A novel in-frame deletion in MYOT causes an early adult onset distal myopathy.

Missense mutations in MYOT encoding the sarcomeric Z-disk protein myotilin cause three main myopathic phenotypes including proximal limb-girdle muscular dystrophy, spheroid body myopathy, and late-onset distal myopathy. We describe a family carrying a heterozygous MYOT deletion (Tyr4_His9del) that clinically was characterized by an early-adult onset distal muscle weakness and pathologically by a myofibrillar myopathy (MFM). Molecular modeling of the full-length myotilin protein revealed that the 4-YERPKH-9 amino acids are involved in local interactions within the N-terminal portion of myotilin. Injection of in vitro synthetized mutated human MYOT RNA or of plasmid carrying its cDNA sequence in zebrafish embryos led to muscle defects characterized by sarcomeric disorganization of muscle fibers and widening of the I-band, and severe motor impairments. We identify MYOT novel Tyr4_His9 deletion as the cause of an early-onset MFM with a distal myopathy phenotype and provide data supporting the importance of the amino acid sequence for the structural role of myotilin in the sarcomeric organization of myofibers.

Combined deficiency of alpha and epsilon sarcoglycan disrupts the cardiac dystrophin complex.

Cardiomyopathy is a puzzling complication in addition to skeletal muscle pathology for patients with mutations in ß-, γ- or δ-sarcoglycan (SG) genes. Patients with mutations in α-SG rarely have associated cardiomyopathy, or their cardiac pathology is very mild. We hypothesize that a fifth SG, ε-SG, may compensate for α-SG deficiency in the heart. To investigate the function of ε-SG in striated muscle, we generated an Sgce-null mouse and a Sgca-;Sgce-null mouse, which lacks both α- and ε-SGs. While Sgce-null mice showed a wild-type phenotype, with no signs of muscular dystrophy or heart disease, the Sgca-;Sgce-null mouse developed a progressive muscular dystrophy and a more anticipated and severe cardiomyopathy. It shows a complete loss of residual SGs and a strong reduction in both dystrophin and dystroglycan. Our data indicate that ε-SG is important in preventing cardiomyopathy in α-SG deficiency.

A missense mutation in CASK causes FG syndrome in an Italian family.

First described in 1974, FG syndrome (FGS) is an X-linked multiple congenital anomaly/mental retardation (MCA/MR) disorder, characterized by high clinical variability and genetic heterogeneity. Five loci (FGS1-5) have so far been linked to this phenotype on the X chromosome, but only one gene, MED12, has been identified to date. Mutations in this gene account for a restricted number of FGS patients with a more distinctive phenotype, referred to as the Opitz-Kaveggia phenotype. We report here that a p.R28L (c.83G-->T) missense mutation in CASK causes FGS phenotype in an Italian family previously mapped to Xp11.4-p11.3 (FGS4). The identified missense mutation cosegregates with the phenotype in this family and is absent in 1000 control X chromosomes of the same ethnic origin. An extensive analysis of CASK protein functions as well as structural and dynamic studies performed by molecular dynamics (MD) simulation did not reveal significant alterations induced by the p.R28L substitution. However, we observed a partial skipping of the exon 2 of CASK, presumably a consequence of improper recognition of exonic splicing enhancers (ESEs) induced by the c.83G-->T transversion. CASK is a multidomain scaffold protein highly expressed in the central nervous system (CNS) with specific localization to the synapses, where it forms large signaling complexes regulating neurotransmission. We suggest that the observed phenotype is most likely a consequence of an altered CASK expression profile during embryogenesis, brain development, and differentiation.

Novel FLNC mutation in a patient with myofibrillar myopathy in combination with late-onset cerebellar ataxia.

INTRODUCTION: Mutations in the gene that encodes filamin C, FLNC, represent a rare cause of a distinctive type of myofibrillar myopathy (MFM). METHODS: We investigated an Italian patient by means of muscle biopsy, muscle and brain imaging and molecular analysis of MFM genes. RESULTS: The patient harbored a novel 7256C>T, p.Thr2419Met mutation in exon 44 of FLNC. Clinical, pathological and muscle MRI findings were similar to the previously described filaminopathy cases. This patient had, in addition, cerebellar ataxia with atrophy of cerebellum and vermis evident on brain MRI scan. Extensive screening failed to establish a cause of cerebellar atrophy. CONCLUSIONS: We report an Italian filaminopathy patient, with a novel mutation in a highly conserved region. This case raises the possibility that the disease spectrum caused by FLNC may include cerebellar dysfunction.

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding ß-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease.

Limb girdle muscular dystrophies: update on genetic diagnosis and therapeutic approaches.

