Computational protocol: Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort

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Protocol publication

[…] Total genomic DNA was isolated from blood samples of the 55 patients and of 300 healthy Spanish individuals (individuals not related to any patient and of the same ethnicity; 600 alleles) using the Puregene DNA purification kit (Gentra Systems, Minneapolis, MI, USA). The genetic study was performed both in patients and in controls, and comprised the direct sequencing of SCN5A (NM_198056.2), CACNA1C (NM_001129827.1), CACNB2 (NM_201596.2), GPD1L (NM_015141.3), SCN1B (NM_001037.4 for isoform a; and NM_199037.3 for isoform b), SCN2B (NM_004588.4), SCN3B (NM_018400.3), SCN4B (NM_174934.3), KCNE3 (NM_005472.4), RANGRF (NM_001177801.1), HCN4 (NM_005477.2), KCNJ8 (NM_004982.3), KCND3 (NM_004980.4), and KCNE1L (NM_012282.2) []. The exons and exon-intron boundaries of each gene were amplified (Verities PCR, Applied Biosystems, Austin, TX, USA), the PCR products were purified (Exosap-IT, Affymetrix, Inc. USB Products, Cleveland, OH, USA) and they were directly sequenced in both directions (Big Dye Terminator v3.1 cycle sequencing kit and 3130XL Genetic Analyzer, both from Applied Biosystems). The DNA sequences obtained were compared with their respective reference sequences (stated above). All variants detected were verified in an independent sequencing reaction from a new PCR product of the DNA of interest. The identified variations were compared with DNA sequences from the control patients, and contrasted with HGMD BioBase [], HapMap [], 1000 genomes project [], NHLBI Exome Sequencing Project [] and The Exome Aggregation Consortium (ExAC) []. Sequence changes altering coding regions were defined as genetic variations. Minor allele frequencies (MAFs) were checked in Exome Variant Server-NHLBI Exome Sequencing Project and dbSNP [] databases. Genetic variations with a MAF in all populations <1% were considered rare variants. Genetic variations with a MAF >1% were considered common variants. Sequence variants were described following the HGVS rules [], and checked in Mutalyzer []. All rare (MAF <1%) variants that had been previously described to be associated with BrS or other cardiac diseases were considered potentially pathogenic variations (PPVs). Stop and frameshift variants were always considered PPVs given their potential effects on ion channel function.Samples were obtained for relatives of the patients who carried PPVs. DNA was extracted and it was screened for the presence of the PPV identified in the patients following the directions described above. […]

Pipeline specifications

Software tools Biobase, Mutalyzer
Databases Exome Variant Server HGMD
Applications Miscellaneous, WES analysis
Organisms Homo sapiens
Diseases Brugada Syndrome
Chemicals Nucleotides, Sodium