Chiara Scotton - Academia.edu (original) (raw)

Papers by Chiara Scotton

Research paper thumbnail of Bovolenta et al. Human Mutation Mar;33(3):572-81. doi: 10.1002/humu.22017

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the... more Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan R low-density array technology and is able to define the fullexon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies. Hum Mutat 33:572-581, 2012. C 2011 Wiley Periodicals, Inc. KEY WORDS: diagnostic biomarker; DMD mutations; fluidic cards; RNA analysis C 2011 WILEY PERIODICALS, INC.

Research paper thumbnail of Validation of genetic modifiers for Duchenne muscular dystrophy: a multicentre study assessing SPP1 and LTBP4 variants

Journal of Neurology, Neurosurgery & Psychiatry, 2014

Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recentl... more Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials. Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates. We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss. This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.

Research paper thumbnail of Muscle Proteomics Reveals Novel Insights into the Pathophysiological Mechanisms of Collagen VI Myopathies

Journal of Proteome Research, 2014

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with s... more Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

Research paper thumbnail of Biomarkers in Rare Diseases

Public Health Genomics, 2013

Nowadays 7,000 rare diseases (RDs) have been identified with a prevalence less than 5/10,000. Des... more Nowadays 7,000 rare diseases (RDs) have been identified with a prevalence less than 5/10,000. Despite of the enormous effort the European Union (EU) has already invested in this field, still 4,000 RDs remain orphan of genetic diagnosis and causative gene identification. The genetic definition of RDs represents a prerequisite for being diagnosed, for having a robust prevention, for entering in a specific standard of care, and ultimately, for being included in clinical trials, often via personalized medicine. It is well established that biomarkers can offer a way to speed up research by understanding the pathophysiological mechanisms of diseases. In particular, biomarkers will offer an invaluable tool for monitoring disease progression, prognosis and response to drug treatment. In this review, we summarize the different types of biomarkers and their importance as well as their translational applications in RDs. We have reviewed the current knowledge on biomarkers state-of-the-art via literature data, specific websites and EU sources regarding past, pending and current projects. Here we provide a comprehensive scenario of biomarkers research, its applications in clinical practice, with special emphasis on translational research applicable to diagnostic and clinical trials. The experience of the EU project BIO-NMD is also mentioned. Biomarkers represent key features in both diagnostics and research on rare diseases and will encounter wide exploitation in translational and personalized medicine.

Research paper thumbnail of The DMD Locus Harbours Multiple Long Non-Coding RNAs Which Orchestrate and Control Transcription of Muscle Dystrophin mRNA Isoforms

PLoS ONE, 2012

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to rough... more The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

Research paper thumbnail of P4.01 ncRNAs originating from the dystrophin gene as biomarker for assessing antisense therapy

Neuromuscular Disorders, 2010

Research paper thumbnail of G.P.13.02 Non-coding RNAs within the DMD gene

Neuromuscular Disorders, 2009

Research paper thumbnail of D.P.12 Whole exome sequencing and RNAseq in a Duchenne-like female with no dystrophin mutations: Search for dystrophin gene modifiers

