Loss of ACTN3 gene function alters mouse muscle metabolism and shows evidence of positive selection in humans

More than a billion humans worldwide are predicted to be completely deficient in the fast skeletal muscle fiber protein α-actinin-3 owing to homozygosity for a premature stop codon polymorphism, R577X, in the ACTN3 gene. The R577X polymorphism is associ

DNA-Dependent Protein Kinase Inhibits AID-Induced Antibody Gene Conversion

Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID)-dependent hypermutation of Ig V(D)J rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(D)J regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(D)J hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion

A comparison of the clinical effectiveness and cost of specialised individually-delivered parent training for preschool attention-deficit/hyperactivity disorder and a generic, group-based programme: a multi-centre, randomised controlled trial of the New Forest Parenting Programme versus Incredible Years

Objective: To compare the efficacy and cost of specialised individually-delivered parent training (PT) for preschool children with attention-deficit/ hyperactivity disorder (ADHD) against generic group-based PT and treatment as usual (TAU). Design: Multi-centre, three-arm parallel group randomised controlled trial. Research Setting: National Health Service Trusts. Participants: Preschool children (33-54 months) fulfilling ADHD research diagnostic criteria. Interventions: New Forest Parenting Programme (NFPP) – 12 week individual, home-delivered ADHD PT programme; Incredible Years (IY) – 12 week group-based, PT programme initially designed for children with behaviour problems. Main outcome measures: Primary outcome - Parent ratings of child’s ADHD symptoms (Swanson, Nolan & Pelham Questionnaire - SNAP-IV). Secondary outcomes - teacher ratings (SNAP-IV) and direct observations of ADHD symptoms and parent/teacher ratings of conduct problems. NFPP, IY and TAU outcomes were measured at baseline (T1) and post-treatment (T2). NFPP and IY outcomes only were measured 6 months post treatment (T3). Researchers, but not therapists or parents, were blind to treatment allocation. Analysis employed mixed effect regression models (multiple imputation). Intervention and other costs were estimated using standardized approaches. Results: NFPP and IY did not differ on parent-rated SNAP-IV, ADHD combined symptoms (mean difference -0.009 95%CI [-0.191, 0.173], p=0.921) or any other measure. Small, non-significant, benefits of NFPP over TAU were seen for parent-rated SNAP-IV, ADHD combined symptoms (-0.189 95%CI [-0.380, 0.003], p=0.053). NFPP significantly reduced parent-rated conduct-problems compared to TAU across scales (p-values.05). The cost per family of providing NFPP in the trial was significantly lower than IY (£1,591 versus £2,103). Conclusions: Although, there were no differences between NFPP and IY with regards clinical effectiveness, individually-delivered NFPP cost less. However, this difference may be reduced when implemented in routine clinical practice. Clinical decisions should take into account parental preferences between delivery approaches. Funding: National Institute of Health Research. Trial Registration: Trial name: COPPI Trial; ISRCTN39288126

2012 ACCF/AHA/ACP/AATS/PCNA/SCAI/STS guideline for the diagnosis and management of patients with stable ischemic heart disease

The recommendations listed in this document are, whenever possible, evidence based. An extensive evidence review was conducted as the document was compiled through December 2008. Repeated literature searches were performed by the guideline development staff and writing committee members as new issues were considered. New clinical trials published in peer-reviewed journals and articles through December 2011 were also reviewed and incorporated when relevant. Furthermore, because of the extended development time period for this guideline, peer review comments indicated that the sections focused on imaging technologies required additional updating, which occurred during 2011. Therefore, the evidence review for the imaging sections includes published literature through December 2011

