Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

Mechanism of RBBP8-mediated homologous recombination repair in gastric cancer synthetic lethal.

Background: It is of great clinical significance to further explore new strategies and potential combined therapeutic targets for gastric cancer. This study aimed to investigate the synthetic lethal effect of RBBP8 molecular intervention combined with a poly ADP ribose polymerase (PARP) inhibitor in non-BRCA mutant gastric cancer and clarify the mechanism by which RBBP8 regulates homologous recombination repair. Methods: The role of RBBP8 in DNA damage repair was observed using bioinformatic analysis, western blot analysis, and immunofluorescence. The synthetic lethal effect was verified using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)and flow cytometry apoptosis experiments. Results: Among the patients with gastric cancer treated with chemotherapy, the prognosis of patients with high RBBP8 expression levels was worse (homologous recombination [HR] = 1.54, p = 0.028). RBBP8 knockdown induced DNA damage and had a synergistic effect with PARP inhibitor treatment on cell viability inhibition and cell apoptosis in AGS (generic code for human gastric adenocarcinoma cells) (t = 11.154, p < 0.001) and N87 (t = 6.362, p < 0.001) cells. RBBP8 knockdown inhibited RAD51 activation and DNA terminal excision in homologous recombination repair. Conclusion: RBBP8 is involved in homologous recombination repair, and molecular intervention into RBBP8 could achieve a synthetic lethal effect with PARP inhibitor treatment in gastric cancer cells.

A novel RBBP8(p.E281*) germline mutation is a predisposing mutation in familial hereditary cancer syndrome.

Screening tumor susceptibility genes helps in identifying powerful biomarkers for hereditary cancer monitoring, prevention, and diagnosis, providing opportunities for understanding potential molecular mechanisms and biomarkers for the precise treatment of hereditary cancer syndromes. Whole-exome sequencing of blood and bioinformatics analysis uncovered a novel RBBP8(p.E281*) germline mutation in a family with hereditary cancer syndrome, which was verified by Sanger sequencing. Cell proliferation, colony formation, cell migration, and in vivo tumorigenesis were investigated by CCK8, colony formation, Transwell, and in vivo xenograft assays. Protein localization and interaction were detected by immunofluorescence, nuclear and cytoplasmic protein extraction kits, and Co-IP. A new heterozygous germline mutation of the RBBP8(p.E281*) gene was found to be associated with familial hereditary cancer syndrome. RBBP8-WT was mainly detected in the nucleus and interacts with BRCA1. In contrast, RBBP8(p.E281*) is mainly located in the cytoplasm, with no interaction with BRCA1. RBBP8(p.E281*) variant plays an oncogenic role in the cytoplasm in addition to its loss of function in the nucleus, which promotes breast cancer proliferation, in vivo tumorigenesis, and migration. Compared with the control group, RBBP8(p.E281*) showed elevated cell death in response to cisplatin and olaparib treatment. A novel RBBP8(p.E281*) germline mutation was identified from familial hereditary cancer syndrome. RBBP8(p.E281*) is not able to enter the nucleus or interact with BRCA1 through the lost binding motif, and RBBP8(p.E281*) variant appears to promote tumorigenesis in the cytoplasm in addition to its loss of function in the nucleus. RBBP8(p.E281*) variant may promote tumor susceptibility and serve as a precision medicine biomarker in familial hereditary cancer syndrome. KEY MESSAGES: RBBP8(p.E281*) is a susceptibility gene in this familial hereditary cancer syndrome RBBP8(p.E281*) lost its ability to enter the nucleus and the BRCA1 binding motif A novel RBBP8(p.E281*) germline mutation promotes breast cancer tumorigenesis Patients with RBBP8(p.E281*) germline mutation may benefit from Olaparib, Cisplatin.

Prognostic Significance of mRNA Expression RBBP8 or Its Methylation in Gliomas.

Retinoblastoma-binding protein 8 (RBBP8) affects the prognosis of patients with malignancies through various mechanisms. However, its function in gliomas is unknown. Our study explored the effects of RBBP8 on the prognosis of glioma patients, as well as its regulatory role in the glioma immune microenvironment. We used various bioinformatics methods to analyze the transcriptional profiles and methylation data of RBBP8 in gliomas from multiple databases. Our results showed that the mRNA and protein expression of RBBP8 in gliomas was higher than that in normal tissues and positively correlated with malignant clinical features such as age and WHO grade. A Kaplan-Meier analysis showed that patients with high RBBP8 expression had a poor prognosis. Cox regression demonstrated that RBBP8 was an independent risk indicator and had good diagnostic value for the poor prognosis of glioma. Importantly, RBBP8 was positively correlated with many well-known immune checkpoints (e.g., CTLA4 and PDL-1). Finally, a gene set enrichment analysis revealed that RBBP8 was remarkably enriched in cancer-related pathways such as cell cycle, DNA replication and so on. In conclusion, this study is the first to elaborate on the value of RBBP8 in the pathological process of glioma for anti-tumor immunotherapy. In addition, the expression of RBBP8 and its methylation site, cg05513509, may provide potential targets for glioma therapy.

