = 0.002; 1RM: r = 0.109, p = 0.002). Age was not related to the training-induced changes in CSA or MVC but was negatively associated with the change in 1RM (r = -0.217, p < 0.001). The study indicates that age does have a significant positive relationship with muscle size and strength in untrained young adults. Although age was negatively associated with improvements in 1RM, the effect of age was small relative to the improvements induced through resistance training, thus suggesting age does not limit response to training in any practical way during early adulthood.

A classic morphogen, bone morphogenetic protein 2 (BMP2) regulates the differentiation of pluripotent mesenchymal cells. High BMP2 levels promote osteogenesis or chondrogenesis and low levels promote adipogenesis. BMP2 inhibits myogenesis. Thus, BMP2 synthesis is tightly controlled. Several hundred nucleotides within the 3' untranslated regions of BMP2 genes are conserved from mammals to fishes indicating that the region is under stringent selective pressure. Our analyses indicate that this region controls BMP2 synthesis by post-transcriptional mechanisms. A common A to C single nucleotide polymorphism (SNP) in the BMP2 gene (rs15705, +A1123C) disrupts a putative post-transcriptional regulatory motif within the human ultra-conserved sequence. In vitro studies indicate that RNAs bearing the A or C alleles have different protein binding characteristics in extracts from mesenchymal cells. Reporter genes with the C allele of the ultra-conserved sequence were differentially expressed in mesenchymal cells. Finally, we analyzed MRI data from the upper arm of 517 healthy individuals aged 18-41 years. Individuals with the C/C genotype were associated with lower baseline subcutaneous fat volumes (P = 0.0030) and an increased gain in skeletal muscle volume (P = 0.0060) following resistance training in a cohort of young males. The rs15705 SNP explained 2-4% of inter-individual variability in the measured parameters. The rs15705 variant is one of the first genetic markers that may be exploited to facilitate early diagnosis, treatment, and/or prevention of diseases associated with poor fitness. Furthermore, understanding the mechanisms by which regulatory polymorphisms influence BMP2 synthesis will reveal novel pharmaceutical targets for these disabling conditions., ((c) 2009 Wiley-Liss, Inc.)

Purpose: We examined associations among myostatin (MSTN) 2379 A > G and 163 G > A and follistatin (FST) -5003 A > T and -833 G > T single nucleotide polymorphisms (SNP) on the muscle size and the strength response to resistance training (RT)., Methods: Subjects (n = 645, age = 24.1 +/- 0.2 yr, body mass index [BMI] = 24.2 +/- 0.2 kg x m(-2)) self-disclosed themselves as Caucasian (78.9%), African American (3.6%), Asian (8.4%), Hispanic (5.0%), or Other (4.2%). They were genotyped for MSTN 2379 A > G (n = 645), MSTN 163 G > A (n = 639), FST -5003 A > T (n = 580), and FST -833 G > T (n = 603). We assessed dynamic (one repetition maximum [1RM]) and isometric (maximum voluntary contraction [MVC]) muscle strength and size (cross-sectional area [CSA]) of the elbow flexors before and after 12 wk of unilateral upper-arm RT. Repeated-measures ANCOVA tested associations among genetic variants and muscle phenotypes with age and BMI as covariates., Results: Baseline MVC was greater among African Americans who were carriers of the MSTN G(2379) allele (AG/GG, n = 15) than the A2379A homozygotes (n = 8; 64.2 +/- 6.8 vs 49.8 +/- 8.7 kg). African Americans who were carriers of the FST T(-5003) allele (n = 12) had greater baseline 1RM (11.9 +/- 0.7 vs 8.8 +/- 0.5 kg) and CSA (24.4 +/- 1.3 vs 19.1 +/- 1.2 cm(2)) than African Americans with the A-5003A genotype (n = 14; P < 0.05). No MSTN or FST genotype and muscle phenotype associations were found among the other ethnic groups (P >or= 0.05)., Conclusion: MSTN 2379 A > G and FST -5003 A > T were associated with baseline muscle strength and size among African Americans only. These ethnic-specific associations are hypothesis generating and should be confirmed in a larger sample of African Americans.

