Prosaposin (PSAP) deficiency is an ultra‐rare, fatal infantile lysosomal storage disorder (LSD) caused by variants in the PSAP gene, with seven subjects reported so far. Here, we provide the ...clinical, biochemical and molecular characterization of two additional PSAP deficiency cases. Lysoplex, a targeted resequencing approach was utilized to identify the variant in the first patient, while quantification of plasma lysosphingolipids (lysoSLs), assessed by liquid chromatography mass spectrometry (LC‐MS/MS) and brain magnetic resonance imaging (MRI), followed by Sanger sequencing allowed to attain diagnosis in the second case. Functional studies were carried out on patients' fibroblast lines to explore the functional impact of variants. The two patients were homozygous for two different truncating PSAP mutations (c.895G>T, p.Glu299*; c.834_835delGA, p.Glu278Aspfs*27). Both variants led to a complete lack of processed transcript. LC‐MS/MS and brain MRI analyses consistently provided a distinctive profile in the two children. Quantification of specific plasma lysoSLs revealed elevated levels of globotriaosylsphingosine (LysoGb3) and glucosylsphingosine (GlSph), and accumulation of autophagosomes, due to a decreased autophagic flux, was observed. This report documents the successful use of plasma lysoSLs profiling in the PSAP deficiency diagnosis, as a reliable and informative tool to obtain a preliminary information in infantile cases with complex traits displaying severe neurological signs and visceral involvement.
A.P.16 Di Fruscio, G; Savarese, M; De Cegli, R ...
Neuromuscular disorders : NMD,
10/2014, Letnik:
24, Številka:
9
Journal Article
Recenzirano
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.
The traditional gene-to-gene approach is too expensive and time-consuming for the molecular diagnosis of genetically heterogeneous disorders. We developed a cost-effective protocol for the DNA ...testing of many genes in large groups of limb-girdle muscular dystrophy (LGMD) patients based on next generation technologies. We defined this process in a flow-chart, composed of four steps: (a) to identify qualitative DNA sequence variations by targeted NGS; (b) to identify copy number variations (such as deletions or duplications >100 bp); (c) to sequence the mRNA and study splicing in diseased muscles; (d) to perform “whole exome sequencing”. For the first step we collected >400 DNA samples from unsolved LGMD patients from a number of Italian reference clinical centres. DNA samples were pooled and enriched for 493,598 bp, covering the exons of 93 genes (Motor Haloplex 3.0). In comparison with the previous design, new genes were added and others removed, considering the ease of differential diagnosis. We obtained >0.1 billion reads/sample and identified a large number of mutations. We preferred an asymmetrical pooling of 5 and 16 samples, in which the 5-plex pools are used to identify the variations, while the 16-plex to assign them to the single DNA samples. Samples negative for causative mutations were also studied by the Agilent MotorChip CGH array version 3.0 to identify deletions and duplications at the single exon resolution. Third, in selected cases, we performed targeted RNA-Seq starting from a muscle biopsy sample. The targeting probes had a 4× coverage with a total target size of 1.41 Mbp of sequences/sample, with a coverage of at 100×. Fourth, we performed whole exome sequencing using the novel Haloplex enrichment. On average, three rare and potentially damaging variations were detected per each patient. Our results confirm that there is a very high genetic heterogeneity, non-penetrance, and complexity in LGMDs.
G.O.7 Savarese, M; Di Fruscio, G; Torella, A ...
Neuromuscular disorders : NMD,
10/2014, Letnik:
24, Številka:
9
Journal Article
Recenzirano
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 naïve 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 (<0.01% in the general population); in about 35% of patients, we identified clearly damaging variants in addition to the causative ones. If we had detected these variants in the context of a single gene testing, we would have considered each of them as causative. In conclusion, our data suggest that “multiple troubles” may lead to complex phenotypes; in some patients, multiple defects in different genes could account for the variable expressivity and phenotypic divergence.
Abstract Next generation sequencing (NGS) is having a tremendous impact on our knowledge of different aspects of biology. It can be also very powerful to study patients with heterogeneous genetic ...conditions, like muscular dystrophies. First, to identify new genes using “exome resequencing”. Second, to diagnose mutations in all the known causative genes, when used as targeted approach. Third to obtain a knowledge of the impact of mutations on mRNA expression and splicing in diseased muscle. We used NGS to identify new genes by whole exome sequencing. We sequenced the whole exome of four family members with LGMD1F separated by up to eleven meioses and identified a single shared novel heterozygous frame-shift variant. This causes a nonstop change in the Transportin 3 (TNPO3) gene that encodes a member of the importin-β super-family. To reach the second task, we first recruited 160 familial cases of nonspecific limb-girdle muscular dystrophies with apparent autosomal inheritance. All DNA samples were first enriched for 486,480 bp, covering 2447 exons of 98 genes by using the Haloplex technology with the use of barcodes. We the performed pooled NGS of all samples and identified a number of mutations, then verified by Sanger sequencing. Cases were also studied by the Agilent MotorChip CGH array version 3.0 to identify deletions or duplications. Finally, in selected cases, we performed the RNA-Seq starting from a muscle biopsy sample. We converted mRNA to cDNA and purified it by a customized SureSelect Target Enrichment System, focused on the same 98 mRNAs. The probes had a 4× coverage with a total target of 1.41 Mb of sequences/sample. These cDNAs were sequenced using barcodes trying to obtain an average sequencing coverage of at least 100×. Our results confirm that there is a very high genetic heterogeneity in muscular dystrophies and that NGS-based DNA and RNA testing are ready for diagnostic use.
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.
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.
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