FLNC-Associated Myofibrillar Myopathy: New Clinical, Functional, and Proteomic Data.

OBJECTIVE: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM). METHODS: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis. RESULTS: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc. CONCLUSIONS: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.

Differential diagnosis of vacuolar myopathies in the NGS era.

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non-inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late-onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr-caveolinopathy and of limb-girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.

A new case expanding the mutation and phenotype spectrum of TMEM5-related alpha-dystroglycanopathy.

Dystroglycanopathies are a diverse group of neuromuscular disorders caused by aberrant glycosylation of alpha-dystroglycan. TMEM5 is one of many glycosyltransferases recently described to be associated with alpha-dystroglycanopathies. We report the case of a 15-year-old boy suffering from a congenital muscular dystrophy with elevated serum creatine kinase levels and an almost complete absence of alpha-dystroglycan in muscle biopsy. The clinical course was milder than any previously reported case and did not include brain or eye defects. Standard next-generation sequencing analysis revealed a homozygous mutation in the donor splice site region of exon 5 in TMEM5 (c.914+6 T>G). Available in-silico prediction tools anticipated a reduced efficiency of the splice site. Subsequent cDNA sequencing confirmed the expression of a truncated transcript of TMEM5 lacking exon 5, hence leading to an in-frame deletion in the exostosin domain of the protein. This report expands the clinical and mutation spectrum of alpha-dystroglycanopathies.

The metabolic and endocrine characteristics in spinal and bulbar muscular atrophy.

OBJECTIVE: Spinal and bulbar muscular atrophy (SBMA) is caused by an abnormal expansion of the CAG repeat in the androgen receptor gene. This study aimed to systematically phenotype a German SBMA cohort (n = 80) based on laboratory markers for neuromuscular, metabolic, and endocrine status, and thus provide a basis for the selection of biomarkers for future therapeutic trials. METHODS: We assessed a panel of 28 laboratory parameters. The clinical course and blood biomarkers were correlated with disease duration and CAG repeat length. A subset of 11 patients was evaluated with body fat MRI. RESULTS: Almost all patients reported muscle weakness (99%), followed by dysphagia (77%), tremor (76%), and gynecomastia (75%) as major complaints. Creatine kinase was the most consistently elevated (94%) serum marker, which, however, did not relate with either the disease duration or the CAG repeat length. Paresis duration and CAG repeat length correlated with dehydroepiandrosterone sulfate after correction for body mass index and age. The androgen insensitivity index was elevated in nearly half of the participants (48%). CONCLUSIONS: Metabolic alterations in glucose homeostasis (diabetes) and fat metabolism (combined hyperlipidemia), and sex hormone abnormalities (androgen insensitivity) could be observed among SBMA patients without association with the neuromuscular phenotype. Dehydroepiandrosterone sulfate was the only biomarker that correlated strongly with both weakness duration and the CAG repeat length after adjusting for age and BMI, indicating its potential as a biomarker for both disease severity and duration and, therefore, its possible use as a reliable outcome measure in future therapeutic studies.

Assessing the influence of age and gender on the phenotype of myotonic dystrophy type 2.

This study aims to provide a detailed clinical characterization of a large cohort of myotonic dystrophy type 2 (DM2) patients investigating the influence of age and gender as modifying factors of DM2 phenotype. A retrospective study was conducted on 307 patients with genetically confirmed DM2. The following data were analyzed: (1) demographics, (2) clinical features (first symptom, muscular complaints, and multisystemic involvement), (3) diagnostics (serological tests, electromyography, and muscle biopsy). In this cohort (186 females, 121 males), a proximal weakness was the leading symptom at onset (55.4%), followed by myalgia (35.5%) and myotonia (25.4%). Proximal weakness was more common in women than men (64.9 vs. 43.8%, p = 0.0006), whereas being male was associated with higher odds for developing myalgia [OR 2.94 (95% CI 1.53-5.67)]. Patients with muscle weakness at onset were older than those with myalgia and myotonia (p < 0.0001), while each additional disease year was associated with 10% decrease in the odds of developing myotonia [OR 0.9 (95% CI 0.87-0.93)] and 6% decrease of myalgia [OR 0.94 (95% CI 0.91-0.97)]. Cataract and thyroid diseases occurred more frequently in women (p = 0.002 and p = 0.002, respectively). Early onset of DM2 is an independent risk factor for the occurrence of multisystemic involvement [OR 0.94 (95% CI 0.90-0.98)]. In this updated clinical description of DM2 emerges a profound gender and age influence on the phenotype, emphasizing that female gender and ageing may be associated with a higher disease burden. These age- and gender-specific differences should be considered in diagnostics, management, and future clinical studies of DM2.

Deep intronic variants introduce DMD pseudoexon in patient with muscular dystrophy.

