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Espen Enerly, Cecilia Olofsson, Mari NygårdDepartment of Research, Cancer Registry of Norway, Oslo, NorwayAbstract: Human papillomavirus (HPV) is the main cause of cervical cancer, and many countries now offer vaccination against HPV to girls by way of government-funded national immunization programs. Monitoring HPV prevalence in adolescents could offer a near-term biological measure of vaccine impact, and urine sampling may be an attractive large-scale method that could be used for this purpose. Our objective was to provide an overview of the literature on HPV DNA detection in urine samples, with an emphasis on adolescents. We searched the PubMed database using the terms “HPV” and “urine” and identified 21 female and 14 male study populations in which HPV prevalence in urine samples was reported, four of which included only asymptomatic female adolescents. We provide herein an overview of the recruitment setting, age, urine sampling procedure, lesion type, HPV assay, and HPV prevalence in urine samples and other urogenital samples for the studies included in this review. In female study populations, concordance for any HPV type and type-specific concordance in paired urine and cervical samples are provided in addition to sensitivity and specificity. We concluded that few studies on HPV prevalence in urine samples have been performed in asymptomatic female adolescent populations but that urine samples may be a useful alternative to cervical samples to monitor changes in HPV prevalence in females in the post-HPV vaccination era. However, care should be taken when extrapolating HPV findings from urine samples to the cervix. In males, urine samples do not seem to be optimal for monitoring HPV prevalence due to a low human genomic DNA content and HPV DNA detection rate compared to other urogenital sites. In each situation the costs and benefits of HPV DNA detection in urine compared to alternative monitoring options should be carefully considered.Keywords: cervical cancer, HPV, surveillance, vaccination, virology, cervix

Introduction: Testing for mutations of epidermal growth factor receptor (EGFR) is crucial to identify non-small cell lung cancer (NSCLC) patients eligible for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs); This study aims to describe EGFR-mutation testing, treatment patterns, and overall survival (OS) in localized NSCLC patients., Materials and Methods: Patients with localized (Stage IB-IIIA) NSCLC registered in the Norwegian Cancer Registry during 2010-2017 were followed from diagnosis until emigration, death, or end of study in 2018. The cohort was linked to data from the Norwegian Patient Registry, the Prescription Database, and the Cause of Death Registry., Results: Of 2367 patients identified with localized NSCLC, 52 % were females and median age at diagnosis was 69 years. Most (66 %) were treated with surgery, while 16 % received curatively-intended radiotherapy (RT). EGFR-mutation testing increased significantly from 58 to 84 % during the study period. Testing frequencies varied across regions and comorbidity levels. Nine-percent of tested patients were EGFR-mutation positive (EGFRm+), of whom 27 % were treated with EGFR-TKIs. There was no correlation between initial treatment with either surgery or RT and EGFR-TKI use. The 3-year OS did not vary considerably by EGFR-mutation testing, but EGFRm+ patients had a higher 3-year OS (78.8 %) than wild-type EGFR (EGFRwt) patients (65.9 %)., Discussion: Although EGFR-mutation testing is increasingly being implemented in the early-stage setting in line with national recommendations, some patients are still not being tested for molecular markers as part of their diagnostic workup-a prerequisite for providing equal access to effective targeted treatments, such as EGFR-TKIs, to eligible patients., Competing Interests: Declaration of competing interest ÅH reports advisory/consultancy/meeting presentation roles for AbbVie, AstraZeneca, BMS, Pfizer, Roche, Janssen, MSD, Sanofi, Bayer, Medicover, and Takeda with honoraria directed to own institution, and receiving research grants from Roche, BMS, Ultimovacs, AstraZeneca, Novartis, InCyte, Eli Lilly, Illumina, Merck, Nanopore, GlaxoSmithKline. TÅM reports no conflict of interest. SC, LEF and JA are employees of the study sponsor, AstraZeneca AB. EE reports receiving research funding from AstraZeneca AB to host institution., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Matching treatment based on tumour molecular characteristics has revolutionized the treatment of some cancers and has given hope to many patients. Although personalized cancer care is an old concept, renewed attention has arisen due to recent advancements in cancer diagnostics including access to high-throughput sequencing of tumour tissue. Targeted therapies interfering with cancer specific pathways have been developed and approved for subgroups of patients. These drugs might just as well be efficient in other diagnostic subgroups, not investigated in pharma-led clinical studies, but their potential use on new indications is never explored due to limited number of patients., Methods: In this national, investigator-initiated, prospective, open-label, non-randomized combined basket- and umbrella-trial, patients are enrolled in multiple parallel cohorts. Each cohort is defined by the patient's tumour type, molecular profile of the tumour, and study drug. Treatment outcome in each cohort is monitored by using a Simon two-stage-like 'admissible' monitoring plan to identify evidence of clinical activity. All drugs available in IMPRESS-Norway have regulatory approval and are funded by pharmaceutical companies. Molecular diagnostics are funded by the public health care system., Discussion: Precision oncology means to stratify treatment based on specific patient characteristics and the molecular profile of the tumor. Use of targeted drugs is currently restricted to specific biomarker-defined subgroups of patients according to their market authorization. However, other cancer patients might also benefit of treatment with these drugs if the same biomarker is present. The emerging technologies in molecular diagnostics are now being implemented in Norway and it is publicly reimbursed, thus more cancer patients will have a more comprehensive genomic profiling of their tumour. Patients with actionable genomic alterations in their tumour may have the possibility to try precision cancer drugs through IMPRESS-Norway, if standard treatment is no longer an option, and the drugs are available in the study. This might benefit some patients. In addition, it is a good example of a public-private collaboration to establish a national infrastructure for precision oncology. Trial registrations EudraCT: 2020-004414-35, registered 02/19/2021; ClinicalTrial.gov: NCT04817956, registered 03/26/2021., (© 2022. The Author(s).)

