Services

Seqüenciació de l’RNA missatger del gen DMD

Presentation

Duchenne muscular dystrophy is the most common X-linked inherited disease in males, affecting 1 in 3500 males born. The debut of the disease occurs between 2-4 years of age with very severe muscle degeneration. There is a form of the disease, Becker muscular dystrophy, which has a later onset and progresses more slowly but can become disabling throughout adulthood. Both Duchenne and Becker muscular dystrophy are caused by mutations in the DMD gene.

The DMD gene, located on the X chromosome, is transcribed to a 14Kb mRNA that gives rise to a subsarcolemmal protein called dystrophin. The 70% of DMD mutations consist of large exonic deletions or duplications. The remaining 30% of mutations are nonsense, frameshift, missense and splicing varaints.

Services

The diagnosis of patients affected by Duchenne or Becker muscular dystrophy is primarily clinical and is complemented by immunohistochemical and/or Western blot analysis of the muscle biopsy with antibodies directed against dystrophin. The molecular study of the DMD gene begins with CNV analysis to determine the presence or absence of an exonic deletion or duplication, and if negative, gDNA sequencing by NGS technology is performed to identify possible point mutations. If the result is also negative, the patient would obtain the clinical and pathologic diagnosis, but not the precise genetic diagnosis, without which the patient cannot benefit from the clinical trials under development, especially those involving gene therapy.

The next step in the diagnostic process is to analyze the intronic sequence by sequencing the mRNA of the DMD gene to identify:

  • Mutations located in the introns that create or destroy splicing sites, or originate the insertion of intronic fragments in the coding sequence.
  • Mutations located in the exons, which are of uncertain significance or which apparently may appear innocuous, but for which RNA sequencing demonstrates their pathogenic effect at the transcriptional level.

The service we offer includes:

  • RNA extraction from the patient's muscle biopsy.
  • Retrotranscription to cDNA
  • Sequencing by the Sanger method in 26 overlapping fragments covering the entire 14kb of the DMD gene cDNA.
  • Analysis of the variants identified: creation/abolition of splicing sites, detection of alternative transcripts, insertion of pseudoexons and assessment of the possible effect that variants of uncertain significance (VUS) may have at the transcriptional level.

The availability of the patient's muscle biopsy is essential for this study.

Responsibles

Contact mail: pgallano@santpau.cat

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Maria Pia Gallano Petit

Scientific Responsible

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Lidia Gonzalez Quereda

Technical Responsible / Manager Responsible

Experience and Trajectory

Our group, associated with the Genetics Service of the Hospital de Sant Pau, has been carrying out the molecular study of genetically based neuromuscular diseases since 1985.

Proof of this long trajectory and experience, regarding mRNA analysis, are the projects awarded and some publications cited below:

  • Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne Muscular Dystrophy.
  • Segarra-Casas A, Domínguez-González C, Natera-de Benito D, Kapetanovic S, Hernández-Laín A, Estévez-Arias B, Llansó L, Ortez C, Jou C, Martí-Carrera I, López-Márquez A, Rodríguez MJ, González-Mera L, Nedkova V, Fernández-Torrón R, Rodríguez-Santiago B, Jimenez-Mallebrera C, Juntas-Morales R, López-de Munain A, Surrallés J, Nascimento A, Gallardo E, Olivé M, Gallano P, González-Quereda L. Translating Muscle RNAseq Into the Clinic for the Diagnosis of Muscle Diseases. Ann Clin Transl Neurol. 2025 Jul;12(7):1465-1479. doi: 10.1002/acn3.70078. Epub 2025 May 25. PMID: 40413734; PMCID: PMC12257123.
  • Segarra-Casas A, Domínguez-González C, Hernández-Laín A, Sanchez-Calvin MT, Camacho A, Rivas E, Campo-Barasoain A, Madruga M, Ortez C, Natera-de Benito D, Nascimento A, Codina A, Rodriguez MJ, Gallano P, Gonzalez-Quereda L. Genetic diagnosis of Duchenne and Becker muscular dystrophy through mRNA analysis: new splicing events. J Med Genet. 2023 Jun;60(6):615-619. doi: 10.1136/jmg-2022-108828. Epub 2022 Dec 19. PMID: 36535754; PMCID: PMC10313949.
  • Aartsma-Rus A, Hegde M, Ben-Omran T, Buccella F, Ferlini A, Gallano P, Howell RR, Leturcq F, Martin AS, Potulska-Chromik A, Saute JA, Schmidt WM, Sejersen T, Tuffery-Giraud S, Uyguner ZO, Witcomb LA, Yau S, Nelson SF.
  • J Pediatr. 2019 Jan;204:305-313.e14. doi: 10.1016/j.jpeds.2018.10.043.
  • PMID: 30579468
  • DMD Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations.
  • Juan-Mateu J, Gonzalez-Quereda L, Rodriguez MJ, Baena M, Verdura E, Nascimento A, Ortez C, Baiget M, Gallano P.
  • PLoS One. 2015 Aug 18;10(8):e0135189. doi: 10.1371/journal.pone.0135189. eCollection 2015.
  • PMID: 26284620
  • Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.
  • Juan-Mateu J, González-Quereda L, Rodríguez MJ, Verdura E, Lázaro K, Jou C, Nascimento A, Jiménez-Mallebrera C, Colomer J, Monges S, Lubieniecki F, Foncuberta ME, Pascual-Pascual SI, Molano J, Baiget M, Gallano P.
  • PLoS One. 2013;8(3):e59916. doi: 10.1371/journal.pone.0059916. Epub 2013 Mar 25.
  • PMID: 23536893
  • Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy.
  • Juan-Mateu J, Rodríguez MJ, Nascimento A, Jiménez-Mallebrera C, González-Quereda L, Rivas E, Paradas C, Madruga M, Sánchez-Ayaso P, Jou C, González-Mera L, Munell F, Roig-Quilis M, Rabasa M, Hernández-Lain A, Díaz-Manera J, Gallardo E, Pascual J, Verdura E, Colomer J, Baiget M, Olivé M, Gallano P.
  • Orphanet J Rare Dis. 2012 Oct 23;7:82. doi: 10.1186/1750-1172-7-82.
  • PMID: 23092449
  • Isolated cardiomyopathy caused by a DMD nonsense mutation in somatic mosaicism: genetic normalization in skeletal muscle.
  • Juan-Mateu J, Paradas C, Olivé M, Verdura E, Rivas E, González-Quereda L, Rodríguez MJ, Baiget M, Gallano P.

Projects:

  • PI18/01585 Análisis del transcriptoma mediante RNA-Seq en pacientes con patología neuromuscular no resuelta tras secuenciación de exoma: optimización del algoritmo diagnóstico. Pia Gallano y Lidia Gonzalez Quereda. (INSTITUT DE RECERCA DE L'HOSPITAL DE LA SANTA CREU I SANT PAU). 01/01/2019-31/12/2021.
  • PI 15/01898 Implementación de la secuenciación masiva en el estudio de las Miopatías Congénitas y los Síndromes Miasténicos congénitos: un modelo de investigación traslacional en enfermedades raras Instituto de Salud Carlos III. Pia Gallano Petit. (INSTITUT DE RECERCA DE L'HOSPITAL DE LA SANTA CREU I SANT PAU). 2015- 2018.
  • PI 11/2586 Elaboración de paneles de diagnóstico molecular en patología monogénica hereditaria mediante el sistema de nanofluidos y  secuenciación masiva Instituto de Salud Carlos III. (INSTITUT DE RECERCA DE L'HOSPITAL DE LA SANTA CREU I SANT PAU). 2012-2015.
  • PI 08/0347 Estudio del RNA mensajero del gen de la distrofia muscular de Duchenne (DMD): Interferencia del mecanismo de degradación de mRNA mediada por mutaciones sin sentido Instituto de Salud Carlos III. Pia Gallano (INSTITUT DE RECERCA DE L'HOSPITAL DE LA SANTA CREU I SANT PAU). 2008-2011.
  • PI 05/2457 Interacción de la disferlina con diferentes proteínas de músculo esquelético y búsqueda de genes modificadores que originan la aparición de tres disferlinopatías diferentes (MM, LGMD2B y DAT) Instituto de Salud Carlos III. Pia Gallano (INSTITUT DE RECERCA DE L'HOSPITAL DE LA SANTA CREU I SANT PAU). 2005-2008.CREU I SANT PAU). 2008-2011.

Equipment

Equipment involved in providing the service::

  • Tissue Ruptor (Qiagen). 
  • Termocicladors Veriti™ 96-Well Thermal Cycler (ThermoFisher 
  • Applied Biosystems 3500 Genetic Analyzer