Dystrophinopathies are X-linked muscle diseases caused by mutations in the large DMD gene. The most common mutations are detected by standard diagnostic techniques. However, some patients remain without detectable mutation, most likely due to changes in the non-coding sequence. We report on a boy with complete absence of dystrophin in muscle biopsy but no causative mutation according to standard diagnostics. To search for deep intronic variations (DIV) in the DMD gene we isolated mRNA from muscle tissue and amplified overlapping cDNA fragments using RT-PCR. One cDNA product revealed an augmented fragment size showing an insertion of 77 bp between the exons 7 and 8 by sequencing. We sequenced the flanking sequences of gDNA and found two hemizygous single nucleotide variants (c.650-39575 A>C and c.650-39498 A>G) surrounding the inserted fragment. Both variants create cryptic splice sites which initiate the formation of a pseudoexon that produces a frameshift in the DMD gene.

The Caveolin-3 G56S sequence variant of unknown significance: Muscle biopsy findings and functional cell biological analysis.

PURPOSE: In the era of next-generation sequencing, we are increasingly confronted with sequence variants of unknown significance. This phenomenon is also known for variations in Caveolin-3 and can complicate the molecular diagnosis of the disease. Here, we aimed to study the ambiguous character of the G56S Caveolin-3 variant. EXPERIMENTAL DESIGN: A comprehensive approach combining genetic and morphological studies of muscle derived from carriers of the G56S Caveolin-3 variant were carried out and linked to biochemical assays (including phosphoblot studies and proteome profiling) and morphological investigations of cultured myoblasts. RESULTS: Muscles showed moderate chronic myopathic changes in all carriers of the variant. Myogenic RCMH cells expressing the G56S Caveolin-3 protein presented irregular Caveolin-3 deposits within the Golgi in addition to a regular localization of the protein to the plasma membrane. This result was associated with abnormal findings on the ultra-structural level. Phosphoblot studies revealed that G56S affects EGFR-signaling. Proteomic profiling demonstrated alterations in levels of physiologically relevant proteins which are indicative for antagonization of G56S Caveolin-3 expression. Remarkably, some proteomic alterations were enhanced by osmotic/mechanical stress. CONCLUSIONS AND CLINICAL RELEVANCE: Our studies suggest that G56S might influence the manifestation of myopathic changes upon the presence of additional cellular stress burden. Results of our studies moreover improve the current understanding of (genetic) causes of myopathic disorders classified as caveolinopathies.

Adult-onset autosomal dominant centronuclear myopathy due to BIN1 mutations.

Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.

Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies.

BACKGROUND: Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement. METHODS: We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n = 43) and particularly focused on the associated multisystemic symptoms. RESULTS: We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p.Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p.Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%). CONCLUSIONS: We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim.

Xp21/A translocation: a rarely considered genetic cause for manifesting carriers of duchenne muscular dystrophy.

Clinically manifesting carriers of Duchenne muscular dystrophy (DMD) are rare among the pediatric population. A standardized diagnostic procedure in supposed DMD carriers entails performing a Multiplex Ligation-dependent Probe Amplification analysis of the DMD gene first, then taking a muscle biopsy to confirm reduced dystrophin levels and/or finally a complete sequencing of the DMD gene. We describe a girl with high-elevated creatine kinase, myalgia, and cardiomyopathy. Muscle biopsy showed a dystrophic pattern and nearly absent expression of dystrophin. Diagnosis could not be confirmed by molecular genetic procedures. Because of a mild mental retardation, a chromosome analysis and molecular karyotyping were performed, revealing a balanced translocation t(X;4)(p21;q31).arr(1-22,X)x2 dn with breakpoint on the X-chromosome within an intron of the DMD gene. The inactivation of the nonderivative X-chromosome was found to be in a nonrandom pattern, resulting in a functionally balanced karyotype and thus leading to a manifesting DMD carrier in this case. Chromosome analysis should be recommended in cases of genetically unsolved DMD carriers as a part of the standard genetic procedures.

Aarskog-Scott syndrome: a novel mutation in the FGD1 gene associated with severe craniofacial dysplasia.

UNLABELLED: Aarskog syndrome (AAS) is an X-linked human disease that affects the skeletal formation and embryonic morphogenesis and is caused by mutations in the FGD1 gene. Patients typically show distinctive skeletal and genital developmental abnormalities, but a broad spectrum of clinical phenotypes has been observed. We report here on the clinical and molecular analysis of a family that reveals a novel FGD1 mutation in a 9-year-old boy displaying extreme craniofacial dysplasia associated with attention deficit hyperactivity disorder. Sequencing of FGD1 revealed a novel mutation in exon 7 at position c.1468 C > T in the index patient, leading to a stop codon in the highly conserved RhoGEF gene domain. His mother and maternal grandmother were also found to be heterozygous for this FGD1 mutation. CONCLUSION: Our results identify a novel mutation of FDG1 in a family with Aarskog syndrome and underscore the phenotypical variability of this condition.