Neuromuscular Disorders, 2012

ABSTRACT We identified a Duchenne-like female clinically evaluated as a severely affected symptom... more ABSTRACT We identified a Duchenne-like female clinically evaluated as a severely affected symptomatic DMD carrier. Carrier status was confirmed by evidence of mosaic pattern of dystrophin expression on muscle biopsy; nevertheless, extensive analysis by MLPA, sequencing, CGH-array and RNA profiling failed to identify any mutation within DMD gene. We performed whole exome sequencing by means of an Illumina GAIIe sequencer in order to identify genetic modifiers that could contribute to the symptomatic phenotype in this female. A total of 23,776 SNPs passed filtering for quality parameters. We further selected variations present in a list of 883 candidate genes belonging to the dystrophin expression regulation network and, applying a dominant model of inheritance for the modifying genes, we considered only heterozygous variations including SNPs already present in the dbSNP database. This allowed us to retain 902 SNPs. We performed RNAseq analysis on her muscle biopsy to restrict the panel of SNPs and integrated exome and transcriptome results to provide critical/confirmatory information about the SNPs identified. Eight out of the 20 SNPs selected in UTR regions were validated by RNAseq data. For non-synonymous variations, 205 out of the 219 SNPs were mapped in genes expressed in muscle and 61 were validated by RNAseq. In total we validated 69 exploratory SNPs. These SNPs are located within genes involved in the sarcolemma and extracellular matrix, cell signalling and, interestingly, chromatin modification. The 69 SNPs are in course of validation in two groups of females: symptomatic (17) and non-symptomatic (18) DMD carriers. Results will be statistically analysed using both multidimensional scaling and discriminate analysis in order to identify those variations able to discriminate between the two phenotypic groups.

Research paper thumbnail of G.P.15 Whole transcriptome expression profiling in COL6a1 null mice shows deregulation of circadian clock genes as exploratory COL6 myopathies biomarkers

Neuromuscular Disorders, 2012

ABSTRACT Collagen VI is an extracellular matrix protein that forms a microfilamentous network in ... more ABSTRACT Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. In humans, mutations in the collagen VI alpha1, 2 and 3 genes cause congenital muscular dystrophies as Bethlem, Ullrich myopathies and autosomal dominant Myosclerosis. Both mitochondrial pore abnormalities and defect in the autophagic pathways have been identified both in patients and in Col6a1−/− knock out mouse model. In order to bridging these findings to the transcriptome signature in mice, we have performed whole expression profile in wild type, Col6a1−/− mice and Col6a1−/− cyclosporine A treated mice. The mice muscles analysed were diaphragm, gastrocnemius and tibialis. We compared three groups: (i) KO mice vs WT mice; (ii) KO mice cyclosporine treated vs KO mice untreated; (iii) KO mice olive oil treated vs KO mice untreated. A total of 3820 genes were found deregulated in WT compared to KO mice but with high variability among muscle type. A pool of 47 genes differentiate KO mice transcriptome following cyclosporine A treatment. The deregulated transcripts belong to the two main circuits of muscle transcription and circadian rhythm. Array data validation by customized fluidic cards allowed us to validate 47 transcripts as truly deregulated in different muscles. Among these 47 validated gene, interestingly, six genes,belonging to the circadian clock mechanisms, are constantly down-regulated in KO mice. This behavior is constant in all muscles analysed, though more prominent in tibialis. The pathway scan analysis of the transcriptomic profiling allowed us to build up an interactome map revealing connections between clock genes and apoptosis and autophagy as well as with muscle regeneration and muscle signaling, opening a previously undisclosed role of circadian rhythm in muscle diseases. These finding make clock genes down-regulation appealing exploratory biomarkers of collagen VI pathology.

Research paper thumbnail of P5.75 High throughput diagnosis of neuromuscular diseases: the NMD-CHIP EU project

Neuromuscular Disorders, 2011

Additionally, the relationship between the head/neck movement and the sonographic muscle appearan... more Additionally, the relationship between the head/neck movement and the sonographic muscle appearance at the resulting head/neck position was assessed. Muscle volume and pattern changes were documented in the splenius muscle of the aged horses. There was a marked difference in longitudinal appearance of the contracted (in head/neck extension) and relaxed (in head/neck flexion) splenius muscles in all horses. While larger muscle volumes were associated with larger EMG activities, sonographic determination of fibrous tissue within the muscle was represented by lower EMG maximum activity during extension only. Increase of connective tissues indicates the aging effect and reduces muscle activity patterns.