Background: Testing for epidermal growth factor receptor mutation (EGFRm) status is a prerequisite to identify eligible patients for tyrosine kinase inhibitors (TKI) treatment. However, EGFR testing of patients with non-small cell lung cancer (NSCLC) is suboptimal in many parts of the world. The aim of this study was to describe real-world EGFR testing practice, EGFRm prevalence, and subsequent TKI treatment patterns in Norway., Patients and Methods: This retrospective, observational, cohort study included all incident locally advanced and metastatic non-squamous NSCLC patients registered in the Norwegian Cancer Registry during 2010-2017. A cohort with follow-up through 2018 was formed with linkage to nationwide registries on comorbidities, prescribed drugs and causes-of-death., Results: A total of 10,717 patients were included, of which 35% (3782) with locally advanced NSCLC and 65% (6935) with metastatic disease. Mean age at diagnosis was 71 years and 47% were female. EGFR testing among patients with metastatic NSCLC increased from 41% to >64% between 2010 and 2017, with a relative stable incidence of EGFRm+ (∼9%). More than 85% of EGFRm+ patients received TKI treatment. Patients with the most dismal prognosis (>80 age, comorbidities) and with diagnosis based on cytology/imaging were less likely to be tested. Differences in testing were observed between regions., Conclusion: Despite increased test rates over the study period, in Norway, a significant proportion of patients with non-squamous metastatic NSCLC are still not tested for EGFR. To maximize the identification of eligible patients for targeted therapies, increased testing is recommended, regardless of age, comorbidity rate and place of residence., Competing Interests: Declaration of Competing Interest AH: advisory/consultancy role/presentations – AbbVie, AstraZeneca, BMS, Pfizer, Pierre Fabre and Takeda. Research grant Roche, BMS, Ultimovacs with honorarium to own institution. Have received research grants / study drug /diagnostic assays from Roche, AstraZeneca, Ultimovacs, BMS, Novartis, InCyte, EliLilly, Illumina. KAA: Employee of Astrazeneca at the time of study TÅM: Nothing to declare TBJ: Nothing to declare JA: Employee of Astrazeneca EE: Research funding from AstraZeneca AB to own institution., (Copyright © 2022. Published by Elsevier Ltd.)