Background: A common SNP upstream of the INSIG2 gene, rs7566605 (g.-10,1025G>C, Chr2:118,552,255, NT&#95;022135.15), was reported to be associated with obesity (Body Mass Index, [BMI]) in a genome-wide association scan using the Framingham Heart Study but has not been reproduced in other cohorts. As BMI is a relatively insensitive measure of adiposity that is subject to many confounding variables, we sought to determine the relationship between the INSIG2 SNP and subcutaneous fat volumes measured by MRI in a young adult population., Methods: We genotyped the INSIG2 SNP rs7566605 in college-aged population enrolled in a controlled resistance-training program, (the Functional Polymorphism Associated with Human Muscle Size and Strength, FAMuSS cohort, n = 752 volunteers 18-40 yrs). In this longitudinal study, we examined the effect of the INSIG2 polymorphism on subcutaneous fat and muscle volumes of the upper arm measured by magnetic resonance imaging (MRI) before and after 12 wks of resistance training. Gene/phenotype associations were tested using an analysis of covariance model with age and weight as covariates. Further, the % variation in each phenotype attributable to genotype was determined using hierarchical models and tested with a likelihood ratio test., Results: Women with a copy of the C allele had higher levels of baseline subcutaneous fat (GG: n = 139; 243473 +/- 5713 mm3 vs. GC/CC: n = 181; 268521 +/- 5003 mm3; p = 0.0011); but men did not show any such association. Men homozygous for the G ancestral allele showed a loss of subcutaneous fat, while those with one or two copies of the C allele gained a greater percentage of subcutaneous fat with resistance training (GG: n = 103; 1.02% +/- 1.74% vs. GC/CC: n = 93; 6.39% +/- 1.82%; p = 0.035)., Conclusion: Our results show that the INSIG2 rs7566605 polymorphism underlies variation in subcutaneous adiposity in young adult women and suppresses the positive effects of resistance training on men. This supports and extends the original finding that there is an association between measures of obesity and INSIG2 rs7566605 and further implicates this polymorphism in fat regulation.

The aims of this study were to examine associations between two SNPs in the human IL-15 gene and three SNPs in the IL-15Ralpha gene with predictors of metabolic syndrome and phenotypes in muscle, strength, and bone at baseline and in response to resistance training (RT). Subjects were Caucasians who had not performed RT in the previous year and consisted of a strength cohort (n=748), volumetric cohort (n=722), and serum cohort (n=544). Subjects completed 12 weeks of unilateral RT of the non-dominant arm, using their dominant arm as an untrained control. ANCOVA analyses revealed gender-specific associations with: (1) IL-15 SNP (rs1589241) and cholesterol (p=0.04), LDL (p=0.02), the homeostasis model assessment (HOMA; p=0.03), and BMI (p=0.002); (2) IL-15 SNP (rs1057972) and the pre- to post-training absolute difference in 1RM strength (p=0.02), BMI (p=0.008), and fasting glucose (p=0.03); (3) IL-15Ralpha SNP (rs2296135) and baseline total bone volume (p=0.04) and the pre- to post-training absolute difference in isometric strength (p=0.01); and 4) IL-15Ralpha SNP (rs2228059) and serum triglycerides (p=0.04), baseline whole muscle volume (p=0.04), baseline cortical bone volume (p=0.04), and baseline muscle quality (p=0.04). All associations were consistent in showing a potential involvement of the IL-15 pathway with muscle and bone phenotypes and predictors of metabolic syndrome.