Research paper thumbnail of A patient with limb girdle muscular dystrophy carries a TRIM32 deletion, detected by a novel CGH array, in compound heterozygosis with a nonsense mutation

Neuromuscular Disorders, 2013

Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically hi... more Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically highly variable, caused by mutations in the TRIM32 gene. Here we describe a 35-years-old who experienced progressive muscle weakness. The muscle biopsy revealed an unspecific pattern of atrophic and hypertrophic fibers; the immunohistochemistry for several proteins was normal. Comparative genomic hybridization (CGH) analysis showed a heterozygous deletion of the entire TRIM32 gene. On the other allele we identified the R316X nonsense mutation. The genetic diagnosis of LGMD2H in this case was reached by using a novel high throughput diagnostic tool.

Research paper thumbnail of D.P.9 Whole exome sequencing filtered by novel candidate genes as tool for gene discovery in a recessive family with Parkinson and ataxia

Neuromuscular Disorders, 2012

ABSTRACT We studied a family of four with a form of juvenile Parkinson, cognitive impairment, ata... more ABSTRACT We studied a family of four with a form of juvenile Parkinson, cognitive impairment, ataxia, and obesity, with variable clinical severity. Many genes associated to these diseases were excluded with conventional sequencing: SCA1, SCA2, SCA3, SCA6 and DRPLA for the spinocerebellar ataxias, GM1 and GM2 for the adrenoleukodystrophy, GCH1 for dopa-responsive dystonia and the linkage analysis excluded the Parkinson genes PARK2, PARK3 and PARK7. For both affected patients we hypothesized a neurological disease with multisystem involvement that was genetically determined. We performed the whole exome sequencing analysis by Illumina GAIIe platform on all family members except the affected sister. We identified a mean of 30,000 SNPs or DIPs passing a quality filters (coverage >10, ambigously mapped reads per variant <5, Phred-scaled consensus quality >50, variant confidence/consensus quality >1.5). We elaborated a list of 143 genes that are frankly or putatively associated to Parkinson, obesity or spinocerebellar ataxia and adopted this list as a filter applied to the WES analysis, considering SNPs only in homozygosity or in compound heterozygosity (dbSNPs not excluded). This analysis allowed us to detect 82 variations within 41 genes in homozygosity and 134 variations in compound heterozygosity within 39 genes. We filtered these identified variations in family’s members or in other samples present in the same run; this investigation greatly reduced the numbers of variations, being now 12 homozygous and 54 the compound heterozygous. The variations identified are in course of technical validation as possible mutations in novel genes causing the peculiar phenotype in this family.

Research paper thumbnail of D.P.7 Whole exome sequencing as genetic diagnostic tool in myofibrillar myopathies

Neuromuscular Disorders, 2012

ABSTRACT Myofibrillar myopathies (MFM) represents a group of neuromuscular disorders either famil... more ABSTRACT Myofibrillar myopathies (MFM) represents a group of neuromuscular disorders either familial or sporadic with clinical and genetic heterogeneity. To date, mutations in six genes are known to cause MFM, but a large number of these diseases are caused by so far unidentified gene defects. Within the NMD-chip project, we had previously studied by CGH arrays 21 sporadic patients with a clinical and immunohistochemical diagnosis of myofibrillar myopathy negative at conventional sequencing for small mutations in desmin, αB-crystallin, myotilin, Z-band alternatively spliced PDZ motif containing protein (ZASP) Bcl2-associated athanogene 3 (BAG3). The CGH analysis did not reveal any rearrangement in myofibrillar myopathy known genes. We have therefore run whole exome sequencing (WES) by Illumina GAIIe platform in seven out our 21 negative patients. This approach has allowed us to identified a mean of 60,000 SNPs or DIPs passing standard quality filters. We applied a further bioinformatics filter consisting in 880 candidate genes selected using the MedScan interactome pathway developed by Ariadne Genomics. We looked at SNPs or DIPs occurring in heterozygosity (according with a dominant model of inheritance) within all these genes. All SNPs already reported in the dbSNP database were excluded. This analysis led to the identification of a mean of 10 variations/patient. We detected (in heterozygosis) a nonsense and a missense variations in Filamin C, and 3 missense variations in three unrelated patients, within the Titin gene. Mutations in this huge gene, are known to cause a recessive form of LGMD (LGMD2J), but have not been previously related to MFMs. We have also identified stop mutations in other three novel possibly causative genes. All variations are in course of validation. We demonstrate here that WES, following facilitation of both molecular and bioinformatics procedures, may represent a “second step” powerful diagnostic tool in NMDs with high genetic heterogeneity.

