BRCA2 Protein Variant

Background and Objective: We have previously reported that BRCA2 N372 H i.a.1342A>C heterozygous variation presented in platinum-resistant patients. This study aimed to further investigate the mechanism of BRCA2 N372 H mutation in the development of platinum resistance in ovarian cancer. Methods: The BRCA2 N372 H i.a.1342A>C was synthesized and used to exchange 1 wildtype allele followed by sequencing to confirm the mutant allele sequence. Plasmids were constructed and transfected into the OVCAR-3 cells after lentiviral packaging. BRCA2 N372 H mRNA was detected by qPCR. BRCA2 protein was assessed by immunoblotting. Binding of the BRCA2 to Rad51 was detected by immunofluorescence staining. Sensitivity of the cells to cisplatin treatment was assessed with CCK-8 assay. Results: It was found that expression of BRCA2 protein in ovarian cancer cells transfected with BRCA2 N372 H i.a.1342A>C gene (2.177 ± 0.003) was significantly increased compared to that of the cells transfected with lenti-EGFP only (1.227 ± 0.003, P < 0.001). Binding of the BRCA2 and Rad51 proteins was significantly increased in the cells with BRCA2 N372 H i.a.1342A>C mutation (3.542 ± 0.24) than that in the cells transfected with lenti-EGFP (1.29 ± 0.32) or empty cells (1.363 ± 0.32, P < 0.001). Cell viability significantly increased in the cells transfected with BRCA2 N372 H mutant gene. The IC50 value was significantly higher in the cells transfected with BRCA2 N372 H mutant gene (1.963 ± 0.04) than that of the cells transfected with lenti-EGFP (0.955 ± 0.03, P < 0.01) or empty cells (1.043 ± 0.007, P < 0.01). Conclusion: Over expression of mRNA and protein of BRCA2 was detected in the cells with BRCA2 N372 H i.a.1342A>C mutation but not in the lentivirus negative control (lenti-EGFP) or the cells without transfection (empty cells), which may lead to resistance to platinum-based drugs in ovarian cancer cells through homologous recombination repair pathway.

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Whole-exome sequencing in eccrine porocarcinoma indicates promising therapeutic strategies

Cancer Gene Therapy ◽

10.1038/s41417-021-00347-z ◽

2021 ◽

Author(s):

Evgeniya Denisova ◽

Dana Westphal ◽

Harald M. Surowy ◽

Friedegund Meier ◽

Barbara Hutter ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Surrounding Tissue ◽

Whole Exome ◽

Brca2 Protein ◽

Break Repair ◽

Eccrine Porocarcinoma

AbstractMalignant sweat gland tumours are rare, with the most common form being Eccrine porocarcinoma (EP). To investigate the mutational landscape of EP, we performed whole-exome sequencing (WES) on 14 formalin-fixed paraffin-embedded samples of matched primary EP and healthy surrounding tissue. Mutational profiling revealed a high overall median mutation rate. This was attributed to signatures of mutational processes related to ultraviolet (UV) exposure, APOBEC enzyme dysregulation, and defective homologous double-strand break repair. All of these processes cause genomic instability and are implicated in carcinogenesis. Recurrent driving somatic alterations were detected in the EP candidate drivers TP53, FAT2, CACNA1S, and KMT2D. The analyses also identified copy number alterations and recurrent gains and losses in several chromosomal regions including that containing BRCA2, as well as deleterious alterations in multiple HRR components. In accordance with this reduced or even a complete loss of BRCA2 protein expression was detected in 50% of the investigated EP tumours. Our results implicate crucial oncogenic driver pathways and suggest that defective homologous double-strand break repair and the p53 pathway are involved in EP aetiology. Targeting of the p53 axis and PARP inhibition, and/or immunotherapy may represent promising treatment strategies.

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Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with pulmonary hypertension

F1000Research ◽

10.12688/f1000research.9254.2 ◽

2016 ◽

Vol 5 ◽

pp. 2003 ◽

Cited By ~ 5

Author(s):

Michael P. Heaton ◽

Timothy P.L. Smith ◽

Jacky K. Carnahan ◽

Veronica Basnayake ◽

Jiansheng Qiu ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Beef Cattle ◽

In Silico ◽

Read Depth ◽

Whole Genome Sequence ◽

Whole Genome ◽

Missense Mutations ◽

Protein Variant ◽

Flight Mass Spectrometry ◽

Protein Variants

The availability of whole genome sequence (WGS) data has made it possible to discover protein variantsin silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in global beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene (EPAS1), a gene associated with pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded byEPAS1(E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The sixEPAS1missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact ofEPAS1alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitatesin silicoidentification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.

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Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears

Biochemical Journal ◽

10.1042/bj20011876 ◽

2002 ◽

Vol 366 (2) ◽

pp. 511-520 ◽

Cited By ~ 63

Author(s):

Benjamin L. SCHULZ ◽

David OXLEY ◽

Nicolle H. PACKER ◽

Niclas G. KARLSSON

Keyword(s):

Molecular Mass ◽

Peptide Mass Fingerprinting ◽

High Molecular Mass ◽

Tear Fluid ◽

Protein Variant ◽

Peptide Mass ◽

Composite Gel ◽

Molecular Masses ◽

Protein Components ◽

Sialyl Lea

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

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A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00327-09 ◽

2009 ◽

Vol 9 (1) ◽

pp. 224-226 ◽

Cited By ~ 41

Author(s):

Chengda Zhang ◽

James B. Konopka

Keyword(s):

Candida Albicans ◽

Green Fluorescent Protein ◽

Codon Usage ◽

Signal Intensity ◽

Fluorescent Protein ◽

Protein Localization ◽

Protein Variant ◽

Green Fluorescent ◽

Green Fluorescent Protein Variant

ABSTRACT Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.

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