Giuseppina Di Fruscio

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 on patients with LGMD and nonspecific myopathies: Findings associated with ANO5 mutations

Highlights • We have carried out the largest screening of the ANO5 gene.• We identified 33 patients (4%) with pathogenic changes in both alleles and 23 heterozygotes (3%).• The identification of a ANO5 carrier is not to be considered an uncommon finding.• The anoctaminopathies have an extremely high genetic and phenotypic heterogeneity.• NGS-based strategies are perfect to dissect the clinical variability in NMDs.

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 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.

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

Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic

Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders.

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.

Familial Exudative Vitreoretinopathy caused by a Homozygous Mutation in TSPAN12 in a Cystic Fibrosis Infant

Abstract Familial exudative vitreoretinopathy (FEVR) is a genetic disease affecting the vascularization of the peripheral retina. The clinical manifestations are very heterogeneous, ranging from mildly affected patients, who could present no visual defects, to severe conditions which can also cause complete blindness at birth or in the first decade. FEVR can be inherited in all the three genetic forms: dominant, recessive and X-linked. To date, four genes have been associated with the condition: TSPAN12. NDP. FDZ4 and LRP5. Interestingly, mutations in TSPAN12 have been considered causative of both a dominant and recessive inheritance and a FEVR phenotype sensitive to the number of TSPAN12 mutations has been supposed. Here we describe a case of a female infant affected by cystic fibrosis and by a severe form of exudative vitreoretinopathy. In particular, we have detected the homozygous missense mutation c.668 T > C in TSPAN12. Neither of the heterozygous parents has ocular manifestations of the disease, suggesting a classic recessive mendelian pattern of inheritance.