Background: The quadrivalent human papillomavirus (qHPV) vaccine prevented vaccine HPV type-related infection and disease in young women in the 4-year FUTURE II efficacy study (NCT00092534). We report long-term effectiveness and immunogenicity at the end of 14 years of follow-up after enrollment in FUTURE II., Methods: Young women (16-23 years of age) from Denmark, Iceland, Norway, and Sweden who received three qHPV vaccine doses during the randomized, double-blind, placebo-controlled FUTURE II base study were followed for effectiveness for an additional ≥10 years through national registries. Tissue samples including but not limited to those collected during organized cervical cancer screening programs were obtained from regional biobanks to be adjudicated for histopathology diagnosis and tested for HPV DNA. The observed incidence of HPV16/18-related high-grade cervical dysplasia (primary outcome) was compared with recent historical background incidence rates in an unvaccinated population. Serum was collected at years 9 and 14 to assess antibody responses., Findings: No cases of HPV16/18-related high-grade cervical dysplasia were observed in the per-protocol effectiveness population ( N  = 2121; 24,099·0 person-years of follow-up) during the entire study. Vaccine effectiveness of 100% (95% CI 94·7-100) was demonstrated for ≥12 years, with a trend toward continued protection through 14 years post-vaccination. Seropositivity rates at study conclusion were >90% (HPV6/11/16) and 52% (HPV18) using competitive Luminex immunoassay, and >90% (all four HPV types) using the more sensitive IgG Luminex immunoassay., Interpretation: Vaccination of young women with qHPV vaccine offers durable protection against HPV16/18-related high-grade cervical dysplasia for ≥12 years, with a trend toward continued protection through 14 years post-vaccination, and induces sustained HPV6/11/16/18 antibody responses for up to 14 years post-vaccination. There was no evidence of waning immunity, suggesting no need for a booster dose during that period., Funding: Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA., Competing Interests: Funding for this research was provided by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD). SKK reports research grants from MSD during the conduct of the study through her affiliating institute and personal fees from MSD outside the submitted work. MN reports research grants from MSD Norway through her affiliating institute during the conduct of the study and outside the scope of this study. KS reports research grants from MSD to her institution for the present work on HPV vaccination in Sweden and research grants to her institution for other register-based studies on HPV vaccination in Sweden. JD reports grants from MSD during the conduct of the study and grants from Genomica outside the submitted work. LT reports that her affiliating institution (ICS) received research grants from MSD Denmark ApS during the conduct of the study. CM reports unrestricted research grants through his affiliating institution from MSD during the conduct of the study. SB reports that her host institution received research grants from MSD Norway during the conduct of the study. EE reports that his affiliating institute received grants from MSD Norway during the conduct of the study. MH reports working on clinical trials sponsored by MSD during the conduct of the study. ÁIÁ has received grants from MSD during the conduct of the study. KB, KF, and IG have nothing to disclose. SWS has received grants from MSD for pathology review of biopsies during the conduct of the study. OB, TG, AL, JBM, DR, YSY, CB, and AS are current or former employees of MSD and may own stock or stock options in Merck & Co., Inc., Kenilworth, NJ, USA., (© 2020 Published by Elsevier Ltd.)

Post-marketing studies are commonly performed to follow-up on the safety and effectiveness of a drug or vaccine after approval has been obtained. These post-marketing studies may involve the collection of real-world data from registries and clinical biobanks in order to obtain real-world evidence. As this approach can monitor the effects of pharmaceutical products over decades, it is particularly necessary for the development of safe and effective vaccines. A long-term follow-up (LTFU) study was initiated as an extension of a phase 3 clinical study (V501-015; NCT00092534) to assess the effectiveness, immunogenicity and safety of the quadrivalent human papillomavirus (qHPV) vaccine for up to 14 years after the start of vaccination. The LTFU study included participants from Denmark, Iceland, Norway, and Sweden, and assessed qHPV vaccine effectiveness against cervical pre-cancers and cancers caused by the oncogenic HPV types 16 and 18. In particular, our study utilized Nordic national health registries, in which individual patient records were linked by a unique Personal Identity Number. Here, we describe the overall implementation and methodology of the qHPV vaccine LTFU study conducted in the Nordic region. The LTFU study format we describe here supported a comprehensive follow-up process, with near-complete retrieval of registry data and specimens from local laboratories achieved in a timely manner; therefore, we have demonstrated that such a collection is feasible and can be used to address stringent post-marketing requirements., Competing Interests: Declaration of Competing Interest EE, SB, KS, SC, CM, and MN have received research grants from MSD through the affiliating institute. SKK has received speaker and advisory board fees from MSD, and a research grant through her institution from MSD. LT reports that her affiliating institution received research grants from MSD Denmark ApS. MH is working on clinical trials of this vaccine, sponsored by Merck & Co., Inc., Kenilworth, NJ, USA. TG, AJ, and AJS are current employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA., and may hold stock and/or stock options in Merck & Co., Inc., Kenilworth, NJ, USA., (Copyright © 2020. Published by Elsevier Inc.)

