Dystrophin analysis in clonal myoblasts derived from a Duchenne muscular dystrophy carrier.

Clonal myogenic cell cultures were established from a potential heterozygote for a mutant Duchenne muscular dystrophy (DMD) gene who was also heterozygous for isozymes of the X-linked enzyme glucose-6-phosphate dehydrogenase. Previous tissue culture studies of this muscle donor demonstrated equal proliferative capacity of myoblasts that had lyonized either the paternal or maternal X-chromosome, indicating that mutation of the DMD gene does not affect growth of myoblasts. If this muscle donor were a gonadal mosaic, this conclusion would be incorrect. In the present study, only those myogenic colonies expressing the glucose-6-phosphate dehydrogenase-A isozyme were found to express dystrophin, indicating that this woman was indeed a heterozygote for DMD. By documenting dystrophin deficiency in a specific population of myogenic cells from this woman, we verify our previous conclusion regarding the normal proliferative capacity of DMD myoblasts. Somatic cell testing of dystrophin expression may offer an alternative to established genetic carrier tests for those women in whom deletions of the DMD are not detectable, whose pedigree structure does not permit linkage analysis, or in whom standard phenotypic analyses are ambiguous.     

Cardiac assessment of patients with late stage Duchenne muscular dystrophy

Background. Duchenne muscular dystrophy (DMD) patients used to die mainly from pulmonary problems. However, as advances in respiratory care increase life expectancy, mortality due to cardiomyopathy rises. Echocardiography remains the standard diagnostic modality for cardiomyopathy in DMD patients, but is hampered by scoliosis and poor echocardiographic acoustic windows in adult DMD patients. Multigated cardiac radionuclide ventriculography (MUGA) does not suffer from these limitations. N-terminal proBNP (NTproBNP) has shown to be a diagnostic factor for heart failure. We present our initial experience with plasma NT-proBNP measurement in the routine screening and diagnosis of cardiomyopathy in adult mechanically ventilated DMD patients. Methods. Retrospective study, 13 patients. Echocardiography classified left ventricular (LV) function as preserved or depressed. NT-proBNP was determined using immunoassay. LV ejection fraction (LVEF) was determined using MUGA. Results. Median (range) NT-proBNP was 73 (25 to 463) ng/l. Six patients had an NT-proBNP >125 ng/l. Seven patients showed an LVEF <45% on MUGA. DMD patients with depressed LV function (n=4) as assessed by echocardiography had significantly higher median NT-proBNP than those (n=9) with preserved LV function: 346 (266 to 463) ng/l versus 69 (25 to 257) ng/l (p=0.003). NT-proBNP significantly correlated with depressed LV function on echocardiogram and with LVEF determined by MUGA. Conclusion. Although image quality of MUGA is superior to echocardiography, the combination of echocardiography and NT-proBNP achieves similar results in the evaluation of left ventricular function and is less time consuming and burdensome for our patients. We advise to add NT-proBNP to echocardiography in the routine cardiac assessment of DMD patients. (Neth Heart J 2009;17:232–7.)     

Birth and population prevalence of Duchenne muscular dystrophy in the Netherlands

Summary Mutations causing Duchenne muscular dystrophy (DMD) have a short survival. Therefore, birth and population prevalence are maintained by new mutations. The present inventory was made to estimate the birth and population prevalence rates of DMD in the Netherlands. Seven methods of case identification were used. Data on 496 definite, probable or possible DMD patients born since 1961, or alive on January 1, 1983, were obtained. Several methods gave an estimated ascertainment of more than 95%. The prevalence rate at birth of DMD was estimated at 23.7×10^–5 (14215) male live births (MLB) yearly. The prevalence rate in the male population on January 1, 1983 was 5.4×10^–5 (118496). About 1% of the males in this study may have autosomal recessive Duchenne-like muscular dystrophy. Until now there has been no convincing evidence for geographic differences in DMD prevalence at birth. A list of frequency studies of Duchenne muscular dystrophy is included. The DMD mutation rate calculated by the indirect method is 7.9×10^–5 genes per generation. However, this may well be an over-estimate, as this method does not account for germline mosaicism. Using a modified sex ratio method the proportion of sporadic DMD among all cases was estimated to be 0.106 (range 0–0.332). High frequency of germline mosaicism in DMD is a likely cause for the apparent lack of sporadic cases as found in previous studies, if mutation rates in male and female gametes are equal. Therefore, methods for estimating the proportion of new mutants in DMD should take germline mosaicism into account. The modified sex ratio method allows incorporation of data on germline mosaicism if available.     