We examined the association of a novel single-nucleotide polymorphism (SNP) in IGF-I (IGF-I -C1245T located in the promoter) and eight SNPs in the IGF-II gene region with indicators of muscle damage [strength loss, muscle soreness, and increases in circulating levels of creatine kinase (CK) and myoglobin] after eccentric exercise. We also examined two SNPs in the IGF binding protein-3 (IGFBP-3). The age, height, and body mass of the 151 subjects studied were 24.1 +/- 5.2 yr, 170.8 +/- 9.9 cm, and 73.3 +/- 17.0 kg, respectively. There were no significant associations of phenotypes with IGF-I. IGF-II SNP (G12655A, rs3213216) and IGFBP-3 SNP (A8618T, rs6670) were not significantly associated with any variable. The most significant finding in this study was that for men, IGF-II (C13790G, rs3213221), IGF-II (ApaI, G17200A, rs680), IGF-II antisense (IGF2AS) (G11711T, rs7924316), and IGFBP-3 (-C1592A, rs2132570) were significantly associated with muscle damage indicators. We found that men who were 1) homozygous for the rare IGF-II C13790G allele and rare allele for the ApaI (G17200A) SNP demonstrated the greatest strength loss immediately after exercise, greatest soreness, and highest postexercise serum CK activity; 2) homozygous wild type for IGF2AS (G11711T, rs7924316) had the greatest strength loss and most muscle soreness; and 3) homozygous wild type for the IGF2AS G11711T SNP showed the greatest strength loss, highest muscle soreness, and greater CK and myoglobin response to exercise. In women, fewer significant associations appeared.

Objective: The biological function of resistin (RST) is unknown, although it may have roles in obesity, diabetes, and insulin resistance. The objective of this study was to examine the effects of single nucleotide polymorphisms (SNPs) in the human RST gene on muscle, bone, and adipose tissue phenotypes and in response to resistance training (RT)., Research Methods and Procedures: Subjects were white and consisted of strength (n = 482) and size (n = 409) cohorts who had not performed RT in the previous year. Subjects completed 12 weeks of structured, unilateral upper arm RT aimed at increasing the size and strength of the non-dominant arm, using their dominant arm as an untrained control. Strength measurements were taken pre- and post-12-week RT and consisted of elbow flexor isometric strength and one-repetition maximum during a biceps curl using free weights. Whole muscle, subcutaneous fat, and cortical bone volumes were measured by magnetic resonance imaging. Six RST SNPs were identified. Analysis of covariance was used to test for effects of the SNPs on pre- and post-muscle strength and whole muscle, fat, and bone volumes independent of gender, age, and body weight., Results: Five RST SNPs (-537 A>C, -420 C>G, 398 C>T, 540 G>A, 980 C>G) were associated with measured phenotypes among subjects when stratified by BMI (<25, >/ or = 25 kg/m(2)). Several gender-specific associations were observed between RST SNPs and phenotypes among individuals with a BMI > or = 25. Conversely, only two associations were observed among individuals with a BMI < 25., Discussion: These data support previous identified associations of RST with adipose tissue and demonstrate additional associations with bone and skeletal muscle that warrant further investigation.

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.

Purpose: To examine associations among the angiotensin I-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism and the response to a 12-wk (2 d.wk) unilateral, upper-arm resistance training (RT) program in the trained (T, nondominant) and untrained (UT, dominant) arms., Methods: Subjects were 631 (mean+/-SEM, 24.2+/-0.2 yr) white (80%) men (42%) and women (58%). The ACE ID genotype was in Hardy-Weinberg equilibrium with frequencies of 23.1, 46.1, and 30.8% for ACE II, ID, and DD, respectively (chi=1.688, P=0.430). Maximum voluntary contraction (MVC) and one-repetition maximum (1RM) assessed peak elbow flexor muscle strength. Magnetic resonance imaging measured biceps muscle cross-sectional area (CSA). Multiple variable and repeated-measures ANCOVA tested whether muscle strength and size differed at baseline and pre- to post-RT among T and UT and ACE ID genotype., Results: Baseline muscle strength and size were greater in UT than T (P<0.001) and did not differ among ACE ID genotype in either arm (P >or= 0.05). In T, MVC increases were greater for ACE II/ID (22%) than DD (17%) (P<0.05), whereas 1RM (51%) and CSA (19%) gains were not different among ACE ID genotype pre- to post-RT (P >or= 0.05). In UT, MVC increased among ACE II/ID (7%) (P<0.001) but was similar among ACE DD (2%) pre- to post-RT (P >or= 0.05). In UT, 1RM (11%) and CSA (2%) increases were greater for ACE DD/ID than ACE II (1RM, 7%; CSA, -0.1%) (P<0.05). ACE ID genotype explained approximately 1% of the MVC response to RT in T and approximately 2% of MVC, 2% of 1RM, and 4% of CSA response in UT (P<0.05)., Conclusion: ACE ID genotype is associated with the contralateral effects of unilateral RT, perhaps more so than with the muscle strength and size adaptations that result from RT.

