Large chromosomal deletions and impaired homologous recombination repairing in HEK293T cells exposed to polychlorinated biphenyl 153

Background Polychlorinated biphenyls (PCBs) are persistent pollutants with carcinogenesis and mutagenesis effects which have been closely associated with PCBs-induced DNA damage. However, the detailed DNA damage events and corresponding pathway alterations under PCBs poisoning is still not well understood. Methods Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) were used to explore genome wide variations and related pathway changes in HEK293T cells that challenged by 15 µM PCB153 for 96 h in vitro. Double strand breaks (DSBs) were measured by 53BP1 foci detection, altered pathways were confirmed by quantitative real-time PCR (qPCR). Results The results indicated that abundant copy number variations (CNVs), including four duplications and 30 deletions, occurred in PCB153-exposed HEK293T cells. Multiple large fragment deletions (>1 Mb) involving up to 245 Mb regions on many chromosomes. Missense mutations were found in six tumor susceptibility genes, two of which are key members participating in homologous recombination (HR) repair response, BRCA1 and BRCA2. RNA-seq data showed that PCB153 poisoning apparently suppressedHR repairing genes. Besides, 15 µM PCB153 exposure significantly increased 53BP1 foci formation and effectively reduced BRCA1, RAD51B and RAD51C expression, indicating an elevated DSBs and impaired HR repairing. Conclusion This study firstly reported multiple large chromosomal deletions and impaired HR repairing in PCB153-exposed HEK293T cells, which provided a new insight into the understanding of early response and the mechanism underlying PCB153 genotoxicity. The chromosomal instabilities might be related to the impaired HR repairing that induced by PCB153; however, further investigations, especially on actual toxic effects of human body, are needed to confirm such speculation.

Engineering large chromosomal deletions by CRISPR-Cas9

Arm-level chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of arm-level deletions are limited. Here we present an effective technique to engineer arm-level deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induce arm-level deletions on chromosome 11q (65 MB) and chromosome 6q (53 MB) in neuroblastoma cell lines. Such isogenic models enable further research on the role of arm-level deletions in tumor development and growth and their possible therapeutic potential.

Severe early onset obesity and hypopituitarism in a child with a novel SIM1 gene mutation

Summary Single-minded homolog 1 (SIM1) is a transcription factor that plays a role in the development of both the hypothalamus and pituitary. SIM1 gene mutations are known to cause obesity in humans, and chromosomal deletions encompassing SIM1 and other genes necessary for pituitary development can cause a Prader–Willi-like syndrome with obesity and hypopituitarism. There have been no reported cases of hypopituitarism linked to a single SIM1 mutation. A 21-month-old male presented to endocrinology clinic with excessive weight gain and severe obesity. History was also notable for excessive drinking and urination. Endocrine workup revealed central hypothyroidism, partial diabetes insipidus, and central adrenal insufficiency. Genetic evaluation revealed a novel mutation in the SIM1 gene. No other genetic abnormalities to account for his obesity and hypopituitarism were identified. While we cannot definitively state this mutation is pathogenic, it is notable that SIM1 plays a role in the development of all three of the patient’s affected hormone axes. He is now 6 years old and remains on treatment for his pituitary hormone deficiencies and continues to exhibit excessive weight gain despite lifestyle interventions. Learning points: Mutations in SIM1 are a well-recognized cause of monogenic human obesity, and there have been case reports of Prader–Willi-like syndrome and hypopituitarism in patients with chromosomal deletions that contain the SIM1 gene. SIM1 is expressed during the development of the hypothalamus, specifically in neuroendocrine lineages that give rise to the hormones oxytocin, arginine vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin. Pituitary testing should be considered in patients with severe obesity and a known genetic abnormality affecting the SIM1 gene, particularly in the pediatric population.

DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and a displaced DNA strand. The regulatory factors required to process this fundamental genetic structure near double-strand DNA breaks (DSBs) are not well understood. We previously reported that cellular depletion of the ATP-dependent DEAD box RNA helicase DDX5 increases R-loops genome-wide causing genomic instability. In this study, we define a pivotal role for DDX5 in clearing R-loops at or near DSBs enabling proper DNA repair to avoid aberrations such as chromosomal deletions. Remarkably, using the non-homologous end joining reporter gene (EJ5-GFP), we show that DDX5-deficient U2OS cells exhibited asymmetric end deletions on the side of the DSBs where there is overlap with a transcribed gene. Cross-linking and immunoprecipitation showed that DDX5 bound RNA transcripts near DSBs and required its helicase domain and the presence of DDX5 near DSBs was also shown by chromatin immunoprecipitation. DDX5 was excluded from DSBs in a transcription- and ATM activation-dependent manner. Using DNA/RNA immunoprecipitation, we show DDX5-deficient cells had increased R-loops near DSBs. Finally, DDX5 deficiency led to delayed exonuclease 1 and replication protein A recruitment to laser irradiation-induced DNA damage sites, resulting in homologous recombination repair defects. Our findings define a role for DDX5 in facilitating the clearance of RNA transcripts overlapping DSBs to ensure proper DNA repair.