Annalaura Torella

MotorPlex provides accurate variant detection across large muscle genes both in single myopathic patients and in pools of DNA samples

Mutations in ~100 genes cause muscle diseases with complex and often unexplained genotype/phenotype correlations. Next-generation sequencing studies identify a greater-than-expected number of genetic variations in the human genome. This suggests that existing clinical monogenic testing systematically miss very relevant information.We have created a core panel of genes that cause all known forms of nonsyndromic muscle disorders (MotorPlex). It comprises 93 loci, among which are the largest and most complex human genes, such as TTN, RYR1, NEB and DMD. MotorPlex captures at least 99.2% of 2,544 exons with a very accurate and uniform coverage. This quality is highlighted by the discovery of 20-30% more variations in comparison with whole exome sequencing. The coverage homogeneity has also made feasible to apply a cost-effective pooled sequencing strategy while maintaining optimal sensitivity and specificity.We studied 177 unresolved cases of myopathies for which the best candidate genes were previously excluded. We have identified known pathogenic variants in 52 patients and potential causative ones in further 56 patients. We have also discovered 23 patients showing multiple true disease-associated variants suggesting complex inheritance. Moreover, we frequently detected other nonsynonymous variants of unknown significance in the largest muscle genes. Cost-effective combinatorial pools of DNA samples were similarly accurate (97-99%).MotorPlex is a very robust platform that overcomes for power, costs, speed, sensitivity and specificity the gene-by-gene strategy. The applicability of pooling makes this tool affordable for the screening of genetic variability of muscle genes also in a larger population. We consider that our strategy can have much broader applications.

Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F.

Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.

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.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

The genetic basis of undiagnosed muscular dystrophies and myopathies Results from 504 patients

Objective: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. Methods: We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. Results: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. Conclusions: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.

Log-PCR: A New Tool for Immediate and Cost-Effective Diagnosis of up to 85% of Dystrophin Gene Mutations

BACKGROUND Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the dystrophin gene. Despite the progress in the technologies of mutation detection, the disease of one third of patients escapes molecular definition because the labor and expense involved has precluded analyzing the entire gene. Novel techniques with higher detection rates, such as multiplex ligation-dependent probe amplification and multiplex amplifiable probe hybridization, have been introduced. METHODS We approached the challenge of multiplexing by modifying the PCR chemistry. We set up a rapid protocol that analyzes all dystrophin exons and flanking introns (57.5 kb). We grouped exons according to their effect on the reading frame and ran 2 PCR reactions for DMD mutations and 2 reactions for BMD mutations under the same conditions. The PCR products are evenly spaced logarithmically on the gel (Log-PCR) in an order that reproduces their chromosomal locations. This strategy enables both simultaneous mapping of all the mutation borders and distinguishing between DMD and BMD. As a proof of principle, we reexamined samples from 506 patients who had received a DMD or BMD diagnosis. RESULTS We observed gross rearrangements in 428 of the patients (84.6%; 74.5% deletions and 10.1% duplications). We also recognized a much broader spectrum of mutations and identified 14.6% additional cases. CONCLUSIONS This study is the first exhaustive investigation of this subject and has made possible the development of a cost-effective test for diagnosing a larger proportion of cases. The benefit of this approach may allow more focused efforts for discovering small or deep-intronic mutations among the few remaining undiagnosed cases. The same protocol can be extended to set up Log-PCRs for other high-throughput applications.

TBCE Mutations Cause Early-Onset Progressive Encephalopathy with Distal Spinal Muscular Atrophy

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration.

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.

Cardiomyopathy in patients with POMT1-related congenital and limb-girdle muscular dystrophy

Protein-o-mannosyl transferase 1 (POMT1) is a glycosyltransferase involved in α-dystroglycan (α-DG) glycosylation. Clinical phenotype in POMT1-mutated patients ranges from congenital muscular dystrophy (CMD) with structural brain abnormalities, to limb-girdle muscular dystrophy (LGMD) with microcephaly and mental retardation, to mild LGMD. No cardiac involvement has until now been reported in POMT1-mutated patients. We report three patients who harbored compound heterozygous POMT1 mutations and showed left ventricular (LV) dilation and/or decrease in myocardial contractile force: two had a LGMD phenotype with a normal or close-to-normal cognitive profile and one had CMD with mental retardation and normal brain MRI. Reduced or absent α-DG immunolabeling in muscle biopsies were identified in all three patients. Bioinformatic tools were used to study the potential effect of POMT1-detected mutations. All the detected POMT1 mutations were predicted in silico to interfere with protein folding and/or glycosyltransferase function. The report on the patients described here has widened the clinical spectrum associated with POMT1 mutations to include cardiomyopathy. The functional impact of known and novel POMT1 mutations was predicted with a bioinformatics approach, and results were compared with previous in vitro studies of protein-o-mannosylase function.

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 β‐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. J. Cell. Physiol. 226: 2894–2900, 2011.