Transcription-terminating mutation in telethonin causing autosomal recessive muscular dystrophy type 2G in a European patient

A 27-year-old woman of Moldavian origin presented at the age of 15 with progressive proximal limb weakness and painful cramps in her calf muscles. Clinical examination revealed prominent Muscle weakness in proximal muscles of the lower extremities and distal anterior compartment of legs, and mild weakness in shoulder girdle muscles. In addition, she had marked calf hypertrophy, Muscle atrophy involving the anterior and posterior compartments of the thighs, and the distal anterior compartment of legs, as well as mild scapular winging and hyperlordosis. A muscle biopsy taken from the biceps brachii showed mild dystrophic changes, absent vacuoles, and abundant lobulated fibers. Immunofluorescence and Western blot assays demonstrated complete telethonin deficiency. Molecular analysis revealed a homozygous Trp25X mutation in the telethonin (TCAP) gene resulting in termination of transcription at an early point. Four families from Brazil with telethonin deficiency have previously been reported and classified as LMD2G, but the actual frequency of this disease is unknown. With this current identification of a case outside the Brazilian Population, telethonin mutation-associated LGMD should be considered worldwide. (C) 2008 Elsevier B.V. All rights reserved

Estudio de la patología molecular en disferlinopatías

Las distrofias musculares de cinturas (LGMD) son un grupo heterogéneo de enfermedades neuromusculares hereditarias, definidas por una debilidad muscular progresiva y atrofia. Se caracterizan por un patrón distrófico que afecta a la musculatura de las cinturas. Las mutaciones en el gen DYSF son las responsables de un amplio espectro de fenotipos caracterizados todos ellos por ausencia de disferlina en el músculo esquelético y un modo de herencia autosómico recesivo. Los tres fenotipos principales por su frecuencia de aparición son: Miopatía de Miyoshi (MM), Distrofia muscular de cinturas de tipo 2B (LGMD2B) y distrofia muscular tibial anterior (DMAT). Los objetivos planteados fueron: • Estudiar el gen DYSF en pacientes de población española, ya que este gen no se había estudiado nunca antes en esta población. • Diseñar una estrategia técnica rápida y eficaz para el estudio de DYSF o Optimizar el proceso diagnóstico mediante el estudio de RNA vs DNA o Analizar grandes reordenamientos del gen (duplicaciones o deleciones). • Caracterizar el espectro mutacional de DYSF en población española • Establecer una relación genotipo-fenotipo • Analizar posibles genes modificadores Los resultados derivados de este trabajo han sido publicados en las siguientes revistas científicas: -Abnormal expresión of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele. M.Meznaric, L. González-Quereda, E. Gallardo, N. de Luna, P. Gallano, M. Fanin, C. Angelini, B. Peterlin and J. Zidar. European Journal of Neurology 2010. Article first published online: 18 OCT 2010 DOI: 10.1111/j.1468-1331.2010.03240.x -A new phenotype of dysferlinopathy with congenital onset. Paradas C, González-Quereda L, de Luna N, Gallardo E, García-Consuegra I, Gómez H, Cabello A, Illa I, Gallano P. *Authors Paradas C and González-Quereda L contributed equally to this work. Neuromuscul Disord. 2009 Jan;19(1):21-5. Epub 2008 Dec 11. doi:10.1016/j.nmd.2008.09.015 Las conclusiones del trabajo son: • Es posible realizar el estudio mutacional en el RNA total extraído de monocitos circulantes en sangre periférica, evitándose por consiguiente el método invasivo de biopsia muscular • Existe una gran heterogeneidad mutacional en el gen DYSF en pacientes de población española con distrofia muscular de cinturas tipo 2B y miopatía distal tipo Miyoshi. • No es posible establecer una relación genotipo-fenotipo en pacientes con disferlinopatía. • La mutación DYSF c.3191_3196dup es la más