Community Structure Analysis of Gene Interaction Networks in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is an important pathology associated with the human skeletal muscle and has been studied extensively. Gene expression measurements on skeletal muscle of patients afflicted with DMD provides the opportunity to understand the underlying mechanisms that lead to the pathology. Community structure analysis is a useful computational technique for understanding and modeling genetic interaction networks. In this paper, we leverage this technique in combination with gene expression measurements from normal and DMD patient skeletal muscle tissue to study the structure of genetic interactions in the context of DMD. We define a novel framework for transforming a raw dataset of gene expression measurements into an interaction network, and subsequently apply algorithms for community structure analysis for the extraction of topological communities. The emergent communities are analyzed from a biological standpoint in terms of their constituent biological pathways, and an interpretation that draws correlations between functional and structural organization of the genetic interactions is presented. We also compare these communities and associated functions in pathology against those in normal human skeletal muscle. In particular, differential enhancements are observed in the following pathways between pathological and normal cases: Metabolic, Focal adhesion, Regulation of actin cytoskeleton and Cell adhesion, and implication of these mechanisms are supported by prior work. Furthermore, our study also includes a gene-level analysis to identify genes that are involved in the coupling between the pathways of interest. We believe that our results serve to highlight important distinguishing features in the structural/functional organization of constituent biological pathways, as it relates to normal and DMD cases, and provide the mechanistic basis for further biological investigations into specific pathways differently regulated between normal and DMD patients. These findings have the potential to serve as fertile ground for therapeutic applications involving targeted drug development for DMD.     

Establishment and maintenance of H19 imprinting in the germline and preimplantation embryo

The mouse H19 and Igf2 genes are oppositely imprinted and share enhancers that reside 3' to the genes. The imprinted expression of these genes is coordinated by a 2-kb regulatory element, the differentially methylated domain (DMD), positioned between the two genes. The methylation status of this region determines the ability of the insulator factor CTCF to bind to its sites in the DMD. Deletions and mutations of the DMD that affect imprinting in the soma have little effect on the methylation pattern of H19 in the germline, suggesting that additional sequences and factors contribute to the earliest stages of imprinting regulation at this locus. Less is known about these initial steps, which include the marking of the parental alleles, the onset of allele-specific expression patterns and maintenance of the imprints in the preimplantation embryo. Here, we will focus on these early steps, summarizing what is known and what questions remain to be addressed.     

Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle

Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and necrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance spectroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (less than 40-50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., a decline in PCr/ATP) observed in adult mdx vs. control mice are qualitatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor.     

Gastrocnemius medialis muscle architecture and physiological cross sectional area in adult males with Duchenne muscular dystrophy

Objectives:

To describe muscle size and architecture of the gastrocnemius medialis (GM) muscle in eleven adult males with Duchenne Muscular Dystrophy (DMD, age 24.5±5.4 years), and a control group of eleven males without DMD (CTRL, age 22.1±0.9 years).

Methods:

GM anatomical cross sectional area (ACSA), volume (VOL), physiological cross sectional area (PCSA), fascicle length (Lf) and pennation angle (θ) were assessed using B-Mode Ultrasonography. GM ACSA was measured at 25, 50 and 75% of muscle length (Lm), from which VOL was calculated. At 50% of Lm, sagittal plane images were analysed to determine GM Lf and θ. GM PCSA was calculated as VOL/Lf. The ratio of Lf and Lm was also calculated.