Differential expression of DNA damage repair genes after chemoradiotherapy and inhibition rate in different bladder cancer cells.

Background: To investigate the potential mechanisms of chemoradiotherapy resistance in patients with bladder cancer. Methods: We assessed three bladder cancer cell lines (5637, J82, and TCCSUP) for their sensitivity to chemoradiotherapy. Reverse transcription quantitative PCR (RT-qPCR) was used to detect the expression of specific genes after chemoradiotherapy or combined with olaparib. The Genome Cancer Atlas (TGCA) database was used to analyze possible radioresistance-related genes and the relationship between their expression and bladder cancer survival and prognostic indicators. Results: The 5637 cell line showed the most significant sensitivity to chemoradiotherapy. The expression levels of DNA damage repair genes in 5637 cells did not significantly change after chemoradiotherapy. In contrast, the expression levels of BRCA1 and RBBP8 genes significantly increased in J82 and TCCSUP cells after chemoradiotherapy. After combination with olaparib, which is a poly (ADP-ribose) polymerase inhibitor that initiates DNA repair, 5637 cells were significantly inhibited by chemoradiotherapy. However, chemoradiotherapy inhibition on J82 cells was weakened when combined with olaparib. A high reactive expression of BRCA1 and RBBP8 after combination with olaparib suggested that olaparib was ineffective because it did not induce synthetic lethality in which inhibiting PARP by olaparib coincided with suppression of BRCA1/2 expression result in cancer cell death. Conclusions: The expression levels of RBBP8 and BRCA1 genes were associated with sensitivity to radiotherapy and chemotherapy in bladder cancer, and an increase in reactive expression after treatment led to worse sensitivity. Therefore, the reactive expression levels of BRCA1 and RBBP8 after chemoradiotherapy may be useful in evaluating the efficacy of olaparib combination therapy.

The mechanism of radiotherapy for lung adenocarcinoma in promoting protein SIRT6-mediated deacetylation of RBBP8 to enhance the sensitivity of targeted therapy.

BACKGROUND: Lung cancer has the fastest increase in morbidity and mortality, and is one of the most threatening malignant tumors to human health and life. Both radiotherapy and targeted therapy are typical treatments after lung cancer surgery. Radiotherapy is a means of locally killing cancer lesions, and it plays an important role in the entire management of lung cancer. Gefitinib is one of the most commonly used targeted therapy drugs in the treatment of lung cancer. The purpose of this project is to explore the mechanism by which deacetylation of RBBP8 mediated by radiotherapy-promoting protein SIRT6 in lung adenocarcinoma enhances the sensitivity of targeted therapy. METHODS: In both the cell experiments and the animal experiments, the samples were divided into five groups: Model group, RT group, CT group, RT+CT group, and RT+CT+inhibitor group. The CCK8 method was used to detect the viability of each group of cells. The flow cytometry experiment was used to analyze the apoptotic characteristics of each group of cells. The scratch test was used to detect the migration ability of each group of cells. Transwell invasion test was used to determine the invasion ability of each group of cells. The lung tumor tissues of each group of mice were collected to analyze the tumor size, volume, and metastasis characteristics. The TUNEL experiment was used to detect the apoptosis characteristics of the cells in the lung cancer tissues of each group mice. Immunohistochemistry experiments were used to analyze the distribution and relative expression characteristics of protein SIRT6 in mouse lung cancer tissues. The colorimetric experiments were used to detect the activity of Caspase 3 and Caspase 8 in each group. Western blot method was used to detect the expression of SIRT6, RBBP8, and MYC in each group. RESULTS: In each experiment, the results of the experiment have mutually proven consistency, and there is no contradiction. In addition to the Model group, the other 4 groups used different treatment methods. The better the curative effect, the lower the cell viability of cancer cells and the higher the apoptotic ratio. This is reflected in the CCK8 test, flow cytometry analysis, cell scratch test, Transwell cell migration test, and TUNEL detection. At the same time, colorimetric detection and Western blot analysis also analyzed the levels of SIRT6, RBBP8 and other cancer-related proteins in each group at the molecular level, implying the importance of SIRT6 protein in the treatment process. CONCLUSION: Our project has proved that radiotherapy can promote the protein SIRT6 to deacetylate RBBP8 proteins, and ultimately enhance targeted therapy drug sensitivity.