prevalente en nuestra población. • La heterogeneidad mutacional en los pacientes de nuestra población, en lo referente a la localización dentro del gen, es elevada y no se han identificado “hotspots” del mismo modo que en otras poblaciones caucásicas. • El estudio de DYSF en mRNA vs DNA genómico permite: o Establecer un diagnóstico molecular de forma más rápida y eficaz. o Detectar un mayor número de mutaciones. o Detectar todas las mutaciones que alteran el proceso de splicing. • No se han identificado mutaciones en el gen MG53, que codifica para mitsugumina-53, en nuestra serie de pacientes con disferlinopatía por lo que hasta la actualidad no puede ser considerado como gen modificador. • Se describe un nuevo fenotipo de disferlinopatía con inicio congénito, ampliándose de este modo el espectro clínico de las disferlinopatías y, añadiendo nuevos datos a la historia natural de la enfermedad.Limb Girdle Muscular Dystrophies (LGMD) are a heterogeneous group of inherited neuromuscular diseases, defined by progressive muscle weakness and atrophy. They are characterized by a progressive dystrophic pattern involving limb-girdle muscles. Mutations in DYSF gene are responsible for a wide range of phenotypes characterized by the absence of dysferlin in skeletal muscle and an autosomal recessive mode of inheritance. So far, three main phenotypes have been reported: Miyoshi Myopathy (MM), Limb Girdle Muscular Dystrophy type 2B (LGMD 2B), and Distal Myopathy with Anterior Tibial onset (DMAT). The objectives of this work were: • To study the DYSF gene in patients from Spanish population, not previously analysed in this population. o To design a technical strategy to analyse the DYSF gene o To optimize the diagnostic process using RNA vs DNA • To analyse gene rearrangements (deletions or duplications). • To characterize the mutational spectrum in our population • To establish a genotype-phenotype relationship • To analyse possible modifiers genes Results from this work had been published: -Abnormal expresión of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele. M.Meznaric, L. González-Quereda, E. Gallardo, N. de Luna, P. Gallano, M. Fanin, C. Angelini, B. Peterlin and J. Zidar. European Journal of Neurology 2010. Article first published online: 18 OCT 2010 DOI: 10.1111/j.1468-1331.2010.03240.x -A new phenotype of dysferlinopathy with congenital onset. Paradas C, González-Quereda L, de Luna N, Gallardo E, García-Consuegra I, Gómez H, Cabello A, Illa I, Gallano P. *Authors Paradas C and González-Quereda L contributed equally to this work. Neuromuscul Disord. 2009 Jan;19(1):21-5. Epub 2008 Dec 11. doi:10.1016/j.nmd.2008.09.015 This thesis drew the conclusions: • It is possible to perform the mutational analysis in RNA isolated from total peripheral blood monocytes, avoiding the invasive method of muscle biopsy. • DYSF gene shows high mutational heterogeneity in patients from Spanish population suffering from Limb Girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. • It is not possible to establish a genotype-phenotype correlation in dysferlinopathy patients. • DYSF c.3191_3196dup is the most prevalent mutation in Spanish population. • Patients from our population show high mutational heterogeneity and it is not possible to identify hotspots as mutations are expanded over the whole sequence of the gene. • The DYSF gene study in mRNA vs genomic DNA allows: o To establish a molecular diagnostic in a quick and efficient way. o To detect a high number of mutations o To detect all mutations changing the splicing process. • Mutations in MG53 have not been identified in our serie of patients, so that, MG53 can not be considered as a modifier gene to date. • It has been described a new phenotype of dysferlinopathy with congenital onset. This new phenotype widens the clinical spectrum of dysferlinopathies and add new data to the natural story of the disease

