Biology subjects

Savage, S. A.

Publications and source records attributed to Savage, S. A..

4 recordsLinked to original sources

bioRxiv2020

RTEL1 Influences the Abundance and Localization of TERRA RNA

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the sub-telomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA at the telomeres is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.

molecular biology↗

bioRxiv2020

Alternative splicing is a developmental switch for hTERT expression

High telomerase activity is restricted to the blastocyst stage of embryonic development when telomere length is reset, and is characteristic of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, the pathways involved in telomerase regulation as a function of pluripotency remain unknown. To explore hTERT transcriptional control, we compare genome-wide interactions (4C-seq) and chromatin accessibility (ATAC-seq) between human ESCs and epithelial cells and identify several putative hTERT cis-regulatory elements. CRISPR/Cas9-mediated deletion of candidate elements in ESCs reduces the levels of hTERT mRNA but does not abolish telomerase expression, thus implicating post-transcriptional processes in telomerase regulation. In agreement with this hypothesis, we find an hTERT splice variant lacking exon-2 and prone to degradation, to be enriched in differentiated cells but absent from ESCs. In addition, we show that forced retention of exon-2 prevents telomerase silencing during differentiation. Lastly, we highlight a role for the splicing co-factor SON in hTERT exon-2 inclusion and identify a SON mutation in a Dyskeratosis congenita patient with short telomeres and decreased telomerase activity. Altogether, our data uncover a novel alternative splice switch that is critical for telomerase activity during development.

molecular biology↗

bioRxiv2020

A pedigree-based prediction model identifies carriers of deleterious de novo mutations in families with Li-Fraumeni syndrome

De novo mutations (DNMs) are increasingly recognized as rare disease causal factors. Identifying DNM carriers will allow researchers to study the likely distinct molecular mechanisms of DNMs. We developed Famdenovo to predict DNM status (DNM or familial mutation (FM)) of deleterious autosomal dominant germline mutations for any syndrome. We introduce Famdenovo.TP53 for Li-Fraumeni syndrome (LFS) and analyze 324 LFS family pedigrees from four US cohorts: a validation set of 186 pedigrees and a discovery set of 138 pedigrees. The concordance index for Famdenovo.TP53 prediction was 0.95 (95% CI: [0.92, 0.98]). Forty individuals (95% CI: [30, 50]) were predicted as DNM carriers, increasing the total number from 42 to 82. We compared clinical and biological features of FM versus DNM carriers: 1) cancer and mutation spectra along with parental ages were similarly distributed; 2) ascertainment criteria like early-onset breast cancer (age 20 to 35 years) provides a condition for an unbiased estimate of the DNM rate: 48% (23 DNMs versus 25 FMs); 3) hotspot mutation R248W was not observed in DNMs, although it was as prevalent as hotspot mutation R248Q in FMs. Furthermore, we introduce Famdenovo.BRCA for hereditary breast and ovarian cancer syndrome, and apply it to a small set of family data from the Cancer Genetics Network. In summary, we introduce a novel statistical approach to systematically evaluate deleterious DNMs in inherited cancer syndromes. Our approach may serve as a foundation for future studies evaluating how new deleterious mutations can be established in the germline, such as those in TP53.

genetics↗

bioRxiv2019

Risk of differential cancer types over age in families with Li-Fraumeni syndrome: a validation study using multi-center cohorts

Li-Fraumeni syndrome (LFS) is a rare hereditary cancer syndrome associated with an autosomal dominant mutation inheritance in the TP53 tumor suppressor gene and a wide spectrum of cancer diagnoses. An accurate estimation of the penetrance of different cancer types in LFS is crucial to improve the clinical characterization and management of high-risk individuals of LFS. Our competing risk-based statistical model incorporates the pedigree structure efficiently into the penetrance estimation and corrects for the ascertainment bias. A set of TP53 penetrance for three cancer types (breast, BR/sarcoma, SA/others, OT) involved in LFS is then estimated from 186 pediatric-sarcoma families collected at MD Anderson Cancer Center. The penetrance was then validated on a mixed cohort of clinically ascertained families with cancer (total number of families=668). The age-dependent onset probability distributions of specific cancer types are different. For breast cancer, the TP53 penetrance goes up at an earlier age than the reported BRCA1/2 penetrance. We validated the prediction performance of the penetrance estimates via two independent cohorts combined (BR=85, SA=540, OT=158). We obtained areas under the ROC curves (AUCs) of 0.92 (BR), 0.75 (SA), and 0.81 (OT). Our R package, LFSPRO, provides risk estimates for the diagnosis of breast cancer, sarcoma, other cancers or death before cancer diagnosis in future.\n\nSignificanceCancer-specific penetrance can facilitate clinical characterization of LFS and will contribute to the management of families at high risk of LFS.

genetics↗