Characterization of TP53 mutations in Pap test DNA of women with and without serous ovarian carcinoma.

OBJECTIVE: Pap tests hold promise as a molecular diagnostic for serous ovarian cancer, but previous studies reported limited sensitivity. Furthermore, the presence of somatic mutations in normal tissue is increasingly recognized as a challenge to the specificity of mutation-based cancer diagnostics. We applied an ultra-deep sequencing method with the goal of improving sensitivity and characterizing the landscape of low-frequency somatic TP53 mutations in Pap tests. METHODS: We used CRISPR-DS to deeply sequence (mean Duplex depth ~3000×) the TP53 gene in 30 Pap tests from 21 women without cancer and 9 women with serous ovarian carcinoma with known TP53 driver mutations. Mutations were annotated and compared to those in the TP53 cancer database. RESULTS: The tumor-derived mutation was identified in 3 of 8 Pap tests from women with ovarian cancer and intact tubes. In addition, 221 low-frequency (≲0.001) exonic TP53 mutations were identified in Pap tests from women with ovarian cancer (94 mutations) and without ovarian cancer (127 mutations). Many of these mutations resembled TP53 mutations found in cancer: they impaired protein activity, were predicted to be pathogenic, and clustered in exons 5 to 8 and hotspot codons. Cancer-like mutations were identified in all women but at higher frequency in women with ovarian cancer. CONCLUSIONS: Pap tests have low sensitivity for ovarian cancer detection and carry abundant low-frequency TP53 mutations. These mutations are more frequently pathogenic in women with ovarian cancer. Determining whether low-frequency TP53 mutations in normal gynecologic tissues are associated with an increased cancer risk warrants further study.

Comprehensive identification and analysis of human accelerated regulatory DNA.

It has long been hypothesized that changes in gene regulation have played an important role in human evolution, but regulatory DNA has been much more difficult to study compared with protein-coding regions. Recent large-scale studies have created genome-scale catalogs of DNase I hypersensitive sites (DHSs), which demark potentially functional regulatory DNA. To better define regulatory DNA that has been subject to human-specific adaptive evolution, we performed comprehensive evolutionary and population genetics analyses on over 18 million DHSs discovered in 130 cell types. We identified 524 DHSs that are conserved in nonhuman primates but accelerated in the human lineage (haDHS), and estimate that 70% of substitutions in haDHSs are attributable to positive selection. Through extensive computational and experimental analyses, we demonstrate that haDHSs are often active in brain or neuronal cell types; play an important role in regulating the expression of developmentally important genes, including many transcription factors such as SOX6, POU3F2, and HOX genes; and identify striking examples of adaptive regulatory evolution that may have contributed to human-specific phenotypes. More generally, our results reveal new insights into conserved and adaptive regulatory DNA in humans and refine the set of genomic substrates that distinguish humans from their closest living primate relatives.