Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort

Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with “private” mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55. Hum Mutat 30:1657–1666, 2009. © 2009 Wiley-Liss, Inc.     

Deletion of PKC�� Selectively Enhances the Amplifying Pathways of Glucose-Stimulated Insulin Secretion via Increased Lipolysis in Mouse ��-Cells

OBJECTIVE

Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of β-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C ε (PKCε) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCε.

RESEARCH DESIGN AND METHODS

We cultured islets isolated from PKCε knockout (PKCεKO) mice in palmitate prior to measuring GSIS, Ca²⁺ responses, palmitate esterification products, lipolysis, lipase activity, and gene expression.

RESULTS

The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca²⁺ influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCεKO β-cells was revealed under conditions in which functional ATP-sensitive K⁺ channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCεKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCεKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCε deletion.

CONCLUSIONS

Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine β-cells, and they suggest an interaction between PKCε and lipolysis. These results further highlight the therapeutic potential of PKCε inhibition to enhance GSIS from the β-cell under conditions of lipid excess.Type 2 diabetes is characterized by hyperglycemia and dyslipidemia, and it results from insufficient insulin secretion from pancreatic β-cells to overcome the resistance of peripheral tissues to the actions of insulin. Insulin resistance is driven by genetic and environmental factors, but it is initially counteracted by an enlargement of β-cell mass and increased insulin output to maintain normal glucose homeostasis. Subsequent loss of β-cell mass and function in susceptible individuals causes impaired glucose homeostasis and progression to overt type 2 diabetes (1,2). The mechanisms underlying β-cell failure are poorly understood (3–5), although there is evidence that the β-cells of predisposed individuals are particularly compromised by exposure to high levels of circulating fatty acids, as occurs during obesity and insulin resistance (6). Moreover, model systems in which β-cells are chronically exposed to fatty acids (lipotoxicity) recapitulate both the loss of β-cell mass and many of the insulin secretory defects that are characteristic of type 2 diabetes (7,8).Glucose-stimulated insulin secretion (GSIS) consists of triggering and amplifying pathways (9). Glucose triggers insulin secretion by generating ATP from oxidative metabolism, which closes ATP-sensitive K⁺ (K_ATP) channels, thereby inducing a membrane depolarization that stimulates Ca²⁺ influx via voltage-dependent Ca²⁺ channels. The amplifying pathways allow glucose to potentiate the secretory response to a given rise in cytosolic Ca²⁺ in a depolarized (triggered) β-cell (9). There is currently no consensus on the mechanism(s) coupling glucose metabolism to the amplification of insulin secretion, although several have been proposed (10–12). One candidate mechanism relates to the regulation by glucose of endogenous lipid signaling in the β-cell (11,12).The protein kinase C (PKC) superfamily consists of conventional, novel, and atypical isoforms of serine-threonine protein kinases. The lipid-regulated subgroup of novel PKCs has been broadly implicated in the development of insulin resistance (13). This is particularly true of the PKCε isoform under conditions of nutrient oversupply (14–16) and in the livers of human type 2 diabetic subjects (17). Functional inhibition of PKCε also improves glucose homeostasis in rodent models of type 2 diabetes (18,19) and reverses acute lipid-induced hepatic insulin resistance (20). Unexpectedly, however, we recently found that enhancement of GSIS was another major means by which inhibition of PKCε function improves glucose homeostasis. This enhancement was observed in both dietary and genetic mouse models of type 2 diabetes and when using ex vivo models of β-cell dysfunction (18). Others have also reported a role for PKCε in the maturation of proinsulin to insulin (21). Because the mechanisms by which PKCε deletion improves GSIS remain unclear, our current aim was to investigate the potential role of alterations in β-cell lipid metabolism. We demonstrate that after chronic lipid exposure, PKCεKO islets show a selective increase of the amplification pathways of GSIS. This was associated with a chronic increase of palmitate tracer incorporation into triglyceride stores, and it was dependent on an increase in the subsequent glucose-stimulated hydrolysis of those stores.     