A paucisymptomatic neuromuscular disease mimicking type III 5q-SMA with complex rearrangements in the SMN gene.

Spinal muscular atrophy is an autosomal-recessive neuromuscular disorder, causing progressive proximal weakness and atrophy of the voluntary muscles. More than 96% of the spinal muscular atrophy patients show a homozygous absence of exons 7 and 8, or exon 7 only, in SMN1, the telomeric copy of the SMN gene. We report a young male patient with neurogenic symptoms and sparse muscle fiber atrophy, suggestive of a mild form of type III spinal muscular atrophy. He was found to be a carrier of intragenic mutations in both copies of the SMN gene, exhibiting a homozygous duplication of exons 7 and 8 in SMN1 and a homozygous deletion of exon 8 as well as a heterozygous deletion of exon 7 in SMN2. However, an intact full-length SMN1 complementary deoxyribonucleic acid was identified, and SMN protein levels in a muscle specimen were identical to that of a healthy control, formally excluding the diagnosis of spinal muscular atrophy III.

Anoctamin 5 muscular dystrophy associated with a silent p.Leu115Leu mutation resulting in exon skipping.

We report a 45year-old patient with an asymmetrical proximal muscle weakness affecting the quadriceps muscle of the right leg starting at the age of 32years. CK was 25-fold increased. MRI of the legs showed signs of fatty degeneration more pronounced in the right side. Biopsy of a thigh muscle showed dystrophic pattern and amyloid deposition in blood vessel walls. The coding region and exon/intron boundaries of the ANO5 gene were amplified and sequenced. The common c.191dupA mutation and a silent novel p.Leu115Leu (c.345G>A) variant were identified. This silent variant was listed neither in the LOVD database nor in the SNP database. To evaluate the pathogenicity of the novel silent mutation in ANO5, cDNA analysis was performed that demonstrated skipping of exon 6. So far, no case with a silent mutation leading to abnormal splicing has been identified in Anoctamin 5 muscular dystrophy. Present findings emphasize that cDNA analysis should be done if a silent variant is not annotated in the databases. In Anoctamin 5 muscular dystrophy a molecular diagnosis is even more important as protein investigation through Western blotting or immunohistochemistry is not yet established.

Facioscapulohumeral muscular dystrophy and Charcot-Marie-Tooth neuropathy 1A - evidence for "double trouble" overlapping syndromes.

BACKGROUND: We report on a patient with genetically confirmed overlapping diagnoses of CMT1A and FSHD. This case adds to the increasing number of unique patients presenting with atypical phenotypes, particularly in FSHD. Even if a mutation in one disease gene has been found, further genetic testing might be warranted in cases with unusual clinical presentation. CASE PRESENTATION: The reported 53 years old male patient suffered from walking difficulties and foot deformities first noticed at age 20. Later on, he developed scapuloperoneal and truncal muscle weakness, along with atrophy of the intrinsic hand and foot muscles, pes cavus, claw toes and a distal symmetric hypoesthesia. Motor nerve conduction velocities were reduced to 20 m/s in the upper extremities, and not educible in the lower extremities, sensory nerve conduction velocities were not attainable. Electromyography showed both, myopathic and neurogenic changes. A muscle biopsy taken from the tibialis anterior muscle showed a mild myopathy with some neurogenic findings and hypertrophic type 1 fibers. Whole-body muscle MRI revealed severe changes in the lower leg muscles, tibialis anterior and gastrocnemius muscles were highly replaced by fatty tissue. Additionally, fatty degeneration of shoulder girdle and straight back muscles, and atrophy of dorsal upper leg muscles were seen. Taken together, the presenting features suggested both, a neuropathy and a myopathy. Patient's family history suggested an autosomal dominant inheritance.Molecular testing revealed both, a hereditary motor and sensory neuropathy type 1A (HMSN1A, also called Charcot-Marie-Tooth neuropathy 1A, CMT1A) due to a PMP22 gene duplication and facioscapulohumeral muscular dystrophy (FSHD) due to a partial deletion of the D4Z4 locus (19 kb). CONCLUSION: Molecular testing in hereditary neuromuscular disorders has led to the identification of an increasing number of atypical phenotypes. Nevertheless, finding the right diagnosis is crucial for the patient in order to obtain adequate medical care and appropriate genetic counseling, especially in the background of arising curative therapies.

Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy.

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

Proteomic characterization of aggregate components in an intrafamilial variable FHL1-associated myopathy.