Results:

GM ACSA at 50% Lm, VOL and PCSA were smaller in DMD males compared to CTRL males by 36, 47 and 43%, respectively (P<0.01). There were no differences in Lf and θ. GM Lm was 29% shorter in DMD compared to CTRL. Lf/Lm was 29% longer in DMD (P<0.01).

Conclusions:

Unlike previous data in children with DMD, our results show significant atrophy in adult males with DMD, and no change in Lf or θ. The shorter Lm may have implications for joint flexibility.     

Diagnose und Therapie der Muskeldystrophie Duchenne und Becker

Duchenne muscular dystrophy (DMD) is the most common neuromuscular disorder during childhood. It shows x-linked inheritance and is clinically characterized by progressive muscle weakness starting during infancy and leading to loss of ambulation during early adolescence. Serum creatine kinase levels are markedly elevated and the diagnosis is confirmed through genetic testing of the dystrophin gene or a muscle biopsy. Therapy of DMD necessitates a multidisciplinary approach. Use of medications, physiotherapy, orthopaedic care and non-invasive ventilation can markedly improve life expectancy and quality of life. Causative treatment strategies are in clinical development, but it is too early for a conclusion about their effectiveness. Becker muscular dystrophy is a much rarer allelic disorder with partial dystrophin deficiency and a milder clinical phenotype.     

Predictive markers of clinical outcome in the GRMD dog model of Duchenne muscular dystrophy

In the translational process of developing innovative therapies for DMD (Duchenne muscular dystrophy), the last preclinical validation step is often carried out in the most relevant animal model of this human disease, namely the GRMD (Golden Retriever muscular dystrophy) dog. The disease in GRMD dogs mimics human DMD in many aspects, including the inter-individual heterogeneity. This last point can be seen as a drawback for an animal model but is inherently related to the disease in GRMD dogs closely resembling that of individuals with DMD. In order to improve the management of this inter-individual heterogeneity, we have screened a combination of biomarkers in sixty-one 2-month-old GRMD dogs at the onset of the disease and a posteriori we addressed their predictive value on the severity of the disease. Three non-invasive biomarkers obtained at early stages of the disease were found to be highly predictive for the loss of ambulation before 6 months of age. An elevation in the number of circulating CD4⁺CD49d^hi T cells and a decreased stride frequency resulting in a reduced spontaneous speed were found to be strongly associated with the severe clinical form of the disease. These factors can be used as predictive tests to screen dogs to separate them into groups with slow or fast disease progression before their inclusion into a therapeutic preclinical trial, and therefore improve the reliability and translational value of the trials carried out on this invaluable large animal model. These same biomarkers have also been described to be predictive for the time to loss of ambulation in boys with DMD, strengthening the relevance of GRMD dogs as preclinical models of this devastating muscle disease.KEY WORDS: GRMD, DMD, Dystrophin, Dog, Predictive biomarker, Lymphocyte, CD49d, Gait analysis, Accelerometry     

Genethics: project accountability via evaluation of teacher and student growth.

Accountability through demonstrated learning is increasingly being demanded by agencies funding science education projects. For example, the National Science Foundation requires evidence of the educational impact of programs designed to increase the scientific understanding and competencies of teachers and their students. The purpose of this paper is to share our human genetics educational experiences and accountability model with colleagues interested in serving the genetics educational needs of in-service secondary school science teachers and their students. Our accountability model is facilitated through (1) identifying the educational needs of the population of teachers to be served, (2) articulating goals and measurable objectives to meet these needs, and (3) then designing and implementing pretest/posttest questions to measure whether the objectives have been achieved. Comparison of entry and exit levels of performance on a 50-item test showed that teacher-participants learned a statistically significant amount of genetics content in our NSF-funded workshops. Teachers, in turn, administered a 25-item pretest/posttest to their secondary school students, and collective data from 121 classrooms across the United States revealed statistically significant increases in student knowledge of genetics content. Methods describing our attempts to evaluate teachers' use of pedagogical techniques and bioethical decision-making skills are briefly addressed.     