Estudio de la patología molecular en disferlinopatías

Las distrofias musculares de cinturas (LGMD) son un grupo heterogéneo de enfermedades neuromusculares hereditarias, definidas por una debilidad muscular progresiva y atrofia. Se caracterizan por un patrón distrófico que afecta a la musculatura de las cinturas. Las mutaciones en el gen DYSF son las responsables de un amplio espectro de fenotipos caracterizados todos ellos por ausencia de disferlina en el músculo esquelético y un modo de herencia autosómico recesivo. Los tres fenotipos principales por su frecuencia de aparición son: Miopatía de Miyoshi (MM), Distrofia muscular de cinturas de tipo 2B (LGMD2B) y distrofia muscular tibial anterior (DMAT). Los objetivos planteados fueron: • Estudiar el gen DYSF en pacientes de población española, ya que este gen no se había estudiado nunca antes en esta población. • Diseñar una estrategia técnica rápida y eficaz para el estudio de DYSF o Optimizar el proceso diagnóstico mediante el estudio de RNA vs DNA o Analizar grandes reordenamientos del gen (duplicaciones o deleciones). • Caracterizar el espectro mutacional de DYSF en población española • Establecer una relación genotipo-fenotipo • Analizar posibles genes modificadores Los resultados derivados de este trabajo han sido publicados en las siguientes revistas científicas: -Abnormal expresión of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele. M.Meznaric, L. González-Quereda, E. Gallardo, N. de Luna, P. Gallano, M. Fanin, C. Angelini, B. Peterlin and J. Zidar. European Journal of Neurology 2010. Article first published online: 18 OCT 2010 DOI: 10.1111/j.1468-1331.2010.03240.x -A new phenotype of dysferlinopathy with congenital onset. Paradas C, González-Quereda L, de Luna N, Gallardo E, García-Consuegra I, Gómez H, Cabello A, Illa I, Gallano P. *Authors Paradas C and González-Quereda L contributed equally to this work. Neuromuscul Disord. 2009 Jan;19(1):21-5. Epub 2008 Dec 11. doi:10.1016/j.nmd.2008.09.015 Las conclusiones del trabajo son: • Es posible realizar el estudio mutacional en el RNA total extraído de monocitos circulantes en sangre periférica, evitándose por consiguiente el método invasivo de biopsia muscular • Existe una gran heterogeneidad mutacional en el gen DYSF en pacientes de población española con distrofia muscular de cinturas tipo 2B y miopatía distal tipo Miyoshi. • No es posible establecer una relación genotipo-fenotipo en pacientes con disferlinopatía. • La mutación DYSF c.3191_3196dup es la más prevalente en nuestra población. • La heterogeneidad mutacional en los pacientes de nuestra población, en lo referente a la localización dentro del gen, es elevada y no se han identificado "hotspots" del mismo modo que en otras poblaciones caucásicas. • El estudio de DYSF en mRNA vs DNA genómico permite: o Establecer un diagnóstico molecular de forma más rápida y eficaz. o Detectar un mayor número de mutaciones. o Detectar todas las mutaciones que alteran el proceso de splicing. • No se han identificado mutaciones en el gen MG53, que codifica para mitsugumina-53, en nuestra serie de pacientes con disferlinopatía por lo que hasta la actualidad no puede ser considerado como gen modificador. • Se describe un nuevo fenotipo de disferlinopatía con inicio congénito, ampliándose de este modo el espectro clínico de las disferlinopatías y, añadiendo nuevos datos a la historia natural de la enfermedad.Limb Girdle Muscular Dystrophies (LGMD) are a heterogeneous group of inherited neuromuscular diseases, defined by progressive muscle weakness and atrophy. They are characterized by a progressive dystrophic pattern involving limb-girdle muscles. Mutations in DYSF gene are responsible for a wide range of phenotypes characterized by the absence of dysferlin in skeletal muscle and an autosomal recessive mode of inheritance. So far, three main phenotypes have been reported: Miyoshi Myopathy (MM), Limb Girdle Muscular Dystrophy type 2B (LGMD 2B), and Distal Myopathy with Anterior Tibial onset (DMAT). The objectives of this work were: • To study the DYSF gene in patients from Spanish population, not previously analysed in this population. o To design a technical strategy to analyse the DYSF gene o To optimize the diagnostic process using RNA vs DNA • To analyse gene rearrangements (deletions or duplications). • To characterize the mutational spectrum in our population • To establish a genotype-phenotype relationship • To analyse possible modifiers genes Results from this work had been published: -Abnormal expresión of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele. M.Meznaric, L. González-Quereda, E. Gallardo, N. de Luna, P. Gallano, M. Fanin, C. Angelini, B. Peterlin and J. Zidar. European Journal of Neurology 2010. Article first published online: 18 OCT 2010 DOI: 10.1111/j.1468-1331.2010.03240.x -A new phenotype of dysferlinopathy with congenital onset. Paradas C, González-Quereda L, de Luna N, Gallardo E, García-Consuegra I, Gómez H, Cabello A, Illa I, Gallano P. *Authors Paradas C and González-Quereda L contributed equally to this work. Neuromuscul Disord. 2009 Jan;19(1):21-5. Epub 2008 Dec 11. doi:10.1016/j.nmd.2008.09.015 This thesis drew the conclusions: • It is possible to perform the mutational analysis in RNA isolated from total peripheral blood monocytes, avoiding the invasive method of muscle biopsy. • DYSF gene shows high mutational heterogeneity in patients from Spanish population suffering from Limb Girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. • It is not possible to establish a genotype-phenotype correlation in dysferlinopathy patients. • DYSF c.3191_3196dup is the most prevalent mutation in Spanish population. • Patients from our population show high mutational heterogeneity and it is not possible to identify hotspots as mutations are expanded over the whole sequence of the gene. • The DYSF gene study in mRNA vs genomic DNA allows: o To establish a molecular diagnostic in a quick and efficient way. o To detect a high number of mutations o To detect all mutations changing the splicing process. • Mutations in MG53 have not been identified in our serie of patients, so that, MG53 can not be considered as a modifier gene to date. • It has been described a new phenotype of dysferlinopathy with congenital onset. This new phenotype widens the clinical spectrum of dysferlinopathies and add new data to the natural story of the disease