Context: Recently, resistin was found to be present in atherosclerotic lesions in apoE(-/-) mice. Resistin may be associated with inflammation and atherosclerosis in humans; however, the role of resistin in human disease remains controversial., Objective: This study assesses cross-sectional relationships of resistin with coronary heart disease (CHD)., Design, Setting, and Participants: Blood samples from the third examination of the Strong Heart Study (SHS)--the largest study of CHD in American Indians--were used. Cases who had suffered previous myocardial infarction (n = 100) were selected randomly from the three SHS sites and matched for study site and sex with controls who had no history of cardiovascular disease (CHD or stroke) (n = 100)., Main Outcome Measure: Resistin levels by enzyme-linked immunosorbent assay method in cases and controls was the main outcome measure., Results: Resistin levels were higher in cases than controls [median (interquartile range): 3.4 (2.5-4.7) vs. 2.8 (2.1-4.0) ng/ml; P = 0.003] and had univariate correlations with age (Spearman r = 0.21; P < 0.002), fasting insulin (r = 0.21; P = 0.003), insulin resistance by homeostasis model (r = 0.22; P = 0.04), albumin to creatinine ratio (r = 0.19; P = 0.01), and fibrinogen (r = 0.34; P < 0.0001). Cases were more likely to have diabetes (cases 67%; controls 41%; P < 0.0001) but had similar body mass index (cases 31.4 +/- 5.4; controls 30.7 +/- 6.3; P = 0.85). Resistin levels were higher in participants with established nephropathy (albumin to creatinine ratio >300 mg/g, n = 26) compared with those with normo- (n = 122) or microalbuminuria (n = 42). In multivariate analysis, nephropathy (P = 0.0013) but not previous myocardial infarction (P = 0.12) was significantly associated with resistin., Conclusions: Resistin is not independently associated with CHD. Resistin is elevated in survivors of myocardial infarction; however, this reflects a novel association of raised resistin with diabetic nephropathy.

Resistin, an adipocyte-derived cytokine linked to insulin resistance and obesity, has recently been shown to activate endothelial cells (ECs). Using microarrays, we found that along with numerous other pro-atherosclerotic genes, resistin expression levels are elevated in the aortas of C57BL/6J apoE-/- mice; these findings led us to further explore the relation between resistin and atherosclerosis. Using TaqMan PCR and immunohistochemistry, we found that ApoE-/- mice had significantly higher resistin mRNA and protein levels in their aortas, and elevated serum resistin levels, compared to C57BL/6J wild-type mice. Incubation of murine aortic ECs with recombinant resistin increased monocyte chemoattractant protein (MCP)-1 and soluble vascular cell adhesion molecule (sVCAM)-1 protein levels in the conditioned medium. Furthermore, human carotid endarterectomy samples stained positive for resistin protein, while internal mammary artery did not show strong staining. Patients diagnosed with premature coronary artery disease (PCAD) were found to have higher serum levels of resistin than normal controls. In summary, resistin protein is present in both murine and human atherosclerotic lesions, and mRNA levels progressively increase in the aortas of mice developing atherosclerosis. Resistin induces increases in MCP-1 and sVCAM-1 expression in murine vascular endothelial cells, suggesting a possible mechanism by which resistin might contribute to atherogenesis. Finally, PCAD patients exhibited increased serum levels of resistin when compared to controls. These findings suggest a possible role of resistin in cardiovascular disease.

