Prevalence of germline variants in consensus moderate-to-high-risk predisposition genes to hereditary breast and ovarian cancer in BRCA1/2-negative Brazilian patients.

PURPOSE: This study aimed to identify and classify genetic variants in consensus moderate-to-high-risk predisposition genes associated with Hereditary Breast and Ovarian Cancer Syndrome (HBOC), in BRCA1/2-negative patients from Brazil. METHODS: The study comprised 126 index patients who met NCCN clinical criteria and tested negative for all coding exons and intronic flanking regions of BRCA1/2 genes. Multiplex PCR-based assays were designed to cover the complete coding regions and flanking splicing sites of six genes implicated in HBOC. Sequencing was performed on HiSeq2500 Genome Analyzer. RESULTS: Overall, we identified 488 unique variants. We identified five patients (3.97%) that harbored pathogenic or likely pathogenic variants in four genes: ATM (1), CHEK2 (2), PALB2 (1), and TP53 (1). One hundred and thirty variants were classified as variants of uncertain significance (VUS), 10 of which were predicted to disrupt mRNA splicing (seven non-coding variants and three coding variants), while other six missense VUS were classified as probably damaging by prediction algorithms. CONCLUSION: A detailed mutational profile of non-BRCA genes is still being described in Brazil. In this study, we contributed to filling this gap, by providing important data on the diversity of genetic variants in a Brazilian high-risk patient cohort. ATM, CHEK2, PALB2 and TP53 are well established as HBOC predisposition genes, and the identification of deleterious variants in such actionable genes contributes to clinical management of probands and relatives.

Familial history and prevalence of BRCA1, BRCA2 and TP53 pathogenic variants in HBOC Brazilian patients from a public healthcare service.

Several studies have demonstrated the cost-effectiveness of genetic testing for surveillance and treatment of carriers of germline pathogenic variants associated with hereditary breast/ovarian cancer syndrome (HBOC). In Brazil, seventy percent of the population is assisted by the public Unified Health System (SUS), where genetic testing is still unavailable. And few studies were performed regarding the prevalence of HBOC pathogenic variants in this context. Here, we estimated the prevalence of germline pathogenic variants in BRCA1, BRCA2 and TP53 genes in Brazilian patients suspected of HBOC and referred to public healthcare service. Predictive power of risk prediction models for detecting mutation carriers was also evaluated. We found that 41 out of 257 tested patients (15.9%) were carriers of pathogenic variants in the analyzed genes. Most frequent pathogenic variant was the founder Brazilian mutation TP53 c.1010G > A (p.Arg337His), adding to the accumulated evidence that supports inclusion of TP53 in routine testing of Brazilian HBOC patients. Surprisingly, BRCA1 c.5266dupC (p.Gln1756fs), a frequently reported pathogenic variant in Brazilian HBOC patients, was not observed. Regarding the use of predictive models, we found that familial history of cancer might be used to improve selection or prioritization of patients for genetic testing, especially in a context of limited resources.

SARS-CoV-2: from its discovery to genome structure, transcription, and replication.

SARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS). SARS-CoV-2 has a single-stranded, positive-sense RNA (+RNA) genome of ~ 29.9 kb and exhibits significant genetic shift from different isolates. After entering the susceptible cells expressing both ACE2 and TMPRSS2, the SARS-CoV-2 genome directly functions as an mRNA to translate two polyproteins from the ORF1a and ORF1b region, which are cleaved by two viral proteases into sixteen non-structural proteins (nsp1-16) to initiate viral genome replication and transcription. The SARS-CoV-2 genome also encodes four structural (S, E, M and N) and up to six accessory (3a, 6, 7a, 7b, 8, and 9b) proteins, but their translation requires newly synthesized individual subgenomic RNAs (sgRNA) in the infected cells. Synthesis of the full-length viral genomic RNA (gRNA) and sgRNAs are conducted inside double-membrane vesicles (DMVs) by the viral replication and transcription complex (RTC), which comprises nsp7, nsp8, nsp9, nsp12, nsp13 and a short RNA primer. To produce sgRNAs, RTC starts RNA synthesis from the highly structured gRNA 3' end and switches template at various transcription regulatory sequence (TRSB) sites along the gRNA body probably mediated by a long-distance RNA-RNA interaction. The TRS motif in the gRNA 5' leader (TRSL) is responsible for the RNA-RNA interaction with the TRSB upstream of each ORF and skipping of the viral genome in between them to produce individual sgRNAs. Abundance of individual sgRNAs and viral gRNA synthesized in the infected cells depend on the location and read-through efficiency of each TRSB. Although more studies are needed, the unprecedented COVID-19 pandemic has taught the world a painful lesson that is to invest and proactively prepare future emergence of other types of coronaviruses and any other possible biological horrors.

HPV18 Utilizes Two Alternative Branch Sites for E6*I Splicing to Produce E7 Protein.

Human papillomavirus 18 (HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6E7 pre-mRNA. The E6 ORF region in the bicistronic E6E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences (BPS) upstream of its 3' splice site, with an identical heptamer AACUAAC, for E6*I splicing. One heptamer has a branch site adenosine (underlined) at nt 384 and the other at nt 388. E6*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6*I splicing prefers the 3'ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment.

Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.

Methylation at 3'LCR of HPV16 can be affected by patient age and disruption of E1 or E2 genes.

CpG methylation at early promoter of HPV16 DNA, in the 3' end of the Long Control Region (3'LCR), has been associated to the presence of episomal forms of viral genome and, consequently, intact E1 and E2 ORFs. The DNA methylation would block the access of E2 viral protein to the E2 binding sites at early-promoter. However, is still unclear if methylation at 3'LCR of HPV16 DNA can also vary depending of other tumor characteristics in addition to viral DNA physical state. In this study, we evaluate whether the methylation level at the five CpG located at 3'LCR of HPV16 is associated to patient age and E1 and/or E2 ORFs integrity. DNA pyrosequencing was used to measure the methylation level in 69 invasive cervical cancer samples obtained from biopsies of patients attended at Brazilian National Institute of Cancer (INCA). PCR amplifications were performed to assess disruption status of E1 and E2 genes of HPV16. The methylation average per sample ranged widely, from <1 to 88.00%. Presence of intact E1/E2 genes and patient age were positively associated with average methylation in both bivariate analyses (p=0.003 and p=0.006, respectively), and multivariate analysis (p=0.002 and p=0.021, respectively), adjusted for tumor type (squamous cell carcinomas or adenocarcinomas) and HPV16 lineage. These findings showed that presence of intact E1/E2 open reading frames was associated with high levels of DNA methylation, and older patients showed higher levels of methylation than younger ones independently of viral genome disruption.

Análise da integração genômica do Papilomavírus Humano e a expressão de seus genes em câncer cervical / Human Papillomavirus genomic integration and gene expression at cervical cancer

O câncer de colo de útero é o terceiro tipo de neoplasia com maior incidência mundial em mulheres, apresentando o Vírus do Papiloma Humano (HPV) como fator necessário mas não suficiente para o seu desenvolvimento. A imortalização e o poder de transformação das células neoplásicas se devem aos oncogenes virais E6 e E7, que geram oncoproteínas capazes de mediar estímulos mitogênicos e antiapoptóticos através de sua interação com proteínas reguladoras do ciclo celular. Esses genes são expressos como um pré-RNAm E6/E7 bicistônico, e por meio da existência de diferentes sítios de splicing, duas ou mais isoformas de mRNA E6/E7 são produzidos. O genoma do HPV é mantido na forma epissomal no estágio inicial das lesões displásicas de baixo grau, porém, nas células pré-cancerosas avançadas e na maioria dos carcinomas, ele está integrado no DNA cromossômico. Essa integração causa o aumento da expressão dos oncogenes E6 e E7, promovendo vantagens seletiva para o crescimento tumoral. O objetivo do presente trabalho foi analisar o padrão de integração do genoma viral no genoma do hospedeiro, a expressão dos genes do HPV e os produtos de splicing do RNAm E6/E7 em tumores cervicais. Para esse fim extraímos o RNA de 25 biópsias (14 infectadas com HPV16, 10 com HPV18 e uma co-infectada pelo HPV16 e HPV18). A partir do RNA das amostras, foi realizado o sequenciamento de RNA (RNA-Seq) na plataforma Hi-Seq 2500 (Illumina). Através da busca por contigs quiméricos pela montagem “de novo” do transcriptoma das células, encontramos integração em 18 amostras. Na maioria das amostras o rompimento do genoma viral ocorreu nos genes E1 ou E2. Cerca de 52,6% das integrações ocorreram em regiões intergênicas do genoma celular, o restante ocorreu dentro dos genes MCL1, PIGR, PXMP4, TP63, MMP13, SLC16A14, HRH1, RAD51B e NRXN1...

Cervical cancer is the third most incident type of cancer in women worldwide, and the presence of Human Papillomavirus (HPV) is necessary but not sufficient for its development. The immortalization and transformation of neoplasic cells are carried out by the viral oncogenes E6 and E7, which will form proteins that can interfere with specific proteins responsible for cell cycle regulation. Those genes are expressed as a bicistronic E6/E7 pre-mRNA, producing two or more different alternative E6/E7 mRNAs due to alternative splicing. At initial phase of cervical infection, the HPV genome is maintained as an episomal DNA, but in tumors, the viral genome is integrated into the nuclear DNA of host cells. Once integrated, E6 and E7 viral oncogenes are overexpressed, what will improve selective advantages to tumoral development. Our goal is to analyze the HPV genome integration pattern at host genome, the HPV gene expression and the E6/E7 splicing products in cervical tumors. To this purpose, we extracted total RNA from 25 cervical cancers samples (14 infected with HPV16, 10 with HPV 18 and 1 co-infected with HPV16 and HPV18). The RNA sequencing (RNA-Seq) at Hi-Seq 2500 (Illumina) was done with the RNA extracted from each sample. To determine the HPV integration, we made a de novo construction of cellular transcriptome and a search for chimeric contigs. We have found integration in 18 samples, and in most samples...