Next generation sequencing in the analysis of an Italian cohort of patients affected by nemaline myopathy

OBJECTIVE: We used next generation sequencing (NGS) to analyze Nebulin (NEB) gene in a cohort of Italian patients with nemaline myopathy (NM). BACKGROUND: NM is a clinical and genetic heterogeneous disorder of skeletal muscle caused by mutations in five different genes encoding thin filament proteins of the striated muscle sarcomere. Mutations in NEB are responsible for about 50% of cases. 70 NEB mutations, mainly small deletions or point mutations, have been published. NEB is the largest and more complex gene among those involved in neuromuscular disorders, making its analysis by traditional sequencing method time consuming and expensive. Given the inherent gene complexity, we have chosen the NGS as an alternative technique. DESIGN/METHODS: We have analysed 10 Italian patients with NM, previously screened for ACTA1, TPM2, TPM3, TNNT1 and CFL2. We have used the Target Region Capture Sequencing in order to investigate the NEB in depth, searching for mutations in all 183 exons, introns and promoter region. Alterations have been confirmed by Sanger. RESULTS: We have found 9 novel NEB variants: missense mutations, affecting conserved amino acids; small indels causing frame shifts; nonsense mutations responsible for premature truncation of nebulin; splice-site mutations. In addition, we have found a deletion in the middle of intron 125 causing the partial inclusion of intron in the coding sequence. CONCLUSIONS: We have found mutations in 7 out of 10 patients, the highest detection rate observed in a selected cohort of NM patients. The application of the Target Region Capture Sequencing allowed a faster determination of recessive missense mutations, compound heterozygote genotypes, small deletions, stop codon and frame shifts. The target analysis has allowed to improve the quality and depth of data. We conclude that this method appears promising for a quicker detection of mutations in complex genes like NEB, with large coding size, exon number and without obvious mutational hotspots. Disclosure: Dr. Piga has nothing to disclose. Dr. Ronchi has nothing to disclose. Dr. Magri has nothing to disclose. Dr. Corti has nothing to disclose. Dr. Ghezzi has nothing to disclose. Dr. Mercuri has received personal compensation for activities with Aceleron Pharma, PTC Therapeutics, Inc., and Genzyme Corporation. Dr. Mercuri has received personal compensation in an editorial capacity for Neuromuscular Disorders, Annals of Neurology, Developmental Medicine & Child Neurology, and Neuropediatric. Dr. Bertini has received research support from Telethon Italy and SMA Europe. Dr. Toscano has nothing to disclose. Dr. Moroni has nothing to disclose. Dr. Moggio has nothing to disclose. Dr. D'Angelo has nothing to disclose. Dr. Bruno has nothing to disclose. Dr. Mora has nothing to disclose. Dr. Bresolin has nothing to disclose. Dr. Comi has received research support from Telethon Italy and SMA Europe.