Background: Many countries have initiated school-based human papillomavirus (HPV) vaccination programs. The real-life effectiveness of HPV vaccines has become increasingly evident, especially among girls vaccinated before HPV exposure in countries with high vaccine uptake. In 2009, Norway initiated a school-based HPV vaccination program for 12-year-old girls using the quadrivalent HPV vaccine (Gardasil®), which targets HPV6, 11, 16, and 18. Here, we aim to assess type-specific vaginal and oral HPV prevalence in vaccinated compared with unvaccinated girls in the first birth cohort eligible for school-based vaccination (born in 1997)., Methods: This observational, cross-sectional study measured the HPV prevalence ratio (PR) between vaccinated and unvaccinated girls in Norway. Facebook advertisement was used to recruit participants and disseminate information about the study. Participants self-sampled vaginal and oral specimens using an Evalyn® Brush and a FLOQSwab™, respectively. Sexual behavior was ascertained through a short questionnaire., Results: Among the 312 participants, 239 (76.6%) had received at least one dose of HPV vaccine prior to sexual debut. 39.1% of vaginal samples were positive for any HPV type, with similar prevalence among vaccinated and unvaccinated girls (38.5% vs 41.1%, PR: 0.93, 95% confidence interval [CI]: 0.62-1.41). For vaccine-targeted types there was some evidence of lower prevalence in the vaccinated (0.4%) compared to the unvaccinated (6.8%) group (PR: 0.06, 95%CI: 0.01-0.52). This difference remained after adjusting for sexual behavior (PR: 0.04, 95%CI: 0.00-0.42). Only four oral samples were positive for any HPV type, and all of these participants had received at least one dose of HPV vaccine at least 1 year before oral sexual debut., Conclusion: There is evidence of a lower prevalence of vaccine-targeted HPV types in the vagina of vaccinated girls from the first birth cohort eligible for school-based HPV vaccination in Norway; this was not the case when considering all HPV types or types not included in the quadrivalent HPV vaccine., Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: M.N. received research grants from MSD/Merck through the affiliating institute.

Increasing attendance to screening offers the best potential for improving the effectiveness of well-established cervical cancer screening programs. Self-sampling at home for human papillomavirus (HPV) testing as an alternative to a clinical sampling can be a useful policy to increase attendance. To determine whether self-sampling improves screening attendance for women who do not regularly attend the Norwegian Cervical Cancer Screening Programme (NCCSP), 800 women aged 25-69 years in the Oslo area who were due to receive a 2nd reminder to attend regular screening were randomly selected and invited to be part of the intervention group. Women in this group received one of two self-sampling devices, Evalyn Brush or Delphi Screener. To attend screening, women in the intervention group had the option of using the self-sampling device (self-sampling subgroup) or visiting their physician for a cervical smear. Self-sampled specimens were split and analyzed for the presence of high-risk (hr) HPV by the CLART® HPV2 test and the digene® Hybrid Capture (HC)2 test. The control group consisted of 2593 women who received a 2nd reminder letter according to the current guidelines of the NCCSP. The attendance rates were 33.4% in the intervention group and 23.2% in the control group, with similar attendance rates for both self-sampling devices. Women in the self-sampling subgroup responded favorably to both self-sampling devices and cited not remembering receiving a call for screening as the most dominant reason for previous non-attendance. Thirty-two of 34 (94.1%) hrHPV-positive women in the self-sampling subgroup attended follow-up. In conclusion, self-sampling increased attendance rates and was feasible and well received. This study lends further support to the proposal that self-sampling may be a valuable alternative for increasing cervical cancer screening coverage in Norway.

Background: The impacts of long-term storage and varying preanalytical factors on the quality and quantity of DNA and miRNA from archived serum have not been fully assessed. Preanalytical and analytical variations and degradation may introduce bias in representation of DNA and miRNA and may result in loss or corruption of quantitative data., Methods: We have evaluated DNA and miRNA quantity, quality, and variability in samples stored up to 40 years using one of the oldest prospective serum collections in the world, the Janus Serumbank, a biorepository dedicated to cancer research., Results: miRNAs are present and stable in archived serum samples frozen at -25°C for at least 40 years. Long-time storage did not reduce miRNA yields; however, varying preanalytical conditions had a significant effect and should be taken into consideration during project design. Of note, 500 μL serum yielded sufficient miRNA for qPCR and small RNA sequencing and on average 650 unique miRNAs were detected in samples from presumably healthy donors. Of note, 500 μL serum yielded sufficient DNA for whole-genome sequencing and subsequent SNP calling, giving a uniform representation of the genomes., Conclusions: DNA and miRNA are stable during long-term storage, making large prospectively collected serum repositories an invaluable source for miRNA and DNA biomarker discovery., Impact: Large-scale biomarker studies with long follow-up time are possible utilizing biorepositories with archived serum and state-of-the-art technology., (©2015 American Association for Cancer Research.)

