Using Ion Torrent PGM semiconductor sequencing (see below) a CNV baseline was established within the Ion Reporter workflow to enable detection of BRCA1 and BRCA2 CNV. Cases with uninformative HISPANEL and BRCA sequencing were further analyzed for CNVs in BRCA1 by MLPA (MRC-Holland, Amsterdam, the Netherlands). For each MLPA reaction, 125 ng of DNA was PCR amplified with probemix P002 according to the manufacturer’s instructions. PCR fragments were size separated on an ABI (Applied Biosystems, Carlsbad, CA) 3130xl capillary sequencer. Dosage calculations and determination of CNV were performed with Coffalyser.Net software v.140701 (MRC-Holland) and/or GeneMarker software (SoftGenetics, State College, PA). CNVs detected by NGS or MLPA, were confirmed with MLPA probe mixes P087 and P045, respectively. BRCA2 was only evaluated for CNV by NGS, in part due to the known 5-fold greater rate of occurrence in BRCA1 relative to BRCA2 of CNVs37 (link).
Herzog J.S., Chavarri-Guerra Y., Castillo D., Abugattas J., Villarreal-Garza C., Sand S., Clague-Dehart J., Alvarez-Gómez R.M., Wegman-Ostrosky T., Mohar A., Mora P., Del Toro-Valero A., Daneri-Navarro A., Rodriguez Y., Cruz-Correa M., Ashton-Prolla P., Alemar B., Mejia R., Gallardo L., Shaw R., Yang K., Cervantes A., Tsang K., Nehoray B., Barrera Saldana H., Neuhausen S, & Weitzel J.N. (2021). Genetic epidemiology of BRCA1- and BRCA2-associated cancer across Latin America. NPJ Breast Cancer, 7, 107.
Other organizations :
City of Hope, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto Nacional de Enfermedades Neoplásicas, Hospital Zambrano Hellion, Tecnológico de Monterrey, Instituto Nacional de Cancerología, Instituto Jalisciense de Cancerología, Universidad de Guadalajara, University of Puerto Rico System, Universidade Federal do Rio Grande do Sul, Hospital de Clínicas de Porto Alegre, Universidad Autónoma de Nuevo León
Type of sequencing: Ion Torrent PGM semiconductor sequencing
MLPA analysis: PCR amplification with probemix P002
Confirmation of CNVs: MLPA probe mixes P087 and P045
dependent variables
Detection of BRCA1 and BRCA2 CNV
Determination of CNV by Coffalyser.Net software v.140701 and/or GeneMarker software
control variables
Quantity of DNA used for MLPA reaction: 125 ng
BRCA2 CNV analysis only performed by NGS, not MLPA, due to lower rate of occurrence compared to BRCA1
positive controls
Not explicitly mentioned
negative controls
Not explicitly mentioned
Annotations
Based on most similar protocols
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