PURPOSE OF REVIEW: This review is an up-to-date analysis of the genetic diagnosis and therapeutic strategies for limb girdle muscular dystrophies (LGMDs). RECENT FINDINGS: LGMDs are an example of both clinical and genetic heterogeneity. Clinically, by the description of non-LGMD phenotypes associated with LGMD genes and of LGMD phenotypes associated with originally non-LGMD disease genes; and genetically, by the description of new LGMD genes that further increase the diagnostic complexity. Moreover, new powerful approaches for DNA analysis, such as exome sequencing, promise to revolutionize the field of heterogeneous genetic diseases, also providing information about the true penetrance of LGMD mutations. The recent inputs on novel pathogenic mechanisms and pathways in LGMD will suggest novel therapeutic approaches and future clinical trials. In addition, therapeutic approaches of gene and cell delivery into animal models show promising results that will be translated into clinical trials. SUMMARY: The genetic diagnosis of LGMD from the present home-made algorithms will move toward high-throughput diagnostic strategies based on next-generation sequencing (NGS) technologies. As therapy, new powerful drug approaches based on recent pathogenetic findings will be pushed to clinical trials. In addition, novel more efficient and safer viral vectors for gene delivery will be proposed.

Motor chip: a comparative genomic hybridization microarray for copy-number mutations in 245 neuromuscular disorders.

BACKGROUND: Array-based comparative genomic hybridization (aCGH) is a reference high-throughput technology for detecting large pathogenic or polymorphic copy-number variations in the human genome; however, a number of quantitative monogenic mutations, such as smaller heterozygous deletions or duplications, are usually missed in most disease genes when proper multiplex ligation-dependent probe assays are not performed. METHODS: We developed the Motor Chip, a customized CGH array with exonic coverage of 245 genes involved in neuromuscular disorders (NMDs), as well as 180 candidate disease genes. We analyzed DNA samples from 26 patients with known deletions or duplications in NMDs, 11 patients with partial molecular diagnoses, and 19 patients with a clinical diagnosis alone. RESULTS: The Motor Chip efficiently confirmed and refined the copy-number mutations in all of the characterized patients, even when only a single exon was involved. In noncharacterized or partially characterized patients, we found deletions in the SETX (senataxin), SGCG [sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein)], and LAMA2 (laminin, alpha 2) genes, as well as duplications involving LAMA2 and the DYSF [dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive)] locus. CONCLUSIONS: The combination of exon-specific gene coverage and optimized platform and probe selection makes the Motor Chip a complementary tool for molecular diagnosis and gene investigation in neuromuscular diseases.

Reliable resequencing of the human dystrophin locus by universal long polymerase chain reaction and massive pyrosequencing.

The X-linked dystrophin gene is well known for its involvement in Duchenne/Becker muscular dystrophies and for its exceptional megabase size. This locus at Xp21 is prone to frequent random molecular changes, including large deletions and duplications, but also smaller variations. To cope with such huge sequence analysis requirements in forthcoming diagnostic applications, we employed the power of the parallel 454 GS-FLX pyrosequencer to the dystrophin locus. We enriched the genomic region of interest by the robust amplification of 62 fragments under universal conditions by the long-PCR protocol yielding 244,707 bp of sequence. Pooled PCR products were fragmented and used for library preparation and DNA sequencing. To evaluate the entire procedure we analyzed four male DNA samples for sequence coverage and accuracy in DNA sequence variation and for any potential bias. We identified 562 known variations and 55 additional variants not yet reported, among which we detected a causative Arg1844Stop mutation in one sample. Sanger sequencing confirmed all changes. Unexpectedly, only 3 x coverage was sufficient for 99.9993% accuracy. Our results show that long PCR combined to massive pyrosequencing is very reliable for the analysis of the biggest gene of the human genome and open the doors to other demanding applications in molecular diagnostics.

Perioral skin biopsy to study skeletal muscle protein expression.

Clinical trials for muscular dystrophy molecular treatment require multiple sampling of skeletal muscle to monitor protein rescue. This practice is invasive and could raise ethical problems. A less invasive tool to obtain sequential muscle sampling is necessary. Using indirect immunofluorescence, we evaluated muscle protein expression in myofiber bundles included in 2-2.5-mm punch skin biopsies from the perioral region from 6 healthy subjects and 6 patients with genetically defined forms of muscular dystrophy. Large intradermal bundles of orbicularis oris muscle were constantly present in skin biopsies. They showed a typical muscular antigenic pattern in controls and the expected protein defect in muscular dystrophy patients. These results demonstrate the feasibility of muscular protein expression analysis using skin biopsy. We propose this minimally invasive technique to follow-up the response to genetic or conventional therapies in muscular dystrophies and to confirm the diagnosis in some special clinical conditions.

Diagnostic value of muscle MRI in differentiating LGMD2I from other LGMDs.