miRNome profiling in Duchenne muscular dystrophy; identification of asymptomatic and manifesting female carriers

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder that occurs due to inactivating mutations in DMD gene, leading to muscular dystrophy. Prediction of pathological complications of DMD and the identification of female carriers are important research points that aim to reduce disease burden. Herein, we describe a case of a late DMD patient and his immediate female family members, who all carry same DMD mutation and exhibited varied degrees of symptoms. In our study, we sequenced the whole miRNome in leukocytes and plasma of the family members and results were validated using real-time PCR. Our results highlighted the role of miR-409-3p, miR-424-5p, miR-144-3p as microRNAs that show correlation with the extent of severity of muscular weakness and can be used for detection of asymptomatic carriers. Cellular and circulating levels of miR-494-3p had shown significant increase in symptomatic carriers, which may indicate significant roles played by this miRNA in the onset of muscular weakness. Interestingly, circulating levels of miR-206 and miR-410-3p were significantly increased only in the severely symptomatic carrier. In conclusion, our study highlighted several miRNA species, which could be used in predicting the onset of muscle and/or neurological complications in DMD carriers.     

Understanding age-specific dispersal in fishes through hydrodynamic modelling, genetic simulations and microsatellite DNA analysis

Many marine species have vastly different capacities for dispersal during larval, juvenile and adult life stages, and this has the potential to complicate the identification of population boundaries and the implementation of effective management strategies such as marine protected areas. Genetic studies of population structure and dispersal rarely disentangle these differences and usually provide only lifetime-averaged information that can be considered by managers. We address this limitation by combining age-specific autocorrelation analysis of microsatellite genotypes, hydrodynamic modelling and genetic simulations to reveal changes in the extent of dispersal during the lifetime of a marine fish. We focus on an exploited coral reef species, Lethrinus nebulosus, which has a circum-tropical distribution and is a key component of a multispecies fishery in northwestern Australia. Conventional population genetic analyses revealed extensive gene flow in this species over vast distances (up to 1,500 km). Yet, when realistic adult dispersal behaviours were modelled, they could not account for these observations, implying adult dispersal does not dominate gene flow. Instead, hydrodynamic modelling showed that larval L. nebulosus are likely to be transported hundreds of kilometres, easily accounting for the observed gene flow. Despite the vast scale of larval transport, juvenile L. nebulosus exhibited fine-scale genetic autocorrelation, which declined with age. This implies both larval cohesion and extremely limited juvenile dispersal prior to maturity. The multidisciplinary approach adopted in this study provides a uniquely comprehensive insight into spatial processes in this marine fish.     

Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle

Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabiolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and ncecrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance sperctroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (< 40–50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., decline in PCr/ATP) observed in adult mdx vs. control mice are quantilatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor.     

Cell based therapy for duchenne muscular dystrophy

Mutations in the dystrophin gene cause an X‐linked genetic disorder: Duchenne muscular dystrophy (DMD). Stem cell therapy is an attractive method to treat DMD because a small number of cells are required to obtain a therapeutic effect. Here, we discussed about multiple types of myogenic stem cells and their possible use to treat DMD. The identification of a stem cell population providing efficient muscle regeneration is critical for the progression of cell therapy for DMD. We speculated that the most promising possibility for the treatment of DMD is a combination of different approaches, such as gene and stem cell therapy. J. Cell. Physiol. 221: 526–534, 2009. © 2009 Wiley‐Liss, Inc.     