Research paper thumbnail of Exome sequencing in a family with intellectual disability, early onset spasticity, and cerebellar atrophy detects a novel mutation in EXOSC3

neurogenetics, 2013

Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, m... more Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, mild intellectual disability, distal amyotrophy, and cerebellar atrophy transmitted as an autosomal recessive trait identified the following two missense mutations in the EXOSC3 gene: a novel p.V80F mutation and a known p.D132A change previously associated with mild variants of pontocerebellar hypoplasia type 1. This study confirms the involvement of RNA processing proteins in disorders with motor neuron and cerebellar degeneration overlapping with spinocerebellar ataxia 36 and rare forms of hereditary spastic paraplegia with cerebellar features.

Research paper thumbnail of SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers

Molecular Genetics and Metabolism, 2013

Brody disease Sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase 1 (SERCA1) Brody syndrome ATP2A1 ... more Brody disease Sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase 1 (SERCA1) Brody syndrome ATP2A1 gene Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

Research paper thumbnail of Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array

Human Mutation, 2012

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the... more Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan R low-density array technology and is able to define the fullexon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies. Hum Mutat 33:572-581, 2012. C 2011 Wiley Periodicals, Inc. KEY WORDS: diagnostic biomarker; DMD mutations; fluidic cards; RNA analysis C 2011 WILEY PERIODICALS, INC.

Research paper thumbnail of Biomarkers in rare neuromuscular diseases

Experimental Cell Research, 2013

Neuromuscular diseases (NMDs) comprise a range of rare disorders that includes both hereditary pe... more Neuromuscular diseases (NMDs) comprise a range of rare disorders that includes both hereditary peripheral neuropathies and myopathies. The heterogeneity and rarity of neuromuscular disorders is a challenge for research seeking to develop effective diagnosis and treatment strategies. In particular, clinical trials of new therapies are made more difficult by the lack of reliable and monitorable clinical outcome measures. Biomarkers could be a way to speed up research in this field, shedding light on the pathophysiological mechanisms behind such diseases and providing invaluable tools for monitoring their progression, prognosis and response to drug treatment. Furthermore, biomarkers could represent a surrogate endpoint for clinical trials, enabling better stratification of patient cohorts through more accurate diagnosis and prognosis prediction.

Research paper thumbnail of Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

BMC Medical Genetics, 2012

Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly a... more Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial.

Research paper thumbnail of Paternal germline mosaicism in collagen VI related myopathies

European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society, Jan 30, 2015

Collagen VI-related disorders are a group of muscular diseases characterized by muscle wasting an... more Collagen VI-related disorders are a group of muscular diseases characterized by muscle wasting and weakness, joint contractures, distal laxity, serious respiratory dysfunction and cutaneous alterations, due to mutations in the COL6A1, COL6A2 and COL6A3 genes, encoding for collagen VI, a critical component of the extracellular matrix. The severe Ullrich congenital muscular dystrophy (UCMD) can be due to autosomal recessive mutations in one of the three genes with a related 25% recurrence risk. In the majority of UCMD cases nevertheless, the underlying mutation is thought to arise de novo and the recurrence risk is considered as low. Here we report a family with recurrence of UCMD in two half-sibs. In both, the molecular analysis revealed heterozygosity for the c.896G > A missense mutation in COL6A1 exon 10 (Gly299Glu) and for the COL6A1 c.1823-8G > A variation within COL6A1 intron 29. The intronic variation was inherited from the father and RNA analysis in skin fibroblasts allo...

