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.

Timothy syndrome (TS) is a multisystem disorder that causes syncope and sudden death from cardiac arrhythmias. Prominent features include congenital heart disease, immune deficiency, intermittent hypoglycemia, cognitive abnormalities, and autism. All TS individuals have syndactyly (webbing of fingers and toes). We discovered that TS resulted from a recurrent, de novo cardiac L-type calcium channel (CaV1.2) mutation, G406R. G406 is located in alternatively spliced exon 8A, encoding transmembrane segment S6 of domain I. Here, we describe two individuals with a severe variant of TS (TS2). Neither child had syndactyly. Both individuals had extreme prolongation of the QT interval on electrocardiogram, with a QT interval corrected for heart rate ranging from 620 to 730 ms, causing multiple arrhythmias and sudden death. One individual had severe mental retardation and nemaline rod skeletal myopathy. We identified de novo missense mutations in exon 8 of CaV1.2 in both individuals. One was an analogous mutation to that found in exon 8A in classic TS, G406R. The other mutation was G402S. Exon 8 encodes the same region as exon 8A, and the two are mutually exclusive. The spliced form of CaV1.2 containing exon 8 is highly expressed in heart and brain, accounting for approximately 80% of CaV1.2 mRNAs. G406R and G402S cause reduced channel inactivation, resulting in maintained depolarizing L-type calcium currents. Computer modeling showed prolongation of cardiomyocyte action potentials and delayed afterdepolarizations, factors that increase risk of arrhythmia. These data indicate that gain-of-function mutations of CaV1.2 exons 8 and 8A cause distinct forms of TS.

Side Population (SP) cells, isolated from murine adult bone marrow (BM) based on the exclusion of the DNA dye Hoechst 33342, exhibit potent hematopoietic stem cell (HSC) activity when compared to Main Population (MP) cells. Furthermore, SP cells derived from murine skeletal muscle exhibit both hematopoietic and myogenic potential in vivo. The multipotential capacity of SP cells isolated from variable tissues is supported by an increasing number of studies. To investigate whether the SP phenotype is associated with a unique transcriptional profile, we characterized gene expression of SP cells isolated from two biologically distinct tissues, bone marrow and muscle. Comparison of SP cells with differentiated MP cells within a tissue revealed that SP cells are in an active transcriptional and translational status and underexpress genes reflecting tissue-specific functions. Direct comparison of gene expression of SP cells isolated from different tissues identified genes common to SP cells as well as genes specific to SP cells within a particular tissue and further define a muscle and bone marrow environment. This study reports gene expression of muscle SP cells, common features and differences between SP cells isolated from muscle and bone marrow, and further identifies common signaling pathways that might regulate SP cell functions.

The origin of the pathogenic endothelial cells in common infantile hemangioma is unknown. We show here that the transcriptomes of human placenta and infantile hemangioma are sufficiently similar to suggest a placental origin for this tumor, expanding on recent immunophenotypical studies that have suggested this possibility [North, P. E., et al. (2001) Arch. Dermatol. 137, 559-570]. The transcriptomes of placenta, hemangioma, and eight normal and diseased tissues were compared by hierarchical and nonhierarchical clustering analysis of >7,800 genes. We found that the level of transcriptome similarity between placenta and hemangioma exceeded that of any other tissue compared and paralleled that observed between a given tissue and its derived tumor, such as normal and cancerous lung. The degree of similarity was even greater when a subset of endothelial cell-specific genes was analyzed. Genes preferentially expressed in both placenta and hemangiomas were identified, including 17-beta hydroxysteroid dehydrogenase type 2 and tissue factor pathway inhibitor 2. These data demonstrate the value of global molecular profiling of tissues as a tool for hypothesis-driven research. Furthermore, it suggests that the unique self-limited growth of infantile hemangioma may, in fact, mirror the lifetime of placental endothelium.

Skeletal muscle differentiation is a complex, highly coordinated process that relies on precise temporal gene expression patterns. To better understand this cascade of transcriptional events, we used expression profiling to analyze gene expression in a 12-day time course of differentiating C2C12 myoblasts. Cluster analysis specific for time-ordered microarray experiments classified 2895 genes and ESTs with variable expression levels between proliferating and differentiating cells into 22 clusters with distinct expression patterns during myogenesis. Expression patterns for several known and novel genes were independently confirmed by real-time quantitative RT-PCR and/or Western blotting and immunofluorescence. MyoD and MEF family members exhibited unique expression kinetics that were highly coordinated with cell-cycle withdrawal regulators. Among genes with peak expression levels during cell cycle withdrawal were Vcam1, Itgb3, Itga5, Vcl, as well as Ptger4, a gene not previously associated with the process of myogenesis. One interesting uncharacterized transcript that is highly induced during myogenesis encodes several immunoglobulin repeats with sequence similarity to titin, a large sarcomeric protein. These data sets identify many additional uncharacterized transcripts that may play important functions in muscle cell proliferation and differentiation and provide a baseline for comparison with C2C12 cells expressing various mutant genes involved in myopathic disorders.