Next-generation sequencing reveals a new mutation in the LTBP2 gene associated with microspherophakia in a Spanish family

Abstract Background Microspherophakia is a rare autosomal recessive eye disorder characterized by small spherical lens. It may present as an isolated finding or in association with other ocular and/or systemic disorders. This clinical and genetic heterogeneity requires the study of large genes (ADAMTSL4, FBN1, LTBP2, ADAMTSL-10 and ADAMTSL17). The purpose of the present study is to identify the genetic cause of this pathology in a consanguineous Spanish family. Methods A clinical exome sequencing experiment was executed by the TruSight One® Sequencing Panel (TSO) from Illumina©. Sanger sequencing was used to validate the NGS results. Results Only the insertion of an adenine in exon 36 of the LTBP2 gene (c.5439_5440insA) was associated with pathogenicity. This new mutation was validated by Sanger sequencing and segregation analysis was also performed. Haplotype analyses using the polymorphic markers D14S1025, D14S43 and D14S999 close to the LTBP2 gene indicated identity by descent in this family. Conclusion We describe the first case of a microspherophakia phenotype associated with a novel homozygous mutation in the LTBP2 gene in a consanguineous Caucasian family by means of NGS technology

Genetic diagnosis of Duchenne and Becker muscular dystrophy through mRNA analysis : New splicing events