Research paper thumbnail of Bovolenta et al. Human Mutation Mar;33(3):572-81. doi: 10.1002/humu.22017

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the... more Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan R low-density array technology and is able to define the fullexon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies. Hum Mutat 33:572-581, 2012. C 2011 Wiley Periodicals, Inc. KEY WORDS: diagnostic biomarker; DMD mutations; fluidic cards; RNA analysis C 2011 WILEY PERIODICALS, INC.

Research paper thumbnail of Validation of genetic modifiers for Duchenne muscular dystrophy: a multicentre study assessing SPP1 and LTBP4 variants

Journal of Neurology, Neurosurgery & Psychiatry, 2014

Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recentl... more Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials. Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates. We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss. This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.

Research paper thumbnail of Muscle Proteomics Reveals Novel Insights into the Pathophysiological Mechanisms of Collagen VI Myopathies

Journal of Proteome Research, 2014

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with s... more Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

Research paper thumbnail of Biomarkers in Rare Diseases

Public Health Genomics, 2013

Nowadays 7,000 rare diseases (RDs) have been identified with a prevalence less than 5/10,000. Des... more Nowadays 7,000 rare diseases (RDs) have been identified with a prevalence less than 5/10,000. Despite of the enormous effort the European Union (EU) has already invested in this field, still 4,000 RDs remain orphan of genetic diagnosis and causative gene identification. The genetic definition of RDs represents a prerequisite for being diagnosed, for having a robust prevention, for entering in a specific standard of care, and ultimately, for being included in clinical trials, often via personalized medicine. It is well established that biomarkers can offer a way to speed up research by understanding the pathophysiological mechanisms of diseases. In particular, biomarkers will offer an invaluable tool for monitoring disease progression, prognosis and response to drug treatment. In this review, we summarize the different types of biomarkers and their importance as well as their translational applications in RDs. We have reviewed the current knowledge on biomarkers state-of-the-art via literature data, specific websites and EU sources regarding past, pending and current projects. Here we provide a comprehensive scenario of biomarkers research, its applications in clinical practice, with special emphasis on translational research applicable to diagnostic and clinical trials. The experience of the EU project BIO-NMD is also mentioned. Biomarkers represent key features in both diagnostics and research on rare diseases and will encounter wide exploitation in translational and personalized medicine.

Research paper thumbnail of The DMD Locus Harbours Multiple Long Non-Coding RNAs Which Orchestrate and Control Transcription of Muscle Dystrophin mRNA Isoforms

PLoS ONE, 2012

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to rough... more The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

Research paper thumbnail of P4.01 ncRNAs originating from the dystrophin gene as biomarker for assessing antisense therapy

Neuromuscular Disorders, 2010

Research paper thumbnail of G.P.13.02 Non-coding RNAs within the DMD gene

Neuromuscular Disorders, 2009

Research paper thumbnail of D.P.12 Whole exome sequencing and RNAseq in a Duchenne-like female with no dystrophin mutations: Search for dystrophin gene modifiers

