TY - JOUR
T1 - High-throughput genotyping robot-assisted method for mutation detection in patients with hypertrophic cardiomyopathy
AU - Bortot, Barbara
AU - Athanasakis, Emmanouil
AU - Brun, Francesca
AU - Rizzotti, Diego
AU - Mestroni, Luisa
AU - Sinagra, Gianfranco
AU - Severini, Giovanni Maria
PY - 2011/9
Y1 - 2011/9
N2 - 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.
AB - 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.
KW - genetic screening
KW - hypertrophic cardiomyopathy
KW - mutation
KW - robotic
KW - sarcomeric gene
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UR - http://www.scopus.com/inward/citedby.url?scp=80052269492&partnerID=8YFLogxK
U2 - 10.1097/PDM.0b013e31820b34fb
DO - 10.1097/PDM.0b013e31820b34fb
M3 - Article
C2 - 21817903
AN - SCOPUS:80052269492
VL - 20
SP - 175
EP - 179
JO - Diagnostic Molecular Pathology
JF - Diagnostic Molecular Pathology
SN - 1052-9551
IS - 3
ER -