Myopathies associated with mutations in FHL1 are rare X-linked dominant myofibrillar myopathies. By clinical examination, histopathology, Sanger sequencing, and laser microdissection combined with quantitative mass spectrometry, we were able to identify the causative gene mutation and protein aggregate composition in two brothers with a late-onset X-linked scapulo-axio-peroneal myopathy. The severely progressive course of the disease revealed a remarkable intrafamilial variability of the clinical presentation. Protein aggregation and reducing bodies were observed in the muscle biopsy. Using quantitative mass spectrometry we identified the FHL1 protein as the component showing highest increased abundance in the aggregates in both patients, however strikingly in a different absolute amount in both brothers. Furthermore, we identified the causative C224W mutation in the fourth LIM-domain of FHL1 in both. Thus, of note is the striking evidence of reducing bodies in the muscle biopsy in both adults, and our proteomic data confirm the underlying gene defect with an intrafamilial variability by the ratio of the total protein content in the aggregates. We suggest that our combined approach has a high potential as a new tool for identification of causative gene mutations and raises hints on possibly intrafamilial variability in protein aggregation disorders. As all clinical subtypes and mutations in each exon of the FHL1 gene are associated with myofibrillar alterations and reducing bodies, we would like to suggest terming the whole group as FHL1-associated myopathies.

ZASPopathy with childhood-onset distal myopathy.

We report on a German family presenting with a predominantly distal myopathy primarily affecting anterior compartments of lower legs in childhood. Proximal lower limb and hip girdle weakness developed later in early adulthood in the female index patient and likewise in her mother. Consecutive muscle biopsy findings were first attributed to a mild congenital myopathy and later on interpreted as neurogenic changes without clear signs of a myopathy. Molecular genetic analysis was performed because of the clinical impression of a distal myopathy combined with dominant inheritance. The heterozygous mutation c.349G>A (p.D117N) in the ZASP gene could be found. This mutation had been previously associated with an adult-onset, isolated, dilated left ventricular non-compaction cardiomyopathy (OMIM*605906.0007), which was not present in our patients. Our data show that this mutation can be associated with an isolated skeletal muscle phenotype. Second, mutation analysis of the ZASP gene is suggested for distal myopathies of any age, even in cases of uncharacteristic muscle biopsy findings on routine analysis.

Impaired neuromuscular transmission and response to acetylcholinesterase inhibitors in centronuclear myopathies.

Many clinical features of autosomal centronuclear myopathies (CNM) and X-linked myotubular myopathy (XLMTM) are common to congenital myasthenic syndromes (CMS). We describe three children whose clinical and electrophysiological findings originally suggested CMS, in whom CNM was diagnosed pathologically, though not yet genetically characterised. A fourth case, with XLMTM, also showed electrophysiological features of a neuromuscular transmission defect. Three (including the XLMTM case) showed improved strength with acetylcholinesterase inhibitor treatment. We also studied neuromuscular junction structure and function in the MTM1 knockdown zebrafish model of XLMTM, demonstrating abnormal neuromuscular junction organization; anticholinesterase therapy resulted in marked clinical response. These observations suggest that a neuromuscular transmission defect may accompany CNM and contribute to muscle weakness. Muscle biopsy should be considered in infants suspected to have CMS, especially if treatment response is incomplete, or no CMS gene mutation is identified. Treatment with acetylcholinesterase inhibitors may benefit some CNM patients. This warrants further confirmation.

Inheritance and variable expression in Rubinstein-Taybi syndrome.

Familial Rubinstein-Taybi syndrome (RTS) is very rare. Here we report on the 6th and 7th case of inherited RTS. Family 1 presents with incomplete or mild RTS over three generations; a 13-year-old girl (proband 1) with mild but typical facial features and learning disabilities, her very mildly affected mother (proband 2), and the maternal grandmother (proband 3). Family 2 includes three females with classical RTS (probands 4-6) and their father (proband 7) with broad thumbs and halluces. Proband 5 also had a brain tumor (ganglioglioma) at the age of 3 years. In probands 1-3, direct sequencing identified a novel CREBBP missense mutation, c.2728A > G (predicting p.Thr910Ala), that was absent in non-affected family members. The p.Thr910Ala variant is outside the crucial histone acetyltransferase domain, and this may explain the mild and variable phenotype. In probands 4-7 we identified another novel CREBBP mutation, c.4134G > T, which alters the consensus splice sequence at position 1 of exon 25. The c.4134G > T mutation was transmitted from the very mildly affected father who displayed somatic mosaicism (with 38% mutated alleles in blood and 31% in buccal smear DNA) to his three daughters. Our findings emphasize that variable expression (family 1) and somatic mosaicism (family 2) contribute to the phenotypic variability of RTS. Somatic mosaicism may be more frequent in RTS than previously assumed. Accumulating data suggest a recurrence risk of approximately 0.5-1% for parents of a child with RTS, exceeding the so far estimated risk of approximately 0.1% for siblings.