Strenuous exercise results in damage to skeletal muscle that is manifested in delayed muscle pain, prolonged strength loss, and increases in muscle proteins in the blood, especially creatine kinase (CK) and myoglobin (Mb). Some individuals experience profound changes in these variables in response to standard laboratory exercise or recreational activities. We proposed that variations in genes coding for two myofibrillar proteins [alpha-actinin 3 (ACTN3) and myosin light chain kinase (MLCK)] may explain the large variability in the response to muscle-damaging exercise. We hypothesized that subjects with specific single nucleotide polymorphisms (SNPs) in ACTN3 and MLCK would show a greater loss in muscle strength and/or a greater increase in blood CK and Mb in response to eccentric exercise. Blood from 157 subjects who performed a standard elbow flexion eccentric exercise protocol was tested for association between genotypes of ACTN3 (1 SNP tested: R577X) and MLCK (2 SNPs tested: C49T and C37885A) and changes in blood CK and Mb and isometric strength. Subjects possessing the ACTN3-deficient genotype (XX) had lower baseline CK compared with the heterozygotes (P = 0.035). After the eccentric exercise, those subjects homozygous for the MLCK 49T rare allele had a significantly greater increase in CK and Mb (P < 0.01) compared with the heterozygotes, and those heterozygous for MLCK C37885A had a significantly greater increase in CK compared with the homozygous wild type (P < 0.05). There was only one subject homozygous for the rare MLCK 37885A allele. MLCK C37885A was also associated with postexercise strength loss (P < 0.05); the heterozygotes demonstrated greater strength loss compared with the homozygous wild type (CC). These results show that variations in genes coding for specific myofibrillar proteins influence phenotypic responses to muscle damaging exercise.

The alpha-actinin 3 (ACTN3) gene encodes a protein of the Z disk of myofibers, and a polymorphism of ACTN3 results in complete loss of the protein. The ACTN3 genotype (R577X) has been found to be associated with performance in Australian elite athletes (Yang N, MacArthur DG, Gulbin JP, Hahn AG, Beggs AH, Easteal S, and North K. Am J Hum Genet 73: 627-631, 2003). We studied associations between ACTN3 genotype and muscle size [cross-sectional area of the biceps brachii via magnetic resonance imaging (MRI)] and elbow flexor isometric (MVC) and dynamic [1-repetition maximum (1-RM)] strength in a large group of men (N = 247) and women (N = 355) enrolled in a 12-wk standardized elbow flexor/extensor resistance training program of the nondominant arm at one of eight study centers. We found no association between ACTN3 R577X genotype and muscle phenotype in men. However, women homozygous for the ACTN3 577X allele (XX) had lower baseline MVC compared with heterozygotes (P < 0.05) when adjusted for body mass and age. Women homozygous for the mutant allele (577X) demonstrated greater absolute and relative 1-RM gains compared with the homozygous wild type (RR) after resistance training when adjusted for body mass and age (P < 0.05). There was a trend for a dose-response with genotype such that gains were greatest for XX and least for RR. Significant associations were validated in at least one ethnic subpopulation (Caucasians, Asians) and were independent of training volume. About 2% of baseline MVC and of 1-RM strength gain after training were attributable to ACTN3 genotype (likelihood-ratio test P value, P = 0.01), suggesting that ACTN3 is one of many genes contributing to genetic variation in muscle performance and adaptation to exercise.