Neuromuscular Disorders, 2012

ABSTRACT We identified a Duchenne-like female clinically evaluated as a severely affected symptom... more ABSTRACT We identified a Duchenne-like female clinically evaluated as a severely affected symptomatic DMD carrier. Carrier status was confirmed by evidence of mosaic pattern of dystrophin expression on muscle biopsy; nevertheless, extensive analysis by MLPA, sequencing, CGH-array and RNA profiling failed to identify any mutation within DMD gene. We performed whole exome sequencing by means of an Illumina GAIIe sequencer in order to identify genetic modifiers that could contribute to the symptomatic phenotype in this female. A total of 23,776 SNPs passed filtering for quality parameters. We further selected variations present in a list of 883 candidate genes belonging to the dystrophin expression regulation network and, applying a dominant model of inheritance for the modifying genes, we considered only heterozygous variations including SNPs already present in the dbSNP database. This allowed us to retain 902 SNPs. We performed RNAseq analysis on her muscle biopsy to restrict the panel of SNPs and integrated exome and transcriptome results to provide critical/confirmatory information about the SNPs identified. Eight out of the 20 SNPs selected in UTR regions were validated by RNAseq data. For non-synonymous variations, 205 out of the 219 SNPs were mapped in genes expressed in muscle and 61 were validated by RNAseq. In total we validated 69 exploratory SNPs. These SNPs are located within genes involved in the sarcolemma and extracellular matrix, cell signalling and, interestingly, chromatin modification. The 69 SNPs are in course of validation in two groups of females: symptomatic (17) and non-symptomatic (18) DMD carriers. Results will be statistically analysed using both multidimensional scaling and discriminate analysis in order to identify those variations able to discriminate between the two phenotypic groups.

Research paper thumbnail of G.P.15 Whole transcriptome expression profiling in COL6a1 null mice shows deregulation of circadian clock genes as exploratory COL6 myopathies biomarkers

Neuromuscular Disorders, 2012

ABSTRACT Collagen VI is an extracellular matrix protein that forms a microfilamentous network in ... more ABSTRACT Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. In humans, mutations in the collagen VI alpha1, 2 and 3 genes cause congenital muscular dystrophies as Bethlem, Ullrich myopathies and autosomal dominant Myosclerosis. Both mitochondrial pore abnormalities and defect in the autophagic pathways have been identified both in patients and in Col6a1−/− knock out mouse model. In order to bridging these findings to the transcriptome signature in mice, we have performed whole expression profile in wild type, Col6a1−/− mice and Col6a1−/− cyclosporine A treated mice. The mice muscles analysed were diaphragm, gastrocnemius and tibialis. We compared three groups: (i) KO mice vs WT mice; (ii) KO mice cyclosporine treated vs KO mice untreated; (iii) KO mice olive oil treated vs KO mice untreated. A total of 3820 genes were found deregulated in WT compared to KO mice but with high variability among muscle type. A pool of 47 genes differentiate KO mice transcriptome following cyclosporine A treatment. The deregulated transcripts belong to the two main circuits of muscle transcription and circadian rhythm. Array data validation by customized fluidic cards allowed us to validate 47 transcripts as truly deregulated in different muscles. Among these 47 validated gene, interestingly, six genes,belonging to the circadian clock mechanisms, are constantly down-regulated in KO mice. This behavior is constant in all muscles analysed, though more prominent in tibialis. The pathway scan analysis of the transcriptomic profiling allowed us to build up an interactome map revealing connections between clock genes and apoptosis and autophagy as well as with muscle regeneration and muscle signaling, opening a previously undisclosed role of circadian rhythm in muscle diseases. These finding make clock genes down-regulation appealing exploratory biomarkers of collagen VI pathology.

Research paper thumbnail of P5.75 High throughput diagnosis of neuromuscular diseases: the NMD-CHIP EU project

Neuromuscular Disorders, 2011

Additionally, the relationship between the head/neck movement and the sonographic muscle appearan... more Additionally, the relationship between the head/neck movement and the sonographic muscle appearance at the resulting head/neck position was assessed. Muscle volume and pattern changes were documented in the splenius muscle of the aged horses. There was a marked difference in longitudinal appearance of the contracted (in head/neck extension) and relaxed (in head/neck flexion) splenius muscles in all horses. While larger muscle volumes were associated with larger EMG activities, sonographic determination of fibrous tissue within the muscle was represented by lower EMG maximum activity during extension only. Increase of connective tissues indicates the aging effect and reduces muscle activity patterns.