Epigenetic inactivation of DNA repair genes as promising prognostic and predictive biomarkers in urothelial bladder carcinoma patients.

We sought to examine epigenetic inactivation of DNA damage repair (DDR) genes as prognostic and predictive biomarkers for urothelial bladder cancer (UBC) as there are currently no reliable prognostic biomarkers that identify UBC patients who would benefit from chemotherapy. Genome-wide DNA methylome using the cancer genome atlas-bladder cancer (TCGA-BLCA) datasets (primary tumors = 374 and normal tissues = 37) was performed for 154 DDR genes. The most two significant differentially methylated genes, Retinoblastoma binding protein 8 (RBBP8) and MutS homologue 4 (MSH4), between primary tumors and normal tissues of TCGA-BLCA were validated by methylation-specific PCR (MSP) in UBC (n = 70) compared to normal tissues (n = 30). RBBP8 and MSH4 expression was measured using qRT-PCR. We developed a predictive model for therapeutic response based on the RBBP8- and MSH4-methylation along with patients' clinical features. Then, we assessed the prognostic significance of RBBP8 and MSH4. RBBP8- and MSH4 methylation and corresponding gene downregulation significantly associated with muscle-invasive phenotype, prolonged progression-free survival (PFS) and increased susceptibility to cisplatin chemotherapy in UBC. Promoter methylation of RBBP8 and MSH4 was positively correlated with each other and with their corresponding gene repression. The best machine-learning classification model predicted UBC patients' response to cisplatin-based chemotherapy with an accuracy of 90.05 ± 4.5%. Epigenetic inactivation of RBBP8 and MSH4 in UBC could sensitize patients to DNA-damaging agents. A predictive machine-learning modeling approach based on the clinical features along with RBBP8- and MSH4-methylation might be a promising tool for stratification of UBC responders from nonresponders to chemotherapy.

Excessive DNA damage mediates ECM degradation via the RBBP8/NOTCH1 pathway in sporadic aortic dissection.

Stanford type A aortic dissection (TA-AD) is a life-threatening disease. Most cases of aortic dissection (AD) are sporadic rather than inherited. Unlike that of inherited AD, the pathogenesis of sporadic AD is still unclear. In the current study, we aimed to explore the pathogenesis of sporadic AD through transcriptome sequencing data analyses. We downloaded sporadic TA-AD transcriptome profiles from Gene Expression Omnibus (GEO) and found response to DNA damage stimulus was activated in AD. Furthermore, by conducting mouse AD tissue single cell RNA sequencing and immunostaining, we found that DNA damage mainly occurred in smooth muscle cells (SMCs) and fibroblasts. Next, we examined the repair patterns in response to DNA damage and found the linker molecules RBBP8/NOTCH1 between DNA damage/repair and extracellular matrix (ECM) organization through protein-protein interaction analysis. Thus, we proposed that DNA damage could contribute to AD by regulating ECM changes. To explore the underlying mechanism, we knocked down the DNA repair-related gene RBBP8 in aortic SMCs, which could exacerbate DNA damage, and observed decreased expression level of NOTCH1. Inhibition of NOTCH1 with crenigacestat in vivo accelerated ß-aminopropionitrile-induced formation of AD and increased mortality. Meanwhile, phenotype switching of SMCs was induced by Notch1 knockdown or inhibition; this switching occurred via a pathway involving downregulation of contractile marker gene expression and upregulation of MMP2 expression, which might aggravate ECM degradation. In conclusion, excessive DNA damage is a characteristic pathological change of sporadic aortic dissection, which might contribute to ECM changes and AD development via action on the NOTCH1 pathway.

Human CtIP: A 'double agent' in DNA repair and tumorigenesis.

Human CtIP was originally identified as an interactor of the retinoblastoma protein and BRCA1, two bona fide tumour suppressors frequently mutated in cancer. CtIP is renowned for its role in the resection of DNA double-strand breaks (DSBs) during homologous recombination, a largely error-free DNA repair pathway crucial in maintaining genome integrity. However, CtIP-dependent DNA end resection is equally accountable for alternative end-joining, a mutagenic DSB repair mechanism implicated in oncogenic chromosomal translocations. In addition, CtIP contributes to transcriptional regulation of G1/S transition, DNA damage checkpoint signalling, and replication fork protection pathways. In this review, we present a perspective on the current state of knowledge regarding the tumour-suppressive and oncogenic properties of CtIP and provide an overview of their relevance for cancer development, progression, and therapy.