Objectives: We sought to understand the genomic program leading to collateral vessel formation., Background: Recently, technology has advanced to the point that it is now possible to elucidate the large array of genes that must be expressed, as well as the temporal expression pattern, for the development of functionally important collateral vessels. In this investigation, we used deoxyribonucleic acid array expression profiling to determine the time course of differential expression of 12,000 genes after femoral artery ligation in C57BL/6 mice., Methods: Ribonucleic acid was extracted from the adductor muscle, which showed no signs of ischemia. Sampling was at baseline, 6 h, and 1, 3, 7, and 14 days after femoral artery ligation or sham operation., Results: Femoral artery ligation caused the differential expression (>2-fold) of 783 genes at one or multiple time points: 518 were induced and 265 were repressed. Cluster analysis generated four temporal patterns: 1) early upregulated (6 to 24 h)-immediate early transcriptional factors, angiogenesis, inflammation, and stress-related genes; 2) mid-phase upregulated (day 3)-cell cycle and cytoskeletal and inflammatory genes; 3) late upregulated (days 7 to 14)-angiostatic, anti-inflammatory, and extracellular matrix-associated genes; and 4) downregulated-genes involved in energy metabolism, water channel, and muscle contraction. Microarray data were validated using quantitative reverse transcription polymerase chain reaction., Conclusions: This study documents the large number of genes whose differential expression and temporal functional clustering appear to contribute to collateral formation. These results can serve as a genomic model for arteriogenesis and as a database for developing new therapeutic strategies.

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin-8 (IL-8) expression via an IL-1 receptor-associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll-like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK-TLR4) express TLR4 mRNA and protein and induce IL-8 promoter activity in response to NE. Treatment of both HEK-TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE-induced IL-8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL-8 expression in bronchial epithelium.

Rett syndrome is a neurodevelopmental disorder that affects females almost exclusively, and in which eight common point mutations on the X-linked MeCP2 gene are knows to cause over 70% of mutation-positive cases. We explored the use of a novel platform to detect the eight common mutations in Rett syndrome patients to expedite and simplify the process of identification of known genotypes. The Nanogen workstation consists of a two-color assay based on electric hybridization and thermal discrimination, all performed on an electronically active NanoChip. This genotyping platform was tested on 362 samples of a pre-determined genotype, which had been previously identified by a combination of DHPLC (denaturing high performance liquid chromatography) and direct sequencing. This genotyping technique proved to be rapid, facile, and displayed a specificity of 100% with 3% ambiguity. In addition, we present consecutive testing of seven mutations on a single pad of the NanoChip. This was accomplished by tagging down two amplimers together and serially hybridizing for seven different loci, allowing us to genotype samples for seven of the eight common Rett mutations on a single pad. This novel method displayed the same level of specificity and accuracy as the single amplimer reactions, and proved to be faster and more economical.

Background: Numerous DNA-based tests are currently in use or under development for the detection of mutations associated with disease. Most of the current methods use PCR amplification technologies and detection after separation or chromatography of the products. We have developed a panel of standard reference materials consisting of 12 plasmid clones containing a 2.0 kb region of the TP53 gene, including exons 5-9. Eleven of these clones contain a single mutation within the mutational hot spots of the TP53 gene, the twelfth is wild-type in this region of the gene. The mutations are amino acid (aa) 128: C to T; aa 175: G to A; aa 237: T to C; aa 245: G to A; aa 248: C to T; aa 248: G to A; aa 249: G to T; aa 273: C to T; aa 273: G to A; aa 282: C to T; and aa 328: T to C. These standard reference materials (SRMs), created by site-directed mutagenesis of wild-type TP53 from a human cell line, include the specific mutations most commonly found to be associated with cancer. Their use will improve disease detection by serving as validation materials to monitor errors in measurement methods, including PCR amplification, amplicon separation, and data analysis from different technology platforms., Methods and Results: The single point mutations of the panel were validated by capillary electrophoresis single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography, as well as full sequence analysis of both DNA strands of the cloned material. For both heteroduplex analysis methods, the presence of the mutations was resolved for each SRM., Conclusion: The generation of a standard TP53 reference panel and demonstration that the panel can successfully validate mutation detection across different mutation scanning technology platforms. Hence, this panel functions as an SRM to normalize results obtained from different laboratories using different techniques.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair-reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers., ((c) 2002 Elsevier Science (USA))