Mutations in the fukutin-related protein (FKRP) have recently been demonstrated to cause limb girdle muscular dystrophy type 2I (LGMD2I), one of the most common forms of the autosomal recessive LGMDs in Europe. We performed a systematic clinical and muscle MRI assessment in 6 LGMD2I patients and compared these findings with those of 14 patients with genetically confirmed diagnosis of other forms of autosomal recessive LGMDs or dystrophinopathies. All LGMD2I patients had a characteristic clinical phenotype with predominant weakness of hip flexion and adduction, knee flexion and ankle dorsiflexion. These findings were also mirrored on MRI of the lower extremities which demonstrated marked signal changes in the adductor muscles, the posterior thigh and posterior calf muscles. This characteristic clinical and MRI phenotype was also seen in LGMD2A. However, in LGMD2A there was a selective involvement of the medial gastrocnemius and soleus muscle in the lower legs which was not seen in LGMD2I. The pattern in LGMD2A and LGMD2I were clearly different from the one seen in alpha-sarcoglycanopathy and dystrophinopathy type Becker which showed marked signal abnormalities in the anterior thigh muscles. Our results indicate that muscular MRI is a powerful tool for differentiating LGMD2I from other forms of autosomal recessive LGMDs and dystrophinopathies.

Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations.

Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a 'nonstop' mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.

One hundred twenty-one dystrophin point mutations detected from stored DNA samples by combinatorial denaturing high-performance liquid chromatography.

Duchenne and Becker muscular dystrophies are caused by a large number of different mutations in the dystrophin gene. Outside of the deletion/duplication "hot spots," small mutations occur at unpredictable positions. These account for about 15 to 20% of cases, with the major group being premature stop codons. When the affected male is deceased, carrier testing for family members and prenatal diagnosis become difficult and expensive. We tailored a cost-effective and reliable strategy to discover point mutations from stored DNA samples in the absence of a muscle biopsy. Samples were amplified in combinatorial pools and tested by denaturing high-performance liquid chromatography analysis. An anomalous elution profile belonging to two different pools univocally addressed the allelic variation to an unambiguous sample. Mutations were then detected by sequencing. We identified 121 mutations of 99 different types. Fifty-six patients show stop codons that represent the 46.3% of all cases. Three non-obvious single amino acid mutations were considered as causative. Our data support combinatorial denaturing high-performance liquid chromatography analysis as a clear-cut strategy for time and cost-effective identification of small mutations when only DNA is available.

Calpain-3 mutations in Turkey.

Autosomal recessive limb-girdle muscular dystrophies (LGMD2s) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement of the proximal limb girdle muscles; the group includes at least 10 different genetic entities. The calpainopathies (LGMD2A), a subgroup of LGMD2s, are estimated to be the most common forms of LGMD2 in all populations so far investigated. LGMD2A is usually characterized by symmetrical and selective atrophy of pelvic, scapular and trunk muscles and a moderate to gross elevation of serum CK. However, the course is highly variable. It is caused by mutations in the CAPN3 gene, which encodes for the calpain-3 protein. Until now, 161 pathogenic mutations have been found in the CAPN3 gene. In the present study, through screening of 93 unrelated LGMD2 families, we identified 29 families with LGMD2A, 21 (22.6%) of which were identified as having CAPN3 gene mutations. We detected six novel (p.K211N, p.D230G, p.Y322H, p.R698S, p.Q738X, c.2257delGinsAA) and nine previously reported mutations (c.550delA, c.19_23del, c.1746-20C>G, p.R49H, p.R490Q, p.Y336N, p.A702V, p.Y537X, p.R541Q) in the CAPN3 gene. There may be a wide variety of mutations, but clustering of specific mutations (c.550delA: 40%, p.R490Q: 10%) could be used in the diagnostic scheme in Turkey.

On symptomatic heterozygous alpha-sarcoglycan gene mutation carriers.

Mutations in the human alpha-sarcoglycan gene on chromosome 17q21.2 have been shown to cause a severe childhood autosomal recessive muscular dystrophy, a less severe limb girdle muscular dystrophy, exercise intolerance, or asymptomatic hyperCKemia. Here, we describe the clinical findings in a German family harboring a 371 T > C (Ile124Thr) missense mutation in the alpha-sarcoglycan gene. Whereas our index patient, an 11-year-old girl homozygous for this mutation, presented with a severe Duchenne-like phenotype, 7 out of 12 heterozygous mutation carriers from three generations showed mild to moderate scapular winging. In analogy to symptomatic female dystrophinopathy carriers, our results suggest that heterozygous alpha-sarcoglycan gene mutation carriers can be symptomatic with selective muscle weakness. This finding may be attributed to an additional negative variation in a yet unknown modifier gene essential to the function of the sarcoglycan complex in shoulder girdle muscles.

Scanning for mutations of the ryanodine receptor (RYR1) gene by denaturing HPLC: detection of three novel malignant hyperthermia alleles.

BACKGROUND: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected. METHODS: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis. RESULTS: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu. CONCLUSION: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.