Disease risk analysis: A paradigm for using health-based data to inform primate conservation and public health

Risk analysis is a multidisciplinary process used to evaluate existing knowledge in order to prioritize risks associated with the spread of disease. A principle aim of risk analysis is to facilitate the development of cost-effective management strategies. Risk analysis calls for a multidisciplinary approach to piece together and integrate the numerous factors that influence disease transmission. The seven papers included in this volume of AJP present current primatological research as viewed through the prism of risk analysis. Issues such as interspecies disease transmission, public health, and conservation of endangered species are addressed, and risk analysis is put forward as a possible paradigm to promote understanding of infectious disease and its impact on nonhuman primate and human populations.     

Duchenne muscular dystrophy: Pathogenetic aspects and genetic prevention

Summary Duchenne muscular dystrophy (DMD) is the most common sex linked lethal disease in man (one case in about 4000 male live births). The patients are wheelchair bound around the age of 8–10 years and usually die before the age of 20 years. The mutation rate, estimated by different methods and from different population studies, is in the order of 7×10^-5, which is higher than for any other X-linked genetic disease. Moreover, unlike other X linked diseases such as hemophilia A or Lesh-Nyhan's disease, there seems to be no sex difference for the mutation rates in DMD. Several observations of DMD in girls bearing X-autosomal translocations and linkage studies on two X chromosomal DNA restriction fragment length polymorphisms indicate that the DMD locus is situated on the short arm of the X chromosome, between Xp11 and Xp22. It may be of considerable length, and perthaps consisting of actively coding and non-active intervening DNA sequences. Thus unequal crossing over during meiosis in females could theoretically account for a considerable proportion of new mutations.However, there is no structurally or functionally abnormal protein known that might represent the primary gene product, nor has any pathogenetic mechanism leading to the observed biochemical and histological alterations been elucidated. Among the numerous pathogenetic concepts the hypothesis of a structural or/and functional defect of the muscular plasma membrane is still the most attractive. It would explain both the excess of muscular constituents found in serum of patients and carriers, such as creatine kinase (CK), as well as the excessive calcium uptake by dystrophic muscle fibres, which, prior to necrosis, could lead to hypercontraction, rupture of myofilaments in adjacent sarcomeres and by excessive Ca uptake to mitochondrial damage causing crucial energy loss.The results of studies on structural and functional memthrane abnormalities in cells other than muscle tissue, e.g., erythrocytes, lymphocytes and cultured fibroblasts, indicate that the DMD mutation is probably demonstrable in these tissues. However, most of the findings are still difficult to reproduce or even controversial.DMD is an incurable disease; therefore most effort, in research as well as in practical medicine, is concentrated upon its prevention. Unfortunately the disease cannot yet be diagnosed prenatally. Potential DMD carriers among female relatives of the patients may be identified by pathological heterozygote tests, of which determination of serum CK activity is probably still the most reliable method, allowing the detection of about 70% of adult and probably up to 90% of carriers at school age. Because of the high mutation rate, assessment of individual heterozygote risks in female relatives of isolated DMD cases is of special importance. For the calculations a maximum of genealogical and phenotype information on unaffected male and on heterozygote tests in female relatives is needed to obtain credible risk figures. However, estimating a consultand's risk and passing on this information is only one aspect of genetic counselling in DMD. At least as important is information on the medical, psychological and social impacts of the disease (burden) and the possibility of maintaining a long-term contact between the couples at risk and the team involved in medical, genetic and social problems of the disease. Neonatal CK screening for DMD, although without any therapeutical consequence, could theoretically lead to the prevention of secondary cases, accounting for some 15% of all DMD patients born, but an almost equal prevention rate of such cases would be achieved if CK examinations were limited to all boys with delayed motor development during the first 2 years of life. Finally, it is believed that the two most important preventive problems in DMD, carrier detection and prenatal diagnosis, will ultimately be solved by means of the rapidly advancing DNA technology.This work was dedicated to Professor P.E. Becker in honour of his 75th birthday     

