New Mutations in NEB Gene Discovered by Targeted Next-Generation Sequencing in Nemaline Myopathy Italian Patients.
Nemaline myopathy represents a group of clinically and genetically heterogeneous neuromuscular disorders. Different clinical-genetic entities have been characterized in the last few years, with implications for diagnostics and genetic counseling. Fifty percent of nemaline myopathy forms are due to NEB mutations, but genetic analysis of this large and complex gene by Sanger sequencing is time consuming and expensive. We selected 10 Italian patients with clinical and biopsy features suggestive for nemaline myopathy and negative for ACTA1, TPM2 and TPM3 mutations. We applied a targeted next-generation sequencing strategy designed to analyse NEB coding regions, the relative full introns and the promoter. We also evaluated copy number variations (by CGH array) and transcriptional changes by RNA Sanger sequencing, whenever possible. This combined strategy revealed 11 likely pathogenic variants in 8 of 10 patients. The molecular diagnosis was fully achieved in 3 of 8 patients, while only one heterozygous mutation was observed in 5 subjects. This approach revealed to be a fast and cost-effective way to analyse the large NEB gene in a small group of patients and might be promising for the detection of pathological variants of other genes featuring large coding regions and lacking mutational hotspots.
Dino Ferrari Centre, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Neurology Unit, I.R.C.C.S. Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.
Daniela
D
Piga
Dino Ferrari Centre, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Neurology Unit, I.R.C.C.S. Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.
Francesca
F
Magri
Dino Ferrari Centre, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Neurology Unit, I.R.C.C.S. Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.
Dario
D
Ronchi
Dino Ferrari Centre, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Neurology Unit, I.R.C.C.S. Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.
Stefania
S
Corti
Molecular Medicine Unit, IRCCS Stella Maris, Pisa, Italy.
Denise
D
Cassandrini
Department of Pediatrics, Policlinico Gemelli, Università Cattolica Sacro Cuore, Roma, Italy.
Eugenio
E
Mercuri
Don Carlo Gnocchi Onlus Foundation, Milan, Italy.
Giorgio
G
Tasca
Department of Laboratories, Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, Rome, Italy.
Enrico
E
Bertini
Department of Laboratories, Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, Rome, Italy.
Fabiana
F
Fattori
Department of Neurosciences, Psychiatry and Anesthesiology, University of Messina, AOU Policlinico G. Martino, Messina, Italy.
Antonio
A
Toscano
Department of Neurosciences, Psychiatry and Anesthesiology, University of Messina, AOU Policlinico G. Martino, Messina, Italy.
Sonia
S
Messina
Pediatric, Neuromuscular Diseases and Neuroimmunology Units, The Foundation "Carlo Besta" IRCCS Neurological Institute, Milan, Italy.
Isabella
I
Moroni
Pediatric, Neuromuscular Diseases and Neuroimmunology Units, The Foundation "Carlo Besta" IRCCS Neurological Institute, Milan, Italy.
Marina
M
Mora
Dino Ferrari Centre, Neuromuscular Unit, IRCCS Foundation Cà Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.
Maurizio
M
Moggio
Dino Ferrari Centre, Neuromuscular Unit, IRCCS Foundation Cà Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.
Irene
I
Colombo
Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy.|Telethon Institute of Genetics and Medicine, Naples, Italy.
Teresa
T
Giugliano
Department of Pediatrics, Policlinico Gemelli, Università Cattolica Sacro Cuore, Roma, Italy.
Marika
M
Pane
Center of Myology and Neurodegenerative Diseases, Giannina Gaslini Institute, Genova, Italy.
Chiara
C
Fiorillo
Department of Laboratories, Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, Rome, Italy.
Adele
A
D'Amico
Center of Myology and Neurodegenerative Diseases, Giannina Gaslini Institute, Genova, Italy.
Claudio
C
Bruno
Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy.|Telethon Institute of Genetics and Medicine, Naples, Italy.
Vincenzo
V
Nigro
Dino Ferrari Centre, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Neurology Unit, I.R.C.C.S. Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.
Nereo
N
Bresolin
Dino Ferrari Centre, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Neurology Unit, I.R.C.C.S. Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy. giacomo.comi@unimi.it.
Giacomo Pietro
GP
Comi
D002648:Child; D056915:DNA Copy Number Variations; D005820:Genetic Testing / Q000379:methods*; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D007223:Infant; D007438:Introns; D009124:Muscle Proteins / Q000235:genetics*; D009154:Mutation; D017696:Myopathies, Nemaline / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D055815:Young Adult
D009124:Muscle Proteins; C035620:nebulin
NEB mutations; Nemaline myopathy; Next-generation sequencing