The effect of α-actinin-3 deficiency on muscle aging

Deficiency of the fast-twitch muscle protein α-actinin-3 due to homozygosity for a nonsense polymorphism (R577X) in the ACTN3 gene is common in humans. α-Actinin-3 deficiency (XX) is associated with reduced muscle strength/power and enhanced endurance performance in elite athletes and in the general population. The association between R577X and loss in muscle mass and function (sarcopenia) has previously been investigated in a number of studies in elderly humans. The majority of studies report loss of ACTN3 genotype association with muscle traits in the elderly, however, there is some indication that the XX genotype may be associated with faster muscle function decline. To further explore these potential age-related effects and the underlying mechanisms, we examined the effect of α-actinin-3 deficiency in aging male and female Actn3 knockout (KO) mice (2, 6, 12, and 18. months). Our findings support previous reports of a diminished influence of ACTN3 genotype on muscle performance in the elderly: genotype differences in intrinsic exercise performance, fast muscle force generation and male muscle mass were lost in aged mice, but were maintained for other muscle function traits such as grip strength. The loss of genotype difference in exercise performance occurred despite the maintenance of some "slower" muscle characteristics in KO muscles, such as increased oxidative metabolism and greater force recovery after fatigue. Interestingly, muscle mass decline in aged 18. month old male KO mice was greater compared to wild-type controls (WT) (α. 12.2% in KO; α. 6.5% in WT). These results provide further support that α-actinin-3 deficient individuals may experience faster decline in muscle function with increasing age

Evidence Based Selection of Commonly Used RT-qPCR Reference Genes for the Analysis of Mouse Skeletal Muscle

<div><p>The ability to obtain accurate and reproducible data using quantitative real-time Polymerase Chain Reaction (RT-qPCR) is limited by the process of data normalization. The use of ‘housekeeping’ or ‘reference’ genes is the most common technique used to normalize RT-qPCR data. However, commonly used reference genes are often poorly validated and may change as a result of genetic background, environment and experimental intervention. Here we present an analysis of 10 reference genes in mouse skeletal muscle (<i>Actb, Aldoa, Gapdh, Hprt1, Ppia, Rer1, Rn18s, Rpl27, Rpl41 and Rpl7L1</i>), which were identified as stable either by microarray or in the literature. Using the MIQE guidelines we compared wild-type (WT) mice across three genetic backgrounds (R129, C57BL/6j and C57BL/10) as well as analyzing the α-actinin-3 knockout (<i>Actn3</i> KO) mouse, which is a model of the common null polymorphism (R577X) in human <i>ACTN3</i>. Comparing WT mice across three genetic backgrounds, we found that different genes were more tightly regulated in each strain. We have developed a ranked profile of the top performing reference genes in skeletal muscle across these common mouse strains. Interestingly the commonly used reference genes; <i>Gapdh, Rn18s</i>, <i>Hprt1</i> and <i>Actb</i> were not the most stable. Analysis of our experimental variant (<i>Actn3</i> KO) also resulted in an altered ranking of reference gene suitability. Furthermore we demonstrate that a poor reference gene results in increased variability in the normalized expression of a gene of interest, and can result in loss of significance. Our data demonstrate that reference genes need to be validated prior to use. For the most accurate normalization, it is important to test several genes and use the geometric mean of at least three of the most stably expressed genes. In the analysis of mouse skeletal muscle, strain and intervention played an important role in selecting the most stable reference genes.</p></div

Models for Ig V Gene Conversion Based on Attack of Both DNA Strands by AID and Attack of One DNA Strand by AID