Improved injection safety after targeted interventions in the Syrian Arab Republic

OBJECTIVES: Concerns about unsafe injection practices and possible infections with blood-borne pathogens in the Syrian Arab Republic motivated an assessment of the injection safety situation in the country in July 2001. In light of the recommendations from this assessment, the Ministry of Health of Syria, with the assistance of WHO, implemented a set of activities under the 'Focus Project', which aims to ensure immunization safety. The first phase of the project ran from May 2002 to February 2004, and consisted of the improved provision of injection safety equipment and supplies, the elaboration and wide distribution of national guidelines on injection safety and safe waste management, a behaviour change and communication campaign targeting the general public, and comprehensive training of healthcare workers. A follow-up survey was carried out in February 2004, 2 years after initiation of the project. METHODS: Two representative surveys were conducted using a standardized assessment tool. A cluster sampling strategy, with probability proportionate to the population size, led to the inclusion of 80 health facilities in eight districts in 2001 and of 120 health facilities in 12 districts in 2004. RESULTS: Injection practices had significantly improved 2 years after the start of the project. The 2001 study had pointed to a low, but non-negligible risk to patients (2% unsafe injections), coupled with a high risk to healthcare workers (61% reported needle-stick injuries in the last 12 months) and to the communities owing to unsafe waste disposal (sharps waste found outside 37% of health facilities, waste disposal considered unsafe in 48% of them). The 2004 survey showed that 90% of Syrian healthcare workers had received training in injection safety. All injections observed were given safely (difference to 2001 not significant), although some problems in preparation and reconstitution prevailed. The risk to healthcare workers was significantly reduced as only 14% of the staff reported needle-stick injuries (p < 0.001). The risk to the communities was notably decreased following improvements in sharps waste management (sharps were found in the surroundings of only 13% of health facilities, p < 0.001). CONCLUSIONS: The example of Syria shows that rapid improvement in injection safety is possible and that the necessary tools and methods to monitor and evaluate progress are at our disposal. Challenges remain in transferring this successful programme from the well-structured immunization programme to the more diverse curative health services.     

Aortic valve leaflet sizer: a new device for aortic valve sparing

In aortic valve-sparing surgery, the native valve of the patient is left in place but may need correction of its dimensions in order to regain full competence. A sizer was designed that can simultaneously measure the leaflet height and free edge length. A prototype was built and successfully used in porcine hearts of various sizes. The sizer is easy to use and provides dependable measurement of the aortic leaflet dimensions. Moreover, it represents a new resource on which surgeons may draw on to perform aortic valve-sparing surgery, with a better outcome for the patients.     

Attention problems among children with a positive family history of alcohol abuse or dependence and controls. Prevalence and course for the period from preteen to early teen years

This longitudinal study investigated the scope and course of attention problems over a period of time from preteen (ages 7-12 years) to early teen years (ages 13-17 years). We compared symptoms in subjects with and without a family history (FH) of alcohol abuse or dependence from among families without evidence of antisocial personality disorder. Evaluations of attention problems for the offspring were based on the Child Behavior Checklist and a validated semistructured interview carried out with the mother. The findings indicate no higher risk for attention problems and attention-deficit hyperactivity disorder (ADHD)-like symptoms in the children of families with an alcohol use disorder. Regarding the course of problems, the ADHD symptom count tended to decrease over time, especially for children without a FH of alcohol abuse or dependence. Further research will be needed to determine whether results can be replicated with families from different social strata and including subjects with the antisocial personality disorder.     

Genetic dissection of neural circuit anatomy underlying feeding behavior in Drosophila: distinct classes of hugin-expressing neurons

The hugin gene of Drosophila encodes a neuropeptide with homology to mammalian neuromedin U. The hugin-expressing neurons are localized exclusively to the subesophageal ganglion of the central nervous system and modulate feeding behavior in response to nutrient signals. These neurons send neurites to the protocerebrum, the ventral nerve cord, the ring gland, and the pharynx and may interact with the gustatory sense organs. In this study, we have investigated the morphology of the hugin neurons at a single-cell level by using clonal analysis. We show that single cells project to only one of the four major targets. In addition, the neurites of the different hugin cells overlap in a specific brain region lateral to the foramen of the esophagus, which could be a new site of neuropeptide release for feeding regulation. Our study reveals novel complexity in the morphology of individual hugin neurons, which has functional implication for how they coordinate feeding behavior and growth.