Autopsy specimens are often used in molecular biological studies of disease pathophysiology. However, few analyses have focused specifically on postmortem changes in skeletal muscles, and almost all of those investigate protein or metabolic changes. Although some structural and enzymatic changes have been described, the sequence of transcriptional events associated with these remains unclear. We analyzed a series of new and preexisting human skeletal muscle data sets on approximately 12,500 genes and expressed sequence tags (ESTs) generated by the Affymetrix U95Av2 GeneChips from seven autopsy and seven surgical specimens. Remarkably, postmortem specimens (up to 46 h) revealed a significant and prominent upregulation of transcripts involved with protein biosynthesis. Additional upregulated transcripts are associated with cellular responses to oxidative stress, hypoxia, and ischemia; however, only a subset of genes in these pathways was affected. Overexpression was also seen for apoptosis-related, cell cycle regulation/arrest-related, and signal transduction-related genes. No major gene expression differences were seen between autopsy specimens with 20-h and 34- to 46-h postmortem intervals or between pediatric and adult cases. These data demonstrate that, likely in response to hypoxia and oxidative stress, skeletal muscle undergoes a highly active transcriptional, and possibly, translational phase during the initial 46-h postmortem interval. Knowledge of these changes is important for proper interpretation of gene expression studies utilizing autopsy specimens.

The genetic basis of Diamond-Blackfan anaemia (DBA), a congenital erythroid hypoplasia that shows marked clinical heterogeneity, remains obscure. However, the fact that nearly one-quarter of patients harbour a variety of mutations in RPS19, a ribosomal protein gene, provides an opportunity to examine whether haplo-insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations. We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations in the 19 probands, and studied mutations in 10 patients at the RNA level and in three patients at the protein level. Characterization of the mutations in 10 probands, including six with novel insertions, nonsense and splice site mutations, showed that the abnormal transcript was detectable in nine cases. The RPS19 mRNA and protein in CD34+ bone marrow cells identified haplo-insufficiency in three cases predicted to have one functional allele. Our data support the notion that, in addition to rare DBA patients with the deletion of one allele, the disease in certain other RPS19 mutant patients is because of RPS19 protein haplo-insufficiency.

We present comparisons of the clinical pictures in a series of 60 patients with nemaline myopathy in whom mutations had been identified in the genes for nebulin or skeletal muscle alpha-actin. In the patients with nebulin mutations, the typical form of nemaline myopathy predominated, while severe, mild or intermediate forms were less frequent. Autosomal recessive inheritance had been verified or appeared likely in all nebulin cases. In the patients with actin mutations, the severe form of nemaline myopathy was the most common, but some had the mild or typical form, and a few showed other associated features such as intranuclear rods or actin accumulation. Most cases were sporadic, but in addition there were instances of both autosomal dominant and autosomal recessive inheritance, while two families showed mosaicism for dominant mutations. Although no specific phenotype was found to be associated with mutations in either gene, clinical and histological features together with pedigree data may be used in guiding mutation detection. Finding the causative mutation(s) determines the mode of inheritance and permits prenatal diagnosis if requested, but will not as such permit prognostication.

Nemaline myopathy (NM) is the most common of several congenital myopathies that present with skeletal muscle weakness and hypotonia. It is clinically heterogeneous and the diagnosis is confirmed by identification of nemaline bodies in affected muscles. The skeletal muscle alpha-actin gene (ACTA1) is one of five genes for thin filament proteins identified so far as responsible for different forms of NM. We have screened the ACTA1 gene in a cohort of 109 unrelated patients with NM. Here, we describe clinical and pathological features associated with 29 ACTA1 mutations found in 38 individuals from 28 families. Although ACTA1 mutations cause a remarkably heterogeneous range of phenotypes, they were preferentially associated with severe clinical presentations (p 0.0001). Most pathogenic ACTA1 mutations were missense changes with two instances of single base pair deletions. Most patients with ACTA1 mutations had no prior family history of neuromuscular disease (24/28). One severe case, caused by compound heterozygous recessive ACTA1 mutations, demonstrated increased alpha-cardiac actin expression, suggesting that cardiac actin might partially compensate for ACTA1 abnormalities in the fetal/neonatal period. This cohort also includes the first instance of an ACTA1 mutation manifesting with adult-onset disease and two pedigrees exhibiting potential incomplete penetrance. Overall, ACTA1 mutations are a common cause of NM, accounting for more than half of severe cases and 26% of all NM cases in this series.

Nemaline myopathy (NM) is a slowly progressive or nonprogressive neuromuscular disorder caused by mutations in genes encoding skeletal muscle sarcomeric thin filament proteins. It is characterized by great heterogeneity at the clinical, histopathological, and genetic level. Although multiple molecular pathways are commonly affected in all NM patients, little is known about the molecular characteristics of muscles from patients in different NM subgroups. We have analyzed a group of global gene expression data sets for transcriptional patterns characteristic of particular nemaline myopathy classes. Differential expression between disease subgroups was primarily seen in mitochondrial-, structural-, and transcription-related genes. Multiple lines of evidence support the hypothesis that muscles from cases with "nontyping" NM, although clinically classified as typical NM, share a unique pathophysiological state and are characterized by distinct patterns of gene expression. Determination of the specific molecular differences in NM subgroups may eventually lead to improved prognostic determinations and treatment of these patients.