Quantitative copy number analysis by multiplex ligation-dependent probe amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness, and as such treatment response.

Lips, Esther H.; Mulder, Lennart; Joosse, Simon A.; Savola, SS; Vollebergh, Marieke A.; Wessels, LF; Wesseling, Jelle; Nederlof, Petra M.; Rodenhuis, S.

doi:10.1200/jco.2011.29.15_suppl.10513

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“…DNA from formalin‐fixed, paraffin‐embedded (FFPE) tumor tissues was isolated using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) after macrodissection. BRCAness was classified by multiplex ligation‐dependent probe amplification (MLPA) with the Probemix P376‐BRCA1ness (MRC‐Holland, Amsterdam, The Netherlands) 34 . BRCAness is defined as a specific chromosomal unstable BC tumor molecular phenotype, which describes the presence of homologous recombination defects and predicts high response rates to cytotoxic chemotherapy (e.g., platinum agents and anthracyclines).…”

Section: Methodsmentioning

confidence: 99%

“…BRCAness was classified by multiplex ligation-dependent probe amplification (MLPA) with the Probemix P376-BRCA1ness (MRC-Holland, Amsterdam, The Netherlands). 34 BRCAness is defined as a specific chromosomal unstable BC tumor molecular phenotype, which describes the presence of homologous recombination defects and predicts high response rates to cytotoxic chemotherapy (e.g., platinum agents and anthracyclines). The BRCA1 Ser282Leu variant was detected in a 30-years-old female (Cancer Genetics and Genomics Unit, TMC-ACTREC) with triple-negative BC with no family history.…”

Section: Brcaness Analysis By Mlpamentioning

confidence: 99%

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Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h‐BRCA1

Mishra,

Dubey,

Kumari

et al. 2023

Proteins

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Preliminary studies have shown BRCA1 (170–1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h‐BRCA1(260–553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small‐angle X‐ray scattering analysis revealed WT scores Rg−32 Å, Dmax−93 Å, and Rflex‐51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder‐to‐order transition in the presence of cruciform DNA and 2,2,2‐Trifluoroethanol (TFE). An increased alpha‐helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT‐DNA complex compared to the Gln356Arg and Ser282Leu protein‐DNA complex. Moreover, mature amyloid‐like fibrils were observed with 30 μM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher‐order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Section: Brcaness Analysis By Mlpamentioning

confidence: 99%