Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

Ageing and ovarian stimulation modulate the relative levels of transcript abundance of oocyte DNA repair genes during the germinal vesicle-metaphase II transition in mice.

PURPOSE: Oocyte quality and reproductive outcome are negatively affected by advanced maternal age, ovarian stimulation and method of oocyte maturation during assisted reproduction; however, the mechanisms responsible for these associations are not fully understood. The aim of this study was to compare the effects of ageing, ovarian stimulation and in-vitro maturation on the relative levels of transcript abundance of genes associated with DNA repair during the transition of germinal vesicle (GV) to metaphase II (MII) stages of oocyte development. METHODS: The relative levels of transcript abundance of 90 DNA repair-associated genes was compared in GV-stage and MII-stage oocytes from unstimulated and hormone-stimulated ovaries from young (5-8-week-old) and old (42-45-week-old) C57BL6 mice. Ovarian stimulation was conducted using pregnant mare serum gonadotropin (PMSG) or anti-inhibin serum (AIS). DNA damage response was quantified by immunolabeling of the phosphorylated histone variant H2AX (γH2AX). RESULTS: The relative transcript abundance in DNA repair genes was significantly lower in MII oocytes compared to GV oocytes in young unstimulated and PMSG stimulated but was higher in AIS-stimulated mice. Interestingly, an increase in the relative level of transcript abundance of DNA repair genes was observed in MII oocytes from older mice in unstimulated, PMSG-stimulated and AIS-stimulated mice. Decreased γH2AX levels were found in both GV oocytes (82.9%) and MII oocytes (37.5%) during ageing in both ovarian stimulation types used (PMSG/AIS; p < 0.05). CONCLUSIONS: In conclusion, DNA repair relative levels of transcript abundance are altered by maternal age and the method of ovarian stimulation during the GV-MII transition in oocytes.

The Thermodynamic Properties of Fat10ylated Proteins Are Regulated by the Fat10ylation Site.

Degradation of proteins by the proteasome is crucial in regulating their levels in the cell. Post-translational modifications, such as ubiquitylation and Fat10ylation, trigger proteasomal degradation of the substrate proteins. While ubiquitylation regulates multiple cellular pathways, Fat10ylation functions explicitly in the inflammatory response pathway. At the proteasome, ubiquitin is recycled after being cleaved from the substrate, while Fat10 is degraded simultaneously with its substrate. Although the thermodynamic properties of the substrate are critical for effective proteasomal degradation, they remain poorly understood for the Fat10-proteasome pathway. We studied the thermodynamic properties of the Fat10∼substrate conjugate to uncover mechanistic details of the pathway. First, the mechanical unfolding of Fat10∼substrate was studied by molecular dynamics simulations, which suggested that the unfolding pathway and unfolding energy of the substrate depend on the site of Fat10 modification. We also investigated different pathways for the entry of the Fat10∼substrate into the proteasome core. Our analysis supports a model where the entry of Fat10, followed by the substrate, is the energetically preferred pathway. Further, we studied Fat10's effect on the thermodynamic properties of distinct substrates, considering their size, flexibility, and surface properties. The results uncovered significant entropic destabilization of substrates due to Fat10ylation, particularly in smaller substrates. For larger substrates, multi-monoFat10ylation is necessary to induce destabilization. Our study further reveals that Fat10 modification at negative patches on substrate surfaces is essential for optimal destabilization and subsequent degradation. These findings provide atomistic insights into the degradation mechanisms in the Fat10 proteasome pathway with potential implications for therapeutic interventions.

Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation.

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

Dietary pattern characterisation among subfertile South Asian women and the impact of vitamin B12 intake on frozen embryo transfer outcomes: a cross-sectional study.

STUDY QUESTION: Is there a difference in dietary patterns among subfertile South Asian women undergoing frozen embryo transfer (FET)? SUMMARY ANSWER: Significant regional differences in dietary pattern exist among subfertile South Asian women undergoing FET. WHAT IS KNOWN ALREADY: Preconception consumption of certain food groups or adopting specific dietary patterns, such as the 'Mediterranean diet', and its level of adherence have been shown to enhance the odds of achieving a successful pregnancy in women undergoing ART. However, differences in geographic location, individual preference, cultural beliefs and local availability contribute to such dietary choices. There is also a predisposition to a vitamin B12 deficiency in those of South Asian ethnicity and a predominant pattern of vegetarian food intake. There is a paucity of studies analysing the type of dietary pattern followed by South Asian women, their vitamin B12 levels and the potential impact on ART treatment outcomes. STUDY DESIGN SIZE DURATION: This is a cross-sectional study of 159 South Asian women aged 21-37 years, belonging to the Eastern (n = 75) and Southern (n = 84) regions of India plus Bangladesh, and undergoing a FET cycle at a tertiary level infertility clinic between February 2019 and March 2020. PARTICIPANTS/MATERIALS SETTING METHODS: Women underwent dietary assessment using '24-hour dietary recall' to capture daily nutrient consumption. A 'Food Frequency Questionnaire' listing commonly consumed foods was used to record frequency of intake. The primary outcome was the characterisation of regional dietary patterns in the cohorts using principal component analysis (PCA). Secondary outcomes included association of vitamin B12 intake and serum levels with clinical and ongoing pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: Four components contributing to overall variance in dietary pattern were identified, namely: meat, poultry and seafood; green leafy vegetables and root tubers; fruits, dairy and sugar; nuts and oilseeds. PCA analysis showed a significantly higher consumption of two components in the East-meat, poultry and fish (P < 0.001); green leafy vegetables and root tubers (P < 0.001). All women reported taking preconception oral folic acid supplementation. The dietary intake of vitamin B12 and serum concentration correlated, showing a good validity of measured dietary intake (r = 0.398; P ≤ 0.001). Compared to the Southern region, participants from the East showed a higher daily median intake of vitamin B12 (1.11 versus 0.28 mcg, respectively; P < 0.001) and a higher serum vitamin B12 levels (441 versus 239 pg/ml, respectively; P < 0.001). Ongoing pregnancy showed no association with dietary vitamin B12 intake (relative risk 0.90; 95% CI, 0.68 to 1.19) or serum vitamin B12 levels (relative risk 0.99; 95% CI, 0.73 to 1.33) after adjustments for female age, body mass index (BMI) and geographic differences. Women belonging to different quartiles of serum vitamin B12 concentration had a similar likelihood of ongoing pregnancy. LIMITATIONS REASONS FOR CAUTION: Self-reported dietary assessment is prone to measurement errors owing to its subjective nature and recall bias. The study was not adequately powered to detect the impact of geographic differences in vitamin B12 intake and serum levels on FET treatment outcomes, the second objective. We adjusted for potential confounders, such as female age and BMI, but it is possible that residual confounders, such as physical activity, stress and use of dietary supplements, may have influenced the results. Extrapolation of the study findings to women undergoing ART in other populations should be made with caution. WIDER IMPLICATIONS OF THE FINDINGS: Our study findings suggest important differences in local dietary patterns within the South Asian region. Hence a personalised approach to dietary assessment and intervention when undergoing ART based on population dynamics is warranted. The geographic differences in the vitamin B12 intake or serum levels did not have an impact on the FET outcomes. There is also a need to further investigate the impact of such dietary differences on ART treatment outcomes in a large study population. STUDY FUNDING/COMPETING INTERESTS: No grant from funding agencies in the public, commercial, or not-for-profit sectors was obtained. The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.