Perlecan, a large heparan sulfate proteoglycan, is a component of the basement membrane and other extracellular matrices and has been implicated in multiple biological functions. Mutations in the perlecan gene (HSPG2) cause two classes of skeletal disorders: the relatively mild Schwartz-Jampel syndrome (SJS) and severe neonatal lethal dyssegmental dysplasia, Silverman-Handmaker type (DDSH). SJS is an autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and chondrodysplasia, and patients with SJS survive. The molecular mechanism underlying the chondrodystrophic myotonia phenotype of SJS is unknown. In the present report, we identify five different mutations that resulted in various forms of perlecan in three unrelated patients with SJS. Heterozygous mutations in two patients with SJS either produced truncated perlecan that lacked domain V or significantly reduced levels of wild-type perlecan. The third patient had a homozygous 7-kb deletion that resulted in reduced amounts of nearly full-length perlecan. Unlike DDSH, the SJS mutations result in different forms of perlecan in reduced levels that are secreted to the extracellular matrix and are likely partially functional. These findings suggest that perlecan has an important role in neuromuscular function and cartilage formation, and they define the molecular basis involved in the difference in the phenotypic severity between DDSH and SJS.

Recently, a Leu309Met mutation in WKL1 (MLC1, KIAA0027), a gene mapped to chromosome 22q13.33, was reported to co-segregate with periodic catatonia, a clinical sub-type of schizophrenia, in seven members of an extended pedigree.(1) WKL1 encodes a putative membrane protein expressed exclusively in the brain, particularly in the amygdala, nucleus caudatus, thalamus, and hippocampus.(1) We screened WKL1 for etiologic mutations in 28 probands from the United States who were given a consensus diagnosis of schizophrenia and met at least one of these criteria: (1) were from multiplex schizophrenia families where at least two schizophrenic subjects were reported to display catatonic behavior at sometime during the course of their illness; or (2) were from multiplex schizophrenia families where, in a genome scan for schizophrenia susceptibility loci, evidence for excess allele sharing among affected family members for markers in the 22q13 region was seen. In addition, 15 affected subjects from 15 German pedigrees were similarly screened for causative mutations. This German cohort exhibited the catatonia phenotype but had ambiguous linkage to 22q13 and included the mutation-positive proband as a positive control. The 43 probands were screened for base changes in WKL1: 15 SNPs in the non-coding regions of the gene, three SNPs in the 3'UTR, four synonymous coding SNPs and two non-synonymous (amino acid changing) SNPs were identified. We were able to rapidly confirm the Leu309Met nucleotide change in the positive control. No missense mutations were detected in any of the other 42 probands studied. These data exclude the role of WKL1 in schizophrenia susceptibility in the subjects studied.

Background: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl CpG binding protein 2 (MeCP2) gene., Methods: One hundred sixteen patients with classical and atypical RTT were studied for mutations of the MeCP2 gene by using DHPLC and direct sequencing., Results: Causative mutations in the MeCP2 gene were identified in 63% of patients, representing a total of 30 different mutations. Mutations were identified in 72% of patients with classical RTT and one third of atypical cases studied (8 of 25). The authors found 17 novel mutations, including a complex gene rearrangement found in one individual involving two deletions and a duplication. The duplication was identical to a region within the 3' untranslated region (UTR), and represents the first report of involvement of the 3' UTR in RTT. The authors also report the identification of MeCP2 mutations in two males; a Klinefelter's male with classic RTT (T158M) and a hemizygous male infant with a Xq27-28 inversion and a novel 32 bp frameshift deletion [1154(del32)]. Studies examining the relationship between mutation type, X-inactivation status, and severity of clinical presentation found significant differences in clinical presentation between different types of mutations. Mutations in the amino-terminus were significantly correlated with a more severe clinical presentation compared with mutations closer to the carboxyl-terminus of MeCP2. Skewed X-inactivation patterns were found in two asymptomatic carriers of MeCP2 mutations and six girls diagnosed with either atypical or classical RTT., Conclusion: This patient series confirms the high frequency of MeCP2gene mutations causative of RTT in females and provides data concerning the molecular basis for clinical variability (mutation type and position and X-inactivation patterns).

