Marco Savarese

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.

Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway

The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease.

The italian limb girdle muscular dystrophy registry: Relative frequency, clinical features, and differential diagnosis

Introduction: Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. Their molecular definition is fundamental for prognostic and therapeutic purposes. Methods: We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. Results: LGMD types 2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next‐generation sequencing (NGS) identified mutations in 36.5% of cases. Conclusion: Detailed clinical characterization combined with muscle tissue analysis is fundamental to guide differential diagnosis and to address molecular tests. NGS is useful for diagnosing forms without specific biomarkers, although, at least in our study cohort, several LGMD disease mechanisms remain to be identified. Muscle Nerve 55: 55–68, 2017

Dominant muscular dystrophy with a novel SYNE1 gene mutation

The synaptic nuclear envelope protein-1 (SYNE1) gene encodes nesprin-1, a protein characterized by the presence of multiple spectrin repeats and highly expressed in striated muscles. In addition to autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) type 4, mutations in the SYNE1 gene cause spinocerebellar ataxia type 8, myogenic multiplex arthrogryposis congenita with features of EDMD, intellectual disability with spastic paraplegia, and axonal neuropathy. We report a family including 3 patients (mother and 2 sons) affected with a dominant form of muscular dystrophy with joint contractures without significant cardiac abnormalities (“EDMD-like” phenotype), in which we identified a novel mutation in the SYNE1 gene. Patient 1 (I-2, Fig. 1) is a woman who, at age 5 years, underwent surgery for congenital pulmonary-valve stenosis; she was subsequently free from heart disturbances. She had onset of progressive muscle weakness at age 6-7 years; foot and elbow joint contractures were present in adolescence. At age 19 she underwent bilateral Achilles tenotomy. She died at age 44 years due to melanoma; she was still able to walk for short distances, but was unable to climb stairs. Patient 2 (II-1, Fig. 1) is a 28-year-old man who had onset of proximal weakness in early childhood, with clumsy gait, and Gowers sign; he had foot, elbow, and knee contractures when he was evaluated at age 7 years. At age 13 he underwent Achilles tenotomy. He is still ambulant but has severe difficulties. Cardiac investigations showed no arrhythmias. Patient 3 (II-2, Fig. 1) is a 26-year-old man who, since age 3 years, had proximal progressive muscle weakness with joint contractures in the foot, elbow, and upper spine. At age 9 he underwent surgery for equinus foot deformity. Clinical cardiac evaluation was normal. The 2 muscle biopsies studied showed dystrophic changes but no vacuolar changes or abnormal nuclear morphology (Fig. 1). Emerin and lamin A/C (LMNA) immunofluorescence (Fig. 1) and lysosomal-associated membrane protein-2 (LAMP-2) immunoblot (Fig. 1) showed normal protein expression. LMNA gene sequencing showed no mutations. Targeted next generation sequencing (NGS) identified a variation in the LAMP2 gene (p.G221R) and a novel heterozygous mutation in the SYNE1 gene (NM_182961.3, c.323C>T, p.N108S) localized in the actinbinding domain of nesprin-1 (isoform 1, http://www.dmd. nl). In silico analysis of the mutations predicted both LAMP2 and SYNE1 gene variations to be damaging. Danon disease in these patients was excluded because the clinical phenotype was not typical, they had normal LAMP-2 protein expression (Danon males usually display absent protein), and the LAMP2 variation we identified was reported as a polymorphism (it may be a genetic modifier). The only potentially causative mutation identified by NGS in our family was the novel mutation in the SYNE1 gene, which has been recognized to cause EDMD in a few kindreds. Although many SYNE1 gene polymorphisms have been identified in individuals at risk for bipolar disorder, ovarian cancer, and autism, and in a reference population, a series of concordant clues suggest a primary role of the SYNE1 mutation identified in our family. These include its predicted deleterious effect, its localization in a functionally crucial domain, its absence in a large series of control chromosomes, and the finding of no other mutations by NGS in this family. Altogether, this SYNE1 gene mutation seems to cause an “EDMD-like” phenotype, characterized by progressive muscular dystrophy with joint contractures, but without heart involvement. One patient with a similar clinical phenotype but a different SYNE1 gene mutation has been reported previously. Table 1. The incidence rate (per million person-years) and prevalence rate (per million persons) of myasthenia gravis in Arab countries.

The Italian LGMD registry: Relative frequency, clinical features, and differential diagnosis

Introduction: Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. Their molecular definition is fundamental for prognostic and therapeutic purposes. Methods: We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. Results: LGMD types 2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next‐generation sequencing (NGS) identified mutations in 36.5% of cases. Conclusion: Detailed clinical characterization combined with muscle tissue analysis is fundamental to guide differential diagnosis and to address molecular tests. NGS is useful for diagnosing forms without specific biomarkers, although, at least in our study cohort, several LGMD disease mechanisms remain to be identified. Muscle Nerve 55: 55–68, 2017

MePR: a novel human mesenchymal progenitor model with characteristics of pluripotency.

Human embryo stem cells or adult tissues are excellent models for discovery and characterization of differentiation processes. The aims of regenerative medicine are to define the molecular and physiological mechanisms that govern stem cells and differentiation. Human mesenchymal stem cells (hMSCs) are multipotent adult stem cells that are able to differentiate into a variety of cell types under controlled conditions both in vivo and in vitro, and they have the remarkable ability of self-renewal. hMSCs derived from amniotic fluid and characterized by the expression of Oct-4 and Nanog, typical markers of pluripotent cells, represent an excellent model for studies on stemness. Unfortunately, the limited amount of cells available from each donation and, above all, the limited number of replications do not allow for detailed studies. Here, we report on the immortalization and characterization of novel mesenchymal progenitor (MePR) cell lines from amniotic fluid-derived hMSCs, whose biological properties are similar to primary amniocytes. Our data indicate that MePR cells display the multipotency potential and differentiation rates of hMSCs, thus representing a useful model to study both mechanisms of differentiation and pharmacological approaches to induce selective differentiation. In particular, MePR-2B cells, which carry a bona fide normal karyotype, might be used in basic stem cell research, leading to the development of new approaches for stem cell therapy and tissue engineering.

Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast

Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression inxa0vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development.

Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements

Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.

Next generation sequencing detection of late onset pompe disease

We report a patient in whom the diagnosis of a treatable disease was delayed for 30 years.