Research paper thumbnail of A patient with limb girdle muscular dystrophy carries a TRIM32 deletion, detected by a novel CGH array, in compound heterozygosis with a nonsense mutation

Neuromuscular Disorders, 2013

Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically hi... more Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically highly variable, caused by mutations in the TRIM32 gene. Here we describe a 35-years-old who experienced progressive muscle weakness. The muscle biopsy revealed an unspecific pattern of atrophic and hypertrophic fibers; the immunohistochemistry for several proteins was normal. Comparative genomic hybridization (CGH) analysis showed a heterozygous deletion of the entire TRIM32 gene. On the other allele we identified the R316X nonsense mutation. The genetic diagnosis of LGMD2H in this case was reached by using a novel high throughput diagnostic tool.

Research paper thumbnail of D.P.9 Whole exome sequencing filtered by novel candidate genes as tool for gene discovery in a recessive family with Parkinson and ataxia

Neuromuscular Disorders, 2012

ABSTRACT We studied a family of four with a form of juvenile Parkinson, cognitive impairment, ata... more ABSTRACT We studied a family of four with a form of juvenile Parkinson, cognitive impairment, ataxia, and obesity, with variable clinical severity. Many genes associated to these diseases were excluded with conventional sequencing: SCA1, SCA2, SCA3, SCA6 and DRPLA for the spinocerebellar ataxias, GM1 and GM2 for the adrenoleukodystrophy, GCH1 for dopa-responsive dystonia and the linkage analysis excluded the Parkinson genes PARK2, PARK3 and PARK7. For both affected patients we hypothesized a neurological disease with multisystem involvement that was genetically determined. We performed the whole exome sequencing analysis by Illumina GAIIe platform on all family members except the affected sister. We identified a mean of 30,000 SNPs or DIPs passing a quality filters (coverage >10, ambigously mapped reads per variant <5, Phred-scaled consensus quality >50, variant confidence/consensus quality >1.5). We elaborated a list of 143 genes that are frankly or putatively associated to Parkinson, obesity or spinocerebellar ataxia and adopted this list as a filter applied to the WES analysis, considering SNPs only in homozygosity or in compound heterozygosity (dbSNPs not excluded). This analysis allowed us to detect 82 variations within 41 genes in homozygosity and 134 variations in compound heterozygosity within 39 genes. We filtered these identified variations in family’s members or in other samples present in the same run; this investigation greatly reduced the numbers of variations, being now 12 homozygous and 54 the compound heterozygous. The variations identified are in course of technical validation as possible mutations in novel genes causing the peculiar phenotype in this family.

Research paper thumbnail of D.P.7 Whole exome sequencing as genetic diagnostic tool in myofibrillar myopathies

Neuromuscular Disorders, 2012

ABSTRACT Myofibrillar myopathies (MFM) represents a group of neuromuscular disorders either famil... more ABSTRACT Myofibrillar myopathies (MFM) represents a group of neuromuscular disorders either familial or sporadic with clinical and genetic heterogeneity. To date, mutations in six genes are known to cause MFM, but a large number of these diseases are caused by so far unidentified gene defects. Within the NMD-chip project, we had previously studied by CGH arrays 21 sporadic patients with a clinical and immunohistochemical diagnosis of myofibrillar myopathy negative at conventional sequencing for small mutations in desmin, αB-crystallin, myotilin, Z-band alternatively spliced PDZ motif containing protein (ZASP) Bcl2-associated athanogene 3 (BAG3). The CGH analysis did not reveal any rearrangement in myofibrillar myopathy known genes. We have therefore run whole exome sequencing (WES) by Illumina GAIIe platform in seven out our 21 negative patients. This approach has allowed us to identified a mean of 60,000 SNPs or DIPs passing standard quality filters. We applied a further bioinformatics filter consisting in 880 candidate genes selected using the MedScan interactome pathway developed by Ariadne Genomics. We looked at SNPs or DIPs occurring in heterozygosity (according with a dominant model of inheritance) within all these genes. All SNPs already reported in the dbSNP database were excluded. This analysis led to the identification of a mean of 10 variations/patient. We detected (in heterozygosis) a nonsense and a missense variations in Filamin C, and 3 missense variations in three unrelated patients, within the Titin gene. Mutations in this huge gene, are known to cause a recessive form of LGMD (LGMD2J), but have not been previously related to MFMs. We have also identified stop mutations in other three novel possibly causative genes. All variations are in course of validation. We demonstrate here that WES, following facilitation of both molecular and bioinformatics procedures, may represent a “second step” powerful diagnostic tool in NMDs with high genetic heterogeneity.