Reliable amplification of short tandem repeat (STR) DNA markers with the polymerase chain reaction (PCR) is dependent on high quality PCR primers. The particular primer combinations and concentrations are especially important with multiplex amplification reactions where multiple STR loci are simultaneously copied. Commercially available kits are now widely used for STR amplification and subsequent DNA typing. We present here the use of high performance liquid chromatography (HPLC) and time-of-flight mass spectrometry (TOF-MS) methods for characterization of commercially available STR kits.

Currently, a major focus of human genetics is the utilization of single-nucleotide polymorphisms for clinical diagnostics, whole-genome linkage disequilibrium screens to identify common disease genes such as Alzheimer disease, determination of the recent evolutionary history of a species, and the process of speciation. We have examined single-nucleotide extension coupled with high-performance liquid chromatography as a method to simultaneously genotype two SNPs occurring in the coding region of the HFE gene that produce clinical effects. This assay allows concurrent genotyping of the C282Y and H63D mutations in 11 min and is 100% concordant with current testing methods for both of these mutations.

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.

Background: Familial polymorphic ventricular tachycardia is an autosomal-dominant, inherited disease with a relatively early onset and a mortality rate of approximately 30% by the age of 30 years. Phenotypically, it is characterized by salvoes of bidirectional and polymorphic ventricular tachycardias in response to vigorous exercise, with no structural evidence of myocardial disease. We previously mapped the causative gene to chromosome 1q42-q43. In the present study, we demonstrate that patients with familial polymorphic ventricular tachycardia have missense mutations in the cardiac sarcoplasmic reticulum calcium release channel (ryanodine receptor type 2 [RyR2])., Methods and Results: In 3 large families studied, 3 different RyR2 mutations (P2328S, Q4201R, V4653F) were detected and shown to fully cosegregate with the characteristic arrhythmic phenotype. These mutations were absent in the nonaffected family members and in 100 healthy controls. In addition to identifying 3 causative mutations, we identified a number of single nucleotide polymorphisms that span the genomic structure of RyR2 and will be useful for candidate-based association studies for other arrhythmic disorders., Conclusions: Our data illustrate that mutations of the RyR2 gene cause at least one variety of inherited polymorphic tachycardia. These findings define a new entity of disorders of myocardial calcium signaling.

Hereditary hemochromatosis is one of the most common hereditary disorders in Caucasians.The disease is linked to two single-nucleotide polymorphisms (SNPs) in the HFE gene.The two point mutations result in a change of Cys to Tyr at position 282 and His to Asp at position 63 in the resultant protein.We have developed a single-nucleotide extension (SNE) assay for hereditary hemochromatosis genetic testing, which employs capillary electrophoresis to simultaneously detect the SNE products generated from the two SNP sites. An upstream or a downstream primer adjacent to the possible mutation site is designed and extended one nucleotide further at the 3' end, complementary to the nucleotide at the possible mutation site.The extended nucleotide is one of four fluorescently labeled dideoxynucleotide triphosphates that also act as terminators. Analysis of the extended products by laser-induced fluorescence capillary electrophoresis (LIF-CE) directly reflects the identity of the possible mutation site. Using one primer upstream or downstream from the possible mutation site, three genotypes at one mutation site can be distinguished. Using both upstream and downstream primers provides a second level of specificity and increases the accuracy of the genetic test. The protocol can also be applied to the study of other SNP analyses and to simultaneous detection of multiple mutation sites.

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.

We describe a novel molecular screening technique for hereditary hemochromatosis through which HFE genotypes at codon positions 282 and 63 are simultaneously detected. The technique combines multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) and allows automated high-throughput analysis. We used this method to genotype 43 previously characterized anonymous DNA specimens in blinded fashion and found multiplex PCR/DHPLC 100% accurate when compared with PCR/restriction enzyme digestion, yet far more efficient.

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.

Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene-divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion-pairing reverse-phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double-stranded DNA sequencing properties. In this study, the IP-RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/ FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak-captured, then cycle-sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 microm and a total length of 47 centimeters. The total analysis time including capillary filling and pre-electrophoresis was less than 30 min for a 220-bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9, 9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised by the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column, hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing, and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time, and cost.

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.