This quadrivalent human papillomavirus (qHPV) (HPV6, -11, -16, and -18) vaccine long-term follow-up (LTFU) study is an ongoing extension of a pivotal clinical study (FUTURE II) taking place in the Nordic region. The LTFU study was designed to evaluate the effectiveness, immunogenicity, and safety of the qHPV vaccine (Gardasil) for at least 10 years following completion of the base study. The current report presents immunogenicity data from testing samples of the year 5 LTFU visit (approximately 9 years after vaccination). FUTURE II vaccination arm subjects, who consented to being followed in the LTFU, donated serum at regular intervals and in 2012. Anti-HPV6, -11, -16, and -18 antibodies were detected by the competitive Luminex immunoassay (cLIA), and in addition, serum samples from 2012 were analyzed by the total IgG Luminex immunoassay (LIA) (n = 1,598). cLIA geometric mean titers (GMTs) remained between 70% and 93% of their month 48 value depending on HPV type. For all HPV types, the lower bound of the 95% confidence interval (CI) for the year 9 GMTs remained above the serostatus cutoff value. The proportion of subjects who remained seropositive based on the IgG LIA was higher than the proportion based on cLIA, especially for anti-HPV18. As expected, the anti-HPV serum IgG and cLIA responses were strongly correlated for all HPV types. Anti-HPV GMTs and the proportion of vaccinated individuals who are seropositive remain high for up to 9 years of follow-up after vaccination., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: A crucial factor concerning the utility of Cancer Registries is the data quality with respect to comparability, completeness, validity and timeliness. However, the data quality of the registration of premalignant lesions has rarely been addressed. High grade vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VaIN) are premalignant lesions which may develop into cancer, and are often associated with infection with the human papillomarvirus (HPV). The aim was to evaluate the quality of registration of VIN and VaIN at the Cancer Registry of Norway (CRN)., Material and Methods: We re-collected all notifications with high grade VIN and VaIN diagnoses during 2002 to 2007 from pathology laboratories, and compared these to the data in the CRN database so as to quantitatively measure the completeness, validity and timeliness of the data., Results: Over the period 2002 to 2007 we estimated the completeness of the 1556 VIN and 297 VaIN notifications to be 95.0% and 92.9%, respectively. The original and reabstracted topography codes showed major discrepancies for 12 of 642 (1.9%) VIN and 7 of 128 (5.5%) VaIN notifications. The original and reabstracted morphology codes for VIN and VaIN were identical for 724 out of 814 notifications. Sixteen notifications had a major discrepancy. For the period 2002 to 2007 the median time elapsed between date of diagnosis and date of registration were 436 and 441 days for VIN and VaIN cases, respectively., Discussion: Based on the present analysis of the comparability, completeness, validity and timeliness of premalignant lesions of vulva and vagina, we conclude that the Cancer Registry of Norway is able to monitor such premalignant lesions satisfactorily.

Introduction: Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information., Methods: We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods., Results: We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process., Conclusion: This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.

Epidermal growth factor (EGF) stimulates cells by launching gene expression programs that are frequently deregulated in cancer. MicroRNAs, which attenuate gene expression by binding complementary regions in messenger RNAs, are broadly implicated in cancer. Using genome-wide approaches, we showed that EGF stimulation initiates a coordinated transcriptional program of microRNAs and transcription factors. The earliest event involved a decrease in the abundance of a subset of 23 microRNAs. This step permitted rapid induction of oncogenic transcription factors, such as c-FOS, encoded by immediate early genes. In line with roles as suppressors of EGF receptor (EGFR) signaling, we report that the abundance of this early subset of microRNAs is decreased in breast and in brain tumors driven by the EGFR or the closely related HER2. These findings identify specific microRNAs as attenuators of growth factor signaling and oncogenesis.

Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies between 5'ss nucleotides as a conserved feature of the entire set of 5'ss. These dependencies are also conserved in human-mouse pairs of orthologous 5'ss. Many disease-associated 5'ss mutations disrupt these dependencies, as can some human SNPs that appear to alter splicing. The consistency of the evidence signifies the relevance of this approach and suggests that 5'ss SNPs play a role in complex diseases.

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild-type p53. These miRNAs are repressed by p53 in an E2F1-mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi-gene transcriptional and post-transcriptional feed-forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.

In humans an estimated 35-60% of genes are alternatively spliced. A large number of genes also show alternative initiation or termination. Regulation of these processes is still poorly understood. For alternative splicing it is believed that the relative concentration of certain proteins and the presence of certain regulatory elements are the key factors determining alterations in splicing pattern. However, there is evidence that antisense RNA might be part of the regulatory processes. Antisense RNA molecules could bind to the target pre-mRNA in a sequence-specific fashion, sterically blocking targeted splice sites and redirecting the spliceosome to available and unhindered splice sites. Here we describe an in silico investigation to identify human sense/antisense pairs with alternative initiation or termination in the sense gene and where only one of the isoforms overlaps the antisense transcript. Alternatively spliced genes with antisense transcripts covering the alternatively used splice site are also identified. Our analyses are based on the ASAP splicing annotation