Promoter methylation of DNA damage repair (DDR) genes in human tumor entities: RBBP8/CtIP is almost exclusively methylated in bladder cancer.

Background: Genome-wide studies identified pan-cancer genes and shared biological networks affected by epigenetic dysregulation among diverse tumor entities. Here, we systematically screened for hypermethylation of DNA damage repair (DDR) genes in a comprehensive candidate-approach and exemplarily identify and validate candidate DDR genes as targets of epigenetic inactivation unique to bladder cancer (BLCA), which may serve as non-invasive biomarkers. Methods: Genome-wide DNA methylation datasets (2755 CpG probes of n = 7819 tumor and n = 659 normal samples) of the TCGA network covering 32 tumor entities were analyzed in silico for 177 DDR genes. Genes of interest were defined as differentially methylated between normal and cancerous tissues proximal to transcription start sites. The lead candidate gene was validated by methylation-specific PCR (MSP) and/or bisulfite-pyrosequencing in different human cell lines (n = 36), in primary BLCA tissues (n = 43), and in voided urine samples (n = 74) of BLCA patients. Urines from healthy donors and patients with urological benign and malignant diseases were included as controls (n = 78). mRNA expression was determined using qRT-PCR in vitro before (n = 5) and after decitabine treatment (n = 2). Protein expression was assessed by immunohistochemistry (n = 42). R 3.2.0. was used for statistical data acquisition and SPSS 21.0 for statistical analysis. Results: Overall, 39 DDR genes were hypermethylated in human cancers. Most exclusively and frequently methylated (37%) in primary BLCA was RBBP8, encoding endonuclease CtIP. RBBP8 hypermethylation predicted longer overall survival (OS) and was found in 2/4 bladder cancer cell lines but not in any of 33 cancer cell lines from entities with another origin like prostate. RBBP8 methylation was inversely correlated with RBBP8 mRNA and nuclear protein expression while RBBP8 was re-expressed after in vitro demethylation. RBBP8 methylation was associated with histological grade in primary BLCA and urine samples. RBBP8 methylation was detectable in urine samples of bladder cancer patients achieving a sensitivity of 52%, at 91% specificity. Conclusions: RBBP8 was identified as almost exclusively hypermethylated in BLCA. RBBP8/CtIP has a proven role in homologous recombination-mediated DNA double-strand break repair known to sensitize cancer cells for PARP1 inhibitors. Since RBBP8 methylation was detectable in urines, it may be a complementary marker of high specificity in urine for BLCA detection.

RBBP8 syndrome with microcephaly, intellectual disability, short stature and brachydactyly.

Primary microcephaly is clinically variable and genetically heterogeneous. Four phenotypically distinct types of autosomal recessive microcephaly syndromes are due to different RBBP8 mutations. We report on a consanguineous Pakistani family with homozygous RBBP8 mutation c.1808_1809delTA (p.Ile603Lysfs*7) manifesting microcephaly and a distinct combination of skeletal, limb and ectodermal defects, mild intellectual disability, minor facial anomalies, anonychia, disproportionate short stature and brachydactyly, and additionally talipes in one patient.

Autophagy-independent senescence and genome instability driven by targeted telomere dysfunction.

Telomere dysfunction plays a complex role in tumorigenesis. While dysfunctional telomeres can block the proliferation of incipient cancer clones by inducing replicative senescence, fusion of dysfunctional telomeres can drive genome instability and oncogenic genomic rearrangements. Therefore, it is important to define the regulatory pathways that guide these opposing effects. Recent work has shown that the autophagy pathway regulates both senescence and genome instability in various contexts. Here, we apply models of acute telomere dysfunction to determine whether autophagy modulates the resulting genome instability and senescence responses. While telomere dysfunction rapidly induces autophagic flux in human fibroblast cell lines, inhibition of the autophagy pathway does not have a significant impact upon the transition to senescence, in contrast to what has previously been reported for oncogene-induced senescence. Our results suggest that this difference may be explained by disparities in the development of the senescence-associated secretory phenotype. We also show that chromosome fusions induced by telomere dysfunction are comparable in autophagy-proficient and autophagy-deficient cells. Altogether, our results highlight the complexity of the senescence-autophagy interface and indicate that autophagy induction is unlikely to play a significant role in telomere dysfunction-driven senescence and chromosome fusions.

