Genetic Analysis
The girl was submitted to a NexSeq (Illumina) analysis of a 286-gene panel for epilepsies and brain malformations. The sequencing was preceded by a selective enrichment of the regions of the DNA analyzed, through hybridation with probes selectively designed (Sureselect, Agilent). Average coverage of the target bases <100X.
The resulting variants were therefore analyzed by the software BWA (V.0.7.7-r441), Picard (v 1.109) and GATK (v 3.1), and annotation performed by Annovar (v. 17June15). In the analysis were considered all the non-synonimous exonic variants and splice-site (+/- 2 nucleotides of the codyging exons) with frequencies lower than 0.1% dominant genes and lower than 1% for recessive genes (ExAC, GnomAD). The data obtained through NGS sequencing have then been analyzed with the tool CoNVaDING in order to identify variants of the copy number of the exons (Johansson et al, 2016) and the variants reported with the Human Genetic Mutation Database (HGMD) nomenclature.
In the same occasion, an array-CGH analysis was also performed in the patient and in her parents recombination breakpoints were detected using high-resolution 8x60K Human Genome CGH microarray (Agilent Technologies, Santa Clara, CA). Array experiments were performed as recommended by the manufacturer (Agilent Technologies, Santa Clara, CA). Proband DNA was labeled with Cy5 and reference DNA (human DNA Promega) with Cy3. Purification, hybridization and washing steps were performed according to the manufacturer’s instructions. Data visualization and analysis were performed with Sure Scan microarray Scanner G2600D (Agilent), Feature Extraction software (Agilent) and Agilent Cytogenomics Software Edition 2.5.