Alvaro Galli

A guide for functional analysis of BRCA1 variants of uncertain significance

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome‐wide pathogenicity assignment. Hum Mutat 33:1526–1537, 2012.

On the mechanism of UV and gamma-ray-induced intrachromosomal recombination in yeast cells synchronized in different stages of the cell cycle.

A genetic system selecting for deletion events (DEL recombination) due to intrachromosomal recombination has previously been constructed in the yeastSaccharomyces cerevisiae. Intrachromosomal recombination is inducible by chemical and physical carcinogens. We wanted to understand better the mechanism of induced DEL recombination and to attempt to determine in which phase of the cell cycle DEL recombination is inducible. Yeast cells were arrested at specific phases of the cell cycle, irradiated with UV or γ-rays, and assayed for DEL recombination and interchromosomal recombination. In addition, the contribution of intrachromatid crossing-over to the number of radiation induced DEL recombination events was directly investigated at different phases of the cell cycle. UV irradiation induced DEL recombination preferentially in S phase, while γ-rays induced DEL recombination in every phase of the cell cycle including G1. UV and γ-radiation induced intrachromatid crossing over preferentially in G1, but it accounted at the most for only 14% of the induced DEL recombination events. The possibility is discussed that single-strand annealing or one-sided invasion events, which can occur in G1 and may be induced by a double-strand break intermediate, may be responsible for a large proportion of the induced DEL recombination events.

Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells

Cell proliferation has been recognized as an important factor in human and experimental carcinogenesis. Point mutations as well as larger chromosomal rearrangements are involved in the initiation of cancer. In this paper we compared the relative potencies of radiation and chemical carcinogens for inducing point mutations vs. deletions in cell cycle arrested with dividing cells of Saccharomyces cerevisiae. Point mutation substrates and deletion (DEL) recombination substrates were constructed with the genes CDC28 and TUB2 that are required for cell cycle progression through G1 and G2, respectively. The carcinogens ionizing radiation, UV, MMS, EMS and 4-NQO induced point mutations in G1 and in G2 arrested as well as in dividing cells. UV, MMS, EMS and 4-NQO caused very weak if any increases in DEL recombination in G1 or G2 arrested cells, but large increases in dividing cells. When cells treated with carcinogen either in G1 or G2 were allowed to progress through the cell cycle, a time-dependent increase in DEL recombination was seen. Ionizing radiation and the site-specific endonuclease I-SceI, which both directly create double-strand breaks, induced DEL recombination in G1 as well as in G2 arrested cells. In conclusion, UV-, MMS-, EMS- and 4-NQO-induced DNA damage was converted during DNA replication to a lesion capable of inducing DEL recombination which is probably a DNA strand break. Thus, cell proliferation is not necessary to turn DNA alkylation or UV damage into a mutagenic lesion but to convert the damage into a lesion that induces DNA deletions. These results are discussed with respect to mechanisms of carcinogenesis.

Functional Assays for Analysis of Variants of Uncertain Significance in BRCA2

Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore, alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication, and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review, we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including variants of uncertain significance analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory‐based methods to assess the impact of the variant on cancer risk.

Hydroxyurea induces recombination in dividing but not in G1 or G2 cell cycle arrested yeast cells

Hydroxyurea, a chemotherapeutic and radiosensitizing agent, inhibits ribonucleotide reductase, arrests cells in the S-phase and is mutagenic and recombinagenic. In this paper we investigated whether the recombinagenic activity of hydroxyurea is due to the same activity that leads to arrest in the S-phase or to a more direct action on DNA. The effect of hydroxyurea on intrachromosomal and interchromosomal recombination was investigated in dividing and in G1 or G2 cell cycle-arrested cells of the yeast Saccharomyces cerevisiae. Treatment of dividing cells with hydroxyurea resulted in a large increase in recombination frequencies, even at low non-toxic doses. In contrast, in cells arrested in the G1 or G2 phase, hydroxyurea failed to induce recombination, even at 60-fold higher toxic doses. The presence of metabolic activation (S9 mix) did not change the effects of hydroxyurea on recombination. The data suggest that the recombinagenic activity of hydroxyurea may not be due to any direct effect of hydroxyurea on DNA, but may be linked to the inhibition of ribonucleotide reductase causing inhibition of DNA synthesis leading to S-phase arrest and possibly causing recombinagenic lesions.

DNA modifications in atherosclerosis: From the past to the future

The role of DNA damage in the pathogenesis of atherosclerosis has been extensively investigated in recent decades. There is now clear that oxidative stress is an important inducer of both DNA damage and telomere attrition which, in turn, can gives rise to genome instability and vascular senescence. This review discusses the role of the DNA damage response, including the key DNA repair pathways (base excision repair, nucleotide excision repair, homologous recombination and non-homologous end joining), deregulated cell cycle and apoptosis in atherosclerosis. We also highlight emerging evidence suggesting that epigenetic changes (DNA methylation and microRNA-mediated mechanisms), not associated with alterations in DNA sequences, may play a critical role in the regulation of the DNA damage response. Nevertheless, further investigation is still required to better understand the complexity of DNA repair and DNA damage response in atherosclerosis, making this topic an exciting and promising field for future investigation. Unraveling these molecular mechanisms provide the rationale for the development of novel efficient therapies to combat the vascular aging process.

Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52

When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies.

Antimutagenic effects of chlorophyllin.

It has been suggested that the majority of human cancers are caused by environmental exposures, but little is known about the effective environmental and occupational risks of cancer. It is likely that the process of mutagenesis and the intricate balance between mutagenesis and antimutagenesis are involved in aging, evolution, and other fundamental life processes (15).

A yeast recombination assay to characterize human BRCA1 missense variants of unknown pathological significance.

The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer‐related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C‐Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild‐type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer‐related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants. Hum Mutat 0, 1–11, 2008.

DNA Damage and Repair in Atherosclerosis: Current Insights and Future Perspectives

Atherosclerosis is the leading cause of morbidity and mortality among Western populations. Over the past two decades, considerable evidence has supported a crucial role for DNA damage in the development and progression of atherosclerosis. These findings support the concept that the prolonged exposure to risk factors (e.g., dyslipidemia, smoking and diabetes mellitus) leading to reactive oxygen species are major stimuli for DNA damage within the plaque. Genomic instability at the cellular level can directly affect vascular function, leading to cell cycle arrest, apoptosis and premature vascular senescence. The purpose of this paper is to review current knowledge on the role of DNA damage and DNA repair systems in atherosclerosis, as well as to discuss the cellular response to DNA damage in order to shed light on possible strategies for prevention and treatment.