Angelo B Cefalù1, Rossella Spina2, Davide Noto3, Valeria Ingrassia2, Vincenza Valenti4, Antonina Giammanco3, Francesca Fayer3, Gabriella Misiano4, Gianfranco Cocorullo5, Chiara Scrimali4, Ornella Palesano3, Grazia I Altieri3, Antonina Ganci3, Carlo M Barbagallo3, Maurizio R Averna6. 1. Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS), University of Palermo, Palermo, Italy; Molecular Biology Diagnostic Laboratory, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), AOUP "Paolo Giaccone", Palermo, Italy. Electronic address: abaldassare.cefalu@unipa.it. 2. Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS), University of Palermo, Palermo, Italy; Molecular Biology Diagnostic Laboratory, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), AOUP "Paolo Giaccone", Palermo, Italy. 3. Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS), University of Palermo, Palermo, Italy. 4. Molecular Biology Diagnostic Laboratory, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), AOUP "Paolo Giaccone", Palermo, Italy. 5. Unit of Emergency and General Surgery, Department of Surgical Oncological and Stomatological Sciences, University of Palermo, Palermo, Italy. 6. Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS), University of Palermo, Palermo, Italy; Molecular Biology Diagnostic Laboratory, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), AOUP "Paolo Giaccone", Palermo, Italy. Electronic address: maurizio.averna@unipa.it.
Abstract
BACKGROUND: Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. OBJECTIVE: The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. METHODS: We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. RESULTS: The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. CONCLUSIONS: We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG.
BACKGROUND: Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. OBJECTIVE: The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. METHODS: We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. RESULTS: The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. CONCLUSIONS: We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG.
Authors: Miklós Péterfy; Candy Bedoya; Carola Giacobbe; Carmen Pagano; Marco Gentile; Paolo Rubba; Giuliana Fortunato; Maria Donata Di Taranto Journal: J Clin Lipidol Date: 2018-07-25 Impact factor: 4.766