Margherita Mutarelli

Identification of microRNA-regulated gene networks by expression analysis of target genes.

MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFβ pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFβ signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs.

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.

The ADAMTS18 gene is responsible for autosomal recessive early onset severe retinal dystrophy

BackgroundInherited retinal dystrophies, including Retinitis Pigmentosa and Leber Congenital Amaurosis among others, are a group of genetically heterogeneous disorders that lead to variable degrees of visual deficits. They can be caused by mutations in over 100 genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying a novel gene for an autosomal recessive form of early onset severe retinal dystrophy in a patient carrying no previously described mutations in known genes.MethodsAn integrated strategy including homozygosity mapping and whole exome sequencing was used to identify the responsible mutation. Functional tests were performed in the medaka fish (Oryzias latipes) model organism to gain further insight into the pathogenic role of the ADAMTS18 gene in eye and central nervous system (CNS) dysfunction.ResultsThis study identified, in the analyzed patient, a homozygous missense mutation in the ADAMTS18 gene, which was recently linked to Knobloch syndrome, a rare developmental disorder that affects the eye and the occipital skull. In vivo gene knockdown performed in medaka fish confirmed both that the mutation has a pathogenic role and that the inactivation of this gene has a deleterious effect on photoreceptor cell function.ConclusionThis study reveals that mutations in the ADAMTS18 gene can cause a broad phenotypic spectrum of eye disorders and contribute to shed further light on the complexity of retinal diseases.

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.

Triple Vectors Expand AAV Transfer Capacity in the Retina

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

A.P.16

The autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous muscular disorders, characterized by the presence of autophagic vacuoles, whose membranes exhibit sarcolemmal features. Although several myopathies with these features have been described, the only one in which the gene defect is known is the Danon disease, due to mutations in the lysosome-associated membrane protein-2 (LAMP2). Given the pathologic features similar to those in Danon disease, the molecular defects in the remaining AVMs could involve proteins related to lysosomal function. Recent evidence has demonstrated lysosomes are important in maintaining cellular processes in skeletal muscle and autophagy acts as contributor to disease pathogenesis and progression. We have developed a “preferential exome” NGS workflow, named LysoPlex, to sequence at high coverage 12,786 human exons of 891 genes. These genes are predicted to be involved in lysosomal function, endocytosis and autophagy pathway and most of them are not yet associated to known genetic disorders. We designed the enrichment probes using a Haloplex custom platform targeting 99.48% of exons. Until now, with Lysoplex, we have been able to perform molecular diagnosis of lysosomal storage disorders (LSDs) and neuronal ceroid lipofuscinoses (NCL), identifying disease-causing mutations in 70 patients and pointing out putative novel causative genes. Moreover, we are recruiting samples from AVM patients in order to elucidate their molecular defects. By using this preferential exome, in fact, we have a complete view of sequence variants in the genes involved in the lysosomal-autophagic pathway. In conclusion, our strategy represents a powerful approach to study the role of lysosomes and autophagy in skeletal muscles and in muscular disorders.

G.O.7

Limb-girdle muscular dystrophies (LGMDs) are a highly heterogeneous group of muscle disorders affecting the pelvic and the shoulder girdle musculature. Until now, over thirty disease genes are known. Even if the age of onset and other clinical features could address the diagnosis towards the specific LGMD form, the phenotypic heterogeneity within each form hampers an easy and rapid molecular identification of causative mutations. We developed a unique and exhaustive platform, based on Next Generation Sequencing on Illumina HiSeq, to analyze 89 muscular disease genes at very high coverage. This regains 20–30% of missing sequences when compared with whole exome sequencing of the same DNA samples. Using this protocol, we have sequenced 312 patients with a LGMD phenotype. Most patients have been previously studied using a gene-by-gene approach without success. Thus, these cases are somewhat enriched for unknown and elusive mutations. Nevertheless, in about 20% of patients we found typical causative mutations in predicted genes, missed by Sanger sequencing or generally poorly studied. An additional 30% of patients carry other novel pathogenic variations. As easily predictable, the diagnostic rate sensibly increases in naive samples not previously screened. Our extensive procedure allowed us to obtain a full comprehensive view of all sequence variants in these samples and to refine the genotype-phenotype correlation. More interestingly, we found in each patient at least five rare mutations (

G.P.220

Hundreds of variants in autosomal genes associated to limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their nonoccurrence in hundreds of healthy controls and/or from segregation studies in small families. We consider that a limited statistics of the genetic variations in the general population may hamper the correct interpretation of the disease-causing effect of variants. To clarify the meaning of low-frequency variants in LGMD genes, we have systematically searched for previously identified missense and nonsense variants described as causative in the Leiden Open Variation Database (LOVD) and the Human Gene Mutation Database (HGMD). To calculate their frequency we used the whole exome data from the NHLBI GO Exome Sequencing Project (ESP) and in our cohort of patients and controls analyzed by Next Generation Sequencing (NGS). Moreover, we predicted the effect of missense changes by several bioinformatic tools. Surprisingly, the ESP already contains 3% of the variants previously associated with autosomal dominant inheritance and about 12% of those associated with recessive inheritance. Moreover, a number of variants (about 20–25%) are predicted in silico to be not damaging. Finally, for specific forms of LGMDs, the putative disease alleles are much more frequent than the calculated disease prevalence. In conclusion, we identified a significant overrepresentation of LGMD-associated variants in large databases, suggesting that a large percentage of these are not the Mendelian cause of autosomal muscular dystrophies or, alternatively, they are pathogenic, but not fully penetrant. This highlights that genetic mechanisms are more complex than often thought. A non-biased testing of more genes in LGMD patients is needed for both genetic counseling and clinical trials.