<div><p>(A) Attack of both DNA strands by AID. In step 1, two complexes containing AID attack opposed strands at the same time. If both complexes directly recruited DNA-PKcs molecules, DNA-PKcs could dimerize and perhaps inhibit base excision by UNG. In step 2, excision by UNG and a lyase or exonuclease creates a staggered DSB. Step 3 shows that gene conversion requires the production of 3′-protruding ends, which may involve a 5′–3′ exonuclease, depending on the relative placement of the nicks. The 3′-protruding ends initiate gene conversion by invading a homologous ΨV gene. This step could be inhibited by the binding of dsDNA ends to the DNA-PK complex. In steps 4–6, nonhomologous (“mutated”) sequences are copied from the ΨV gene by mismatched end trimming, primer extension, template switch, further end trimming, and ligation.</p> <p>(B) Attack of one DNA strand by AID. In step 1, AID deamination in G1-phase may be ignored until S-phase, based on the model in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050080#pbio-0050080-b011" target="_blank">11</a>]. In S-phase, shown in step 2, the dU base produced by AID is encountered by a replication fork, enabling access by UNG, which then creates an abasic site. Step 3 shows the abasic site is excised to create a dsDNA end, which can recruit the DNA-PK complex. In step 4, if DNA-PK is not recruited, the dsDNA end promotes gene conversion with an upstream ΨV gene.</p> <p>(C) Attack of one DNA strand by AID. As in (B), AID deamination is ignored until S-phase. In step 2, as in step 2 of (B), the dU base is encountered by a replication fork, but in this case lagging strand nicks (Okazaki fragments) are still present in the upstream ΨV genes. Step 3 shows the abasic site generated by UNG stalls the replication fork, without inducing nicking, and promotes strand invasion into an upstream ΨV gene. Step 4 shows a dsDNA end is created when primer extension encounters a lagging strand nick due to Okazaki fragments in the template ΨV gene. The replication fork stalls again. If DNA-PK binds the dsDNA end it inhibits the completion of HDR. In step 5, a second reconfiguration of the stalled replication fork occurs, and in step 6, resolution of the double cross-over completes gene conversion.</p></div

Surface Ig Reversion of DT40 Clones

<p>Accumulation (top) of sIg^+ve cells in sIg^−ve DT40 clones, or (bottom) of sIg^−ve cells in sIg^+ve DT40 clones after (A) 50 d of culture, or (B) 24 d of culture in two independent experiments (A or B) is depicted. The protein missing from each cell line is indicated. Control cells were (top) sIg^−ve DT40-CL18 cells, or (bottom) sIg^+ve DT40-CL18 cells. Each circle represents the frequency of sIg-reverted cells (on a log scale) detected in each clone by FACS after the culture period. The bar indicates the median reversion frequency. The numbers above each dataset give the number of clones analyzed. Asterisks indicate significant difference of the median reversion frequency from the control population: * <i>p</i> < 0.05, *** <i>p</i> < 0.001. Note: lack of surface Ig-expression in the <i>XRCC3^−/−</i> founder clones was due to Ig V(D)J point mutations and not due to the canonical CL18 frame shift carried by the control, <i>Ku70^−/−</i>, and <i>DNA-PKcs^−/−/−</i> clones (unpublished data). Thus, the reduced rate of Ig V gene conversion known to exist in these cells [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050080#pbio-0050080-b043" target="_blank">43</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050080#pbio-0050080-b045" target="_blank">45</a>] did not significantly reduce their rate of surface Ig gain.</p

α-Actinin-3 deficiency results in reduced glycogen phosphorylase activity and altered calcium handling in skeletal muscle

Approximately one billion people worldwide are homozygous for a stop codon polymorphism in the ACTN3 gene (R577X) which results in complete deficiency of the fast fibre muscle protein alpha-actinin-3. ACTN3 genotype is associated with human athletic performance and alpha-actinin-3 deficient mice [Actn3 knockout (KO) mice] have a shift in the properties of fast muscle fibres towards slower fibre properties, with increased activity of multiple enzymes in the aerobic metabolic pathway and slower contractile properties. alpha-Actinins have been shown to interact with a number of muscle proteins including the key metabolic regulator glycogen phosphorylase (GPh). In this study, we demonstrated a link between alpha-actinin-3 and glycogen metabolism which may underlie the metabolic changes seen in the KO mouse. Actn3 KO mice have higher muscle glycogen content and a 50% reduction in the activity of GPh. The reduction in enzyme activity is accompanied by altered post-translational modification of GPh, suggesting that alpha-actinin-3 regulates GPh activity by altering its level of phosphorylation. We propose that the changes in glycogen metabolism underlie the downstream metabolic consequences of alpha-actinin-3 deficiency. Finally, as GPh has been shown to regulate calcium handling, we examined calcium handling in KO mouse primary mouse myoblasts and find changes that may explain the slower contractile properties previously observed in these mice. We propose that the alteration in GPh activity in the absence of alpha-actinin-3 is a fundamental mechanistic link in the association between ACTN3 genotype and human performance