Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.
Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.
Laboratoire de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique, Hôpitaux de Paris, Paris, France.
Laure
L
Raymond
Bertrand
B
Diebold
Céline
C
Leroux
Hélène
H
Maurey
Valérie
V
Drouin-Garraud
Andre
A
Delahaye
Olivier
O
Dulac
Julia
J
Metreau
Gia
G
Melikishvili
Annick
A
Toutain
François
F
Rivier
Nadia
N
Bahi-Buisson
Thierry
T
Bienvenu
D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017346:Protein Serine-Threonine Kinases; C114482:CDKL5 protein, human