Research paper thumbnail of Exome sequencing in a family with intellectual disability, early onset spasticity, and cerebellar atrophy detects a novel mutation in EXOSC3

neurogenetics, 2013

Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, m... more Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, mild intellectual disability, distal amyotrophy, and cerebellar atrophy transmitted as an autosomal recessive trait identified the following two missense mutations in the EXOSC3 gene: a novel p.V80F mutation and a known p.D132A change previously associated with mild variants of pontocerebellar hypoplasia type 1. This study confirms the involvement of RNA processing proteins in disorders with motor neuron and cerebellar degeneration overlapping with spinocerebellar ataxia 36 and rare forms of hereditary spastic paraplegia with cerebellar features.

Research paper thumbnail of SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers

Molecular Genetics and Metabolism, 2013

Brody disease Sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase 1 (SERCA1) Brody syndrome ATP2A1 ... more Brody disease Sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase 1 (SERCA1) Brody syndrome ATP2A1 gene Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

Research paper thumbnail of Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array

Human Mutation, 2012

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the... more Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan R low-density array technology and is able to define the fullexon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies. Hum Mutat 33:572-581, 2012. C 2011 Wiley Periodicals, Inc. KEY WORDS: diagnostic biomarker; DMD mutations; fluidic cards; RNA analysis C 2011 WILEY PERIODICALS, INC.

Research paper thumbnail of Biomarkers in rare neuromuscular diseases

Experimental Cell Research, 2013

Neuromuscular diseases (NMDs) comprise a range of rare disorders that includes both hereditary pe... more Neuromuscular diseases (NMDs) comprise a range of rare disorders that includes both hereditary peripheral neuropathies and myopathies. The heterogeneity and rarity of neuromuscular disorders is a challenge for research seeking to develop effective diagnosis and treatment strategies. In particular, clinical trials of new therapies are made more difficult by the lack of reliable and monitorable clinical outcome measures. Biomarkers could be a way to speed up research in this field, shedding light on the pathophysiological mechanisms behind such diseases and providing invaluable tools for monitoring their progression, prognosis and response to drug treatment. Furthermore, biomarkers could represent a surrogate endpoint for clinical trials, enabling better stratification of patient cohorts through more accurate diagnosis and prognosis prediction.

Research paper thumbnail of Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

BMC Medical Genetics, 2012

Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly a... more Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial.

Research paper thumbnail of Paternal germline mosaicism in collagen VI related myopathies

European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society, Jan 30, 2015

Collagen VI-related disorders are a group of muscular diseases characterized by muscle wasting an... more Collagen VI-related disorders are a group of muscular diseases characterized by muscle wasting and weakness, joint contractures, distal laxity, serious respiratory dysfunction and cutaneous alterations, due to mutations in the COL6A1, COL6A2 and COL6A3 genes, encoding for collagen VI, a critical component of the extracellular matrix. The severe Ullrich congenital muscular dystrophy (UCMD) can be due to autosomal recessive mutations in one of the three genes with a related 25% recurrence risk. In the majority of UCMD cases nevertheless, the underlying mutation is thought to arise de novo and the recurrence risk is considered as low. Here we report a family with recurrence of UCMD in two half-sibs. In both, the molecular analysis revealed heterozygosity for the c.896G > A missense mutation in COL6A1 exon 10 (Gly299Glu) and for the COL6A1 c.1823-8G > A variation within COL6A1 intron 29. The intronic variation was inherited from the father and RNA analysis in skin fibroblasts allo...