Background Up to 7% of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) remain genetically undiagnosed after routine genetic testing. These patients are thought to carry deep intronic variants, structural variants or splicing alterations not detected through multiplex ligation-dependent probe amplification or exome sequencing. Methods RNA was extracted from seven muscle biopsy samples of patients with genetically undiagnosed DMD/BMD after routine genetic diagnosis. RT-PCR of the DMD gene was performed to detect the presence of alternative transcripts. Droplet digital PCR and whole-genome sequencing were also performed in some patients. Results We identified an alteration in the mRNA level in all the patients. We detected three pseudoexons in DMD caused by deep intronic variants, two of them not previously reported. We also identified a chromosomal rearrangement between Xp21.2 and 8p22. Furthermore, we detected three exon skipping events with unclear pathogenicity. Conclusion These findings indicate that mRNA analysis of the DMD gene is a valuable tool to reach a precise genetic diagnosis in patients with a clinical and anatomopathological suspicion of dystrophinopathy that remain genetically undiagnosed after routine genetic testing

Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements

Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy

<p>Abstract</p> <p>Background</p> <p>Between 8% and 22% of female carriers of <it>DMD</it> mutations exhibit clinical symptoms of variable severity. Development of symptoms in <it>DMD</it> mutation carriers without chromosomal rearrangements has been attributed to skewed X-chromosome inactivation (XCI) favouring predominant expression of the <it>DMD</it> mutant allele. However the prognostic use of XCI analysis is controversial. We aimed to evaluate the correlation between X-chromosome inactivation and development of clinical symptoms in a series of symptomatic female carriers of dystrophinopathy.</p> <p>Methods</p> <p>We reviewed the clinical, pathological and genetic features of twenty-four symptomatic carriers covering a wide spectrum of clinical phenotypes. <it>DMD</it> gene analysis was performed using MLPA and whole gene sequencing in blood DNA and muscle cDNA. Blood and muscle DNA was used for X-chromosome inactivation (XCI) analysis thought the <it>AR</it> methylation assay in symptomatic carriers and their female relatives, asymptomatic carriers as well as non-carrier females.</p> <p>Results</p> <p>Symptomatic carriers exhibited 49.2% more skewed XCI profiles than asymptomatic carriers. The extent of XCI skewing in blood tended to increase in line with the severity of muscle symptoms. Skewed XCI patterns were found in at least one first-degree female relative in 78.6% of symptomatic carrier families. No mutations altering XCI in the <it>XIST</it> gene promoter were found.</p> <p>Conclusions</p> <p>Skewed XCI is in many cases familial inherited. The extent of XCI skewing is related to phenotype severity. However, the assessment of XCI by means of the <it>AR</it> methylation assay has a poor prognostic value, probably because the methylation status of the <it>AR</it> gene in muscle may not reflect in all cases the methylation status of the <it>DMD</it> gene.</p

Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy

Background: Between 8% and 22% of female carriers of DMD mutations exhibit clinical symptoms of variable severity. Development of symptoms in DMD mutation carriers without chromosomal rearrangements has been attributed to skewed X-chromosome inactivation (XCI) favouring predominant expression of the DMD mutant allele. However the prognostic use of XCI analysis is controversial. We aimed to evaluate the correlation between X-chromosome inactivation and development of clinical symptoms in a series of symptomatic female carriers of dystrophinopathy. Methods: We reviewed the clinical, pathological and genetic features of twenty-four symptomatic carriers covering a wide spectrum of clinical phenotypes. DMD gene analysis was performed using MLPA and whole gene sequencing in blood DNA and muscle cDNA. Blood and muscle DNA was used for X-chromosome inactivation (XCI) analysis thought the AR methylation assay in symptomatic carriers and their female relatives, asymptomatic carriers as well as non-carrier females. Results: Symptomatic carriers exhibited 49.2% more skewed XCI profiles than asymptomatic carriers. The extent of XCI skewing in blood tended to increase in line with the severity of muscle symptoms. Skewed XCI patterns were found in at least one first-degree female relative in 78.6% of symptomatic carrier families. No mutations altering XCI in the XIST gene promoter were found. Conclusions: Skewed XCI is in many cases familial inherited. The extent of XCI skewing is related to phenotype severity. However, the assessment of XCI by means of the AR methylation assay has a poor prognostic value, probably because the methylation status of the AR gene in muscle may not reflect in all cases the methylation status of the DMD gene