title: High-throughput genotyping robot-assisted method for mutation detection in patients with hypertrophic cardiomyopathy.
abstract: Hypertrophic cardiomyopathy (HCM) is the most frequent autosomal dominant genetic heart muscle disease and the most common cause of sudden cardiac death in young people (under 30 y of age), who are often unaware of their underlying condition. Genetic screening is now considered a fundamental tool for clinical management of HCM families. However, the high genetic heterogeneity of HCM makes genetic screening very expensive. Here, we propose a new high-throughput genotyping method based on a HCM 96-well sequencing plate for the analysis of 8 of the most frequent HCM-causing sarcomeric genes by automating several processes required for direct sequencing, using a commercially available robotic systems and routinely used instruments. To assess the efficiency of the robot-assisted method, we have analyzed the entire coding sequence and flanking intronic sequences of the 8 sarcomeric genes in samples from 18 patients affected by HCM and their relatives, which revealed 9 different mutations, 3 of which were novel. The automated, robot-assisted assembling of polymerase chain reaction, purification of polymerase chain reaction products, and assembly of sequencing reactions resulted in a substantial saving of time, reagent costs, and reduction of human errors, and can therefore be proposed as a primary strategy for mutation identification in HCM genetic screening in many medical genetic laboratories.
authors.affiliation: Department of Molecular Medicine and Laboratory, Medical Genetics Laboratory, Institute of Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bortot
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Athanasakis
authors.forename: Francesca
authors.initials: F
authors.lastname: Brun
authors.forename: Diego
authors.initials: D
authors.lastname: Rizzotti
authors.forename: Luisa
authors.initials: L
authors.lastname: Mestroni
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Sinagra
authors.forename: Giovanni Maria
authors.initials: GM
authors.lastname: Severini
mesh_terms: D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics*; D004252:DNA Mutational Analysis; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D012371:Robotics
chemical_list: D009124:Muscle Proteins