The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged ...forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased gamma-H2AX foci. Because the increased gamma-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess gamma-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we ...evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.
Homologous recombination (HR) is a genetic mechanism in somatic cells that repairs DNA double-strand breaks and restores productive DNA synthesis following disruption of replication forks. Although ...HR is indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes. HR-associated genomic instabilities arise in three human genetic disorders, Bloom syndrome (BS), Werner syndrome (WS), and Rothmund–Thomson syndrome (RTS), which are caused by defects in three individual proteins of the RecQ family of helicases, BLM, WRN, and RECQL4, respectively. Cells derived from persons with these syndromes display varying types of genomic instability as evidenced by the presence of different kinds of chromosomal abnormalities and different sensitivities to DNA damaging agents. Persons with these syndromes exhibit a variety of developmental defects and are predisposed to a wide range of cancers. WS and RTS are further characterized by premature aging. Recent research has shown many connections between all three proteins and the regulation of excess HR. Here, we illustrate the elaborate networks of BLM, WRN, and RECQL4 in regulating HR, and the potential mechanistic linkages to cancer and aging.
Polymerase stalling results in uncoupling of DNA polymerase and the replicative helicase, which generates single-stranded DNA (ssDNA). After stalling, RAD51 accumulates at stalled replication forks ...to stabilize the fork and to repair by homologous recombination (HR) double-strand breaks (DSBs) that accumulate there. We showed recently that SUMO modification of the BLM helicase is required in order for RAD51 to accumulate at stalled forks. In order to investigate how BLM SUMOylation controls RAD51 accumulation, we characterized the function of HR proteins and ssDNA-binding protein RPA in cells that stably expressed either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM). In HU-treated SM-BLM cells, mediators BRCA2 and RAD52, which normally substitute RAD51 for RPA on ssDNA, failed to accumulate normally at stalled forks; instead, excess RPA accumulated. SM-BLM cells also exhibited higher levels of HU-induced chromatin-bound RPA than BLM+ cells did. The excess RPA did not result from excessive intrinsic BLM helicase activity, because in vitro SUMOylated BLM unwound similar amounts of replication-fork substrate as unSUMOylated BLM. Nor did BLM SUMOylation inhibit binding of RPA to BLM in vitro; however, in immunoprecipitation experiments, more BLM-RPA complex formed in HU-treated SM-BLM cells, indicating that BLM SUMOylation controls the amount of BLM-RPA complex normally formed at stalled forks. Together, these results showed that BLM SUMOylation regulates the amount of ssDNA that accumulates during polymerase stalling. We conclude that BLM SUMOylation functions as a licensing mechanism that permits and regulates HR at damaged replication forks.
Background
Clinicians ordering multi-gene next-generation sequencing panels for hereditary breast cancer risk have a variety of test panel options. Many panels include lesser known breast cancer ...genes or genes associated with other cancers. The authors hypothesized that using broader gene panels increases the identification of clinically significant findings, some relevant and others incidental to the testing indication. They examined clinician ordering patterns and compared the yield of pathogenic or likely pathogenic (P/LP) variants in non-
BRCA
genes of female breast cancer patients.
Methods
This study analyzed de-identified personal and family histories in 1085 breast cancer cases with P/LP multi-gene panel findings in non-
BRCA
cancer genes and sorted them into three groups by the panel used for testing: group A (breast cancer genes only), group B (commonly assessed cancers: breast, gynecologic, and gastrointestinal), and group C (a more expanded set of tumors). The frequency of P/LP variants in genes with established management guidelines was compared and evaluated for consistency with personal and family histories.
Results
This study identified 1131 P/LP variants and compared variants in clinically actionable genes for breast and non-breast cancers. Overall, 91.5% of these variants were in genes with management guidelines. Nearly 12% were unrelated to personal or family history.
Conclusion
Broader panels were used for 85.6% of our cohort (groups B and C). Although pathogenic variants in non-
BRCA
genes are reportedly rare, the study found that most were in clinically actionable genes. Expanded panel testing improved the identification of hereditary cancer risk. Small, breast-limited panels may miss clinically relevant findings in genes associated with other heritable cancers.
Guidelines for variant interpretation include criteria for incorporating phenotype evidence, but this evidence is inconsistently applied. Systematic approaches to using phenotype evidence are needed. ...We developed a method for curating disease phenotypes as highly or moderately predictive of variant pathogenicity based on the frequency of their association with disease‐causing variants. To evaluate this method's accuracy, we retrospectively reviewed variants with clinical classifications that had evolved from uncertain to definitive in genes associated with curated predictive phenotypes. To demonstrate the clinical validity and utility of this approach, we compared variant classifications determined with and without predictive phenotype evidence. The curation method was accurate for 93%–98% of eligible variants. Among variants interpreted using highly predictive phenotype evidence, the percentage classified as pathogenic or likely pathogenic was 80%, compared with 46%–54% had the evidence not been used. Positive results among individuals harboring variants with highly predictive phenotype‐guided interpretations would have been missed in 25%–37% of diagnostic tests and 39%–50% of carrier screens had other approaches to phenotype evidence been used. In summary, predictive phenotype evidence associated with specific curated genes can be systematically incorporated into variant interpretation to reduce uncertainty and increase the clinical utility of genetic testing.
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