Dataset Information

Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm.

ABSTRACT: Homologous Recombination (HR) repair is essential for repairing DNA double strand breaks (DSB) in dividing cells and preventing tumorigenesis. BRCA2 plays an important role in HR by recruiting the DNA recombinase RAD51 to the DSB. Despite being a popular model organism in genetic and cancer research, knowledge on the conservation of the HR pathway and function of zebrafish Brca2 is limited. To evaluate this, we developed a Rad51 foci assay in zebrafish embryos. We identified the zebrafish embryonic intestinal tissue as an ideal target for Rad51 immunostaining. After inducing DSB through irradiation, Rad51 foci were present in irradiated embryos but not in unirradiated controls. We present a method for accurate quantification of HR. Both morpholino-induced knockdown and knockout of Brca2 lead to almost complete absence of Rad51 foci in irradiated embryos. These findings indicate conserved function of Brca2 in zebrafish. Interestingly, a statistically significant decrease in Rad51 foci was observed in Brca2 heterozygous carriers compared to wild types, indicative of haploinsufficiency, a hypothesised cause of some tumours in patients with a germline BRCA2 mutation. In conclusion, we demonstrated the suitability of zebrafish as an excellent in vivo model system for studying the HR pathway and its functionality.

SUBMITTER: Vierstraete J

PROVIDER: S-EPMC5705637 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm.

Vierstraete Jeroen J Willaert Andy A Vermassen Petra P Coucke Paul J PJ Vral Anne A Claes Kathleen B M KBM

Scientific reports 20171128 1

Homologous Recombination (HR) repair is essential for repairing DNA double strand breaks (DSB) in dividing cells and preventing tumorigenesis. BRCA2 plays an important role in HR by recruiting the DNA recombinase RAD51 to the DSB. Despite being a popular model organism in genetic and cancer research, knowledge on the conservation of the HR pathway and function of zebrafish Brca2 is limited. To evaluate this, we developed a Rad51 foci assay in zebrafish embryos. We identified the zebrafish embryo ...[more]

PMID: 29184099

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Project description:BackgroundUterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of < 10%. Mutations in the homologous recombination (HR) DNA repair pathway have been observed in ~ 10% of uLMS cases, with reports of some individuals benefiting from poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy, which targets this DNA repair defect. In this report, we screened individuals with uLMS, accrued nationally, for mutations in the HR repair pathway and explored new approaches to therapeutic targeting.MethodsA cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting.ResultsAll 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs.ConclusionsOur work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.

Dataset Information

Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm.

Publications

Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm.

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