Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.

Myoblast fusion is a highly regulated process that is important during muscle development and myofiber repair and is also likely to play a key role in the incorporation of donor cells in myofibers for cell-based therapy. Although several proteins involved in muscle cell fusion in Drosophila are known, less information is available on the regulation of this process in vertebrates, including humans. To identify proteins that are regulated during fusion of human myoblasts, microarray studies were performed on samples obtained from human fetal skeletal muscle of seven individuals. Primary muscle cells were isolated, expanded, induced to fuse in vitro, and gene expression comparisons were performed between myoblasts and early or late myotubes. Among the regulated genes, melanoma cell adhesion molecule (M-CAM) was found to be significantly downregulated during human fetal muscle cell fusion. M-CAM expression was confirmed on activated myoblasts, both in vitro and in vivo, and on myoendothelial cells (M-CAM(+) CD31(+)), which were positive for the myogenic markers desmin and MyoD. Lastly, in vitro functional studies using M-CAM RNA knockdown demonstrated that inhibition of M-CAM expression enhances myoblast fusion. These studies identify M-CAM as a novel marker for myogenic progenitors in human fetal muscle and confirm that downregulation of this protein promotes myoblast fusion.

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles.

In the last few decades, the importance of selenium in human health has been the subject of numerous studies. It is believed that the physiological effects of selenium occur mainly through the function of selenoproteins, which incorporate selenium in the form of one or more selenocysteine residues. Recent advances in understanding the complex regulation of selenoprotein synthesis and functional characterization of several members of the selenoprotein family have contributed to an improved comprehension of the role(s) of selenium in human health and the great diversity of physiological pathways influenced by this trace element.

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

CONTEXT: The serotonergic (5-hydroxytryptamine [5-HT]) neurons in the medulla oblongata project extensively to autonomic and respiratory nuclei in the brainstem and spinal cord and help regulate homeostatic function. Previously, abnormalities in 5-HT receptor binding in the medullae of infants dying from sudden infant death syndrome (SIDS) were identified, suggesting that medullary 5-HT dysfunction may be responsible for a subset of SIDS cases. OBJECTIVE: To investigate cellular defects associated with altered 5-HT receptor binding in the 5-HT pathways of the medulla in SIDS cases. DESIGN, SETTING, AND PARTICIPANTS: Frozen medullae from infants dying from SIDS (cases) or from causes other than SIDS (controls) were obtained from the San Diego Medical Examiner's office between 1997 and 2005. Markers of 5-HT function were compared between SIDS cases and controls, adjusted for postconceptional age and postmortem interval. The number of samples available for each analysis ranged from 16 to 31 for SIDS cases and 6 to 10 for controls. An exploratory analysis of the correlation between markers and 6 recognized risk factors for SIDS was performed. MAIN OUTCOME MEASURES: 5-HT neuron count and density, 5-HT(1A) receptor binding density, and 5-HT transporter (5-HTT) binding density in the medullary 5-HT system; correlation between these markers and 6 recognized risk factors for SIDS. RESULTS: Compared with controls, SIDS cases had a significantly higher 5-HT neuron count (mean [SD], 148.04 [51.96] vs 72.56 [52.36] cells, respectively; P.001) and 5-HT neuron density (P.001), as well as a significantly lower density of 5-HT(1A) receptor binding sites (P

The identification of dystrophin as the defective protein in patients with Duchenne and Becker muscular dystrophies (DMD and BMD) has allowed the development of sensitive and specific tests to establish a diagnosis and to aid in genetic counseling and prenatal diagnosis. The Basic Protocol describes three complementary multiplex PCR assays that detect 26 dystrophin gene exons. The multiplex nature of these assays allows the detection of up to ten different exons in a single reaction. At least one of these exons is missing in >95% of deletions. The Support Protocol describes preparation and storage of stock PCR reaction mixes with primers for each of the three diagnostic assays. The Alternate Protocol is a modification of the Basic Protocol for radioactive detection of duplications in males and deletions in carrier females.

Recent work has significantly enhanced our understanding of the centronuclear myopathies and, in particular, myotubular myopathy. These myopathies share similar morphologic appearances with other diseases, namely the presence of hypotrophic myofibers with prominent internalized or centrally placed nuclei. Early workers suggested that this alteration represented an arrest in myofiber maturation, while other hypotheses implicated either failure in myofiber maturation or neurogenic causes. Despite similarities in morphology, distinct patterns of inheritance and some differences in clinical features have been recognized among cases. A severe form, known as X-linked myotubular myopathy (XLMTM), presents at or near birth. Affected males have profound global hypotonia and weakness, accompanied by respiratory difficulties that often require ventilation. Most of these patients die in infancy or early childhood, but some survive into later childhood or even adulthood. The responsible gene (MTM1) has been cloned; it encodes a phosphoinositide lipid phosphatase known as myotubularin that appears to be important in muscle maintenance. In autosomal recessive centronuclear myopathy (AR CNM), the onset of weakness typically occurs in infancy or early childhood. Some investigators have divided AR CNM into 3 subgroups: 1) an early-onset form with ophthalmoparesis, 2) an early-onset form without ophthalmoparesis, and 3) a late-onset form without ophthalmoparesis. Clinically, autosomal dominant CNM (AD CNM) is relatively mild and usually presents in adults with a diffuse weakness that is slowly progressive and may be accompanied by muscle hypertrophy. Overall, the autosomal disorders are not as clinically uniform as XLMTM, which has made their genetic characterization more difficult. Currently the responsible gene(s) remain unknown. This review will explore the historical evolution in understanding of these myopathies and give an update on their histopathologic features, genetics and pathogenesis.

There is a consistent variation in the response of different skeletal muscle groups to mutations in genes known to cause muscular dystrophy, yet these muscles appear histologically similar. To better understand these phenotypic differences, we analyzed gene expression patterns in control muscle specimens obtained from four sites at autopsy: deltoid, quadriceps, gastrocnemius, and tibialis anterior (TA). A total of 35 muscle samples from nine individuals (four pediatric and five geriatric) were studied. Factors potentially influencing gene expression in the different samples included individuality, age, muscle type, gender, cause of death, postmortem interval, and ethnicity. The first three factors, in decreasing order, were found to have a significant impact on the stratification of muscle specimens. A novel analytic method, using a second round of normalization, was used to elicit differences between muscle types. This approach may be extended to a broader survey, potentially elucidating a molecular classification of the skeletal muscles.

Autosomal dominant centronuclear myopathy is a rare congenital myopathy characterized by delayed motor milestones and muscular weakness. In 11 families affected by centronuclear myopathy, we identified recurrent and de novo missense mutations in the gene dynamin 2 (DNM2, 19p13.2), which encodes a protein involved in endocytosis and membrane trafficking, actin assembly and centrosome cohesion. The transfected mutants showed reduced labeling in the centrosome, suggesting that DNM2 mutations might cause centronuclear myopathy by interfering with centrosome function.