High-throughput DNA Sequencing Identifies Novel CtIP (RBBP8) Variants in Muscle-invasive Bladder Cancer Patients.

BACKGROUND: Germline mutations in DNA damage signalling and repair genes predispose individuals to cancer. Rare germline variants may also increase cancer risk and be predictive of outcomes following cancer treatments, but require high-throughput sequencing (HTS) for detection in large cohorts. OBJECTIVE: To use a dual indexing system on a HTS platform to detect novel variants in CtIP (RBBP8) which may be associated with clinical outcomes following radiotherapy treatment for bladder cancer. METHODS: All exons and flanking introns of CtIP were amplified from germline DNA from bladder cancer patients using seven primer pairs by automated long-range PCR. Amplicons were pooled, fragmented and ligated to adaptor sequences. One of 96 tag sequences was introduced at each end by PCR. Sequencing was performed on a single flow cell of an Illumina MiSeq. Reads were mapped by Stampy and variants called by Platypus. For phasing experiments, target regions were amplified and cloned for Sanger sequencing. RESULTS: Of 201 samples, 160 were successfully amplified. Eleven CtIP variants were called, within the exons and 15 bp adjacent intronic DNA, including eight known variants from the 1000 Genomes project, plus three previously unreported variants now confirmed by Sanger sequencing. In two individuals, phasing experiments showed two variants of interest to be on separate alleles, likely to result in stronger impairment of gene function. CONCLUSIONS: We have demonstrated proof of principle for dual indexing on 160 samples on one MiSeq flow cell sequencing surface, and show that for the CtIP gene multiplexing of up to 720 samples would provide sufficient coverage to achieve >98% detection power for rare germline variation, reducing HTS costs substantially.

A complex microcephaly syndrome in a Pakistani family associated with a novel missense mutation in RBBP8 and a heterozygous deletion in NRXN1.

We report on a consanguineous Pakistani family with a severe congenital microcephaly syndrome resembling the Seckel syndrome and Jawad syndrome. The affected individuals in this family were born to consanguineous parents of whom the mother presented with mild intellectual disability (ID), epilepsy and diabetes mellitus. The two living affected brothers presented with microcephaly, white matter disease of the brain, hyponychia, dysmorphic facial features with synophrys, epilepsy, diabetes mellitus and ID. Genotyping with a 250K SNP array in both affected brothers revealed an 18 MB homozygous region on chromosome 18 p11.21-q12.1 encompassing the SCKL2 locus of the Seckel and Jawad syndromes. Sequencing of the RBBP8 gene, underlying the Seckel and Jawad syndromes, identified the novel mutation c.919A>G, p.Arg307Gly, segregating in a recessive manner in the family. In addition, in the two affected brothers and their mother we have also found a heterozygous 607kb deletion, encompassing exons 13-19 of NRXN1. Bidirectional sequencing of the coding exons of NRXN1 did not reveal any other mutation on the other allele. It thus appears that the phenotype of the mildly affected mother can be explained by the NRXN1 deletion, whereas the more severe and complex microcephalic phenotype of the two affected brothers is due to the simultaneous deletion in NRXN1 and the homozygous missense mutation affecting RBBP8.

Prognostic value of CtIP/RBBP8 expression in breast cancer.

CtIP/RBBP8 is a multifunctional protein involved in transcription, DNA replication, DNA repair by homologous recombination and the G1 and G2 checkpoints. Its multiple roles are controlled by its interaction with several specific factors, including the tumor suppressor proteins BRCA1 and retinoblastoma. Both its functions and interactors point to a putative oncogenic potential of CtIP/RBBP8 loss. However, CtIP/RBBP8 relevance in breast tumor appearance, development, and prognosis has yet to be established. We performed a retrospective analysis of CtIP/RBBP8 and RB1 levels by immunohistochemistry using 384 paraffin-embedded breast cancer biopsies obtained during tumor removal surgery. We have observed that low or no expression of CtIP/RBBP8 correlates with high-grade breast cancer and with nodal metastasis. Reduction on CtIP/RBBP8 is most common in hormone receptor (HR)-negative, HER2-positive, and basal-like tumors. We observed lower levels of RB1 on those tumors with reduced CtIP/RBBP8 levels. On luminal tumors, decreased but not absence of CtIP/RBBP8 levels correlate with increased disease-free survival when treated with a combination of hormone, radio, and chemo therapies.