Maxillofacial fractures in the elderly: a comparative study

Previous maxillofacial trauma research has dealt primarily with facial bone fractures in the general population. Very few studies have specifically addressed maxillofacial fractures in the elderly. We compared 45 elderly (65 years of age or older) and 201 younger adult (16 to 64 years of age) patients admitted to our hospital with maxillofacial fractures. The percentage of patients admitted with nasal bone fractures was much greater in the elderly population, while mandibular fractures were more common in the adult group. Motor vehicle accidents accounted for over half the injuries in both groups, while falls were more prevalent in the elderly. Management of the elderly patient may be complicated by their associated injuries or underlying medical problems, perhaps partially accounting for their longer median length of hospital stay. The elderly are a unique subpopulation of maxillofacial fracture patients and deserve further study regarding their injuries and optimal methods for treatment.     

Biomolecular computing: Is it ready to take off?

Biomolecular computing is an emerging field at the interface of computer science, biological science and engineering. It uses DNA and other biological materials as the building blocks for construction of living computational machines to solve difficult combinatorial problems. In this article, notable advances in the biomolecular computing are reviewed and challenges associated with this multidisciplinary research are addressed. Finally, several perspectives are given based on the review of biomolecular computing.     

应用胎儿特异性抗体HbF（γ链）标记法无创性 产前基因诊断DMD

以孕8~26周孕妇外周血为材料,经过Percoll密度梯度离心初步富集,胎儿细胞特异性抗体—HbF标记、识别胎儿有核红细胞,母体和胎儿有核红细胞的精确区分是以胎儿和成人血红蛋白的组成差异为基础的。胎儿细胞胞浆黄染,而具有成人血红蛋白的母体细胞没有颜色。显微操作法获取全部阳性细胞后,以其全基因组扩增（PEP）的产物为模板,进行性别检测、DMD基因的多重PCR检测和STR连锁分析。结果,20名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC。并完成7例DMD的产前基因诊断。HbF抗体标记法能有效识别胎儿有核红细胞,是无创性产前基因诊断中很有应用前景的标记方法。 Abstract： Maternal blood was obtained at 8-26weeks of gestation.After discontinuous density gradient centrifugation with Percoll ,HbF antibody was used to identify fetal NRBC.The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb).Fetal cells appear yellow cytoplasmic staining,while adult cells colorless. NRBCs were collected by micromanipulation andwhole genome amplification was performed. DMD was prenatally diagnosed by using the combination of sex determination,multiplex PCR and linkage analysis of several STR sites of dystrophin. NRBCs stained with HbF were found in all of 20 maternal blood samples with gestations, and 7 fetuses with risk of DMD were diagnosed. We concluded that HbF antibody could identify fetal NRBC efficaciously,and this is a kind of more prospective application method.     

Pheromone production by male Tribolium castaneum (Coleoptera: Tenebrionidae) is influenced by diet quality

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), the red flour beetle, is a common cosmopolitan pest exploiting a variety of stored products. We experimentally manipulated diet nutritional quality by using non-nutritive filler to examine how this influenced pheromone production and olfactory attractiveness of T. castaneum adult males. Volatiles released by individual males reared on high versus low nutrition diets were collected using solid phase microextraction, and gas chromatography coupled to mass spectrometry was used to identify and quantify the Tribolium aggregation pheromone 4, 8-dimethyldecanal (DMD). Males kept on high nutrition diet showed a three-fold increase in daily DMD production, which suggests the possibility that this pheromone could act as a condition-dependent mating signal. In pitfall trap assays, there was no significant difference in the mean response of virgin females to discs kept with low versus high nutrition males, although discs carrying male cues were significantly more attractive than blank discs. These results suggest that DMD production rates by T. castaneum males will depend on the nutritional quality of various stored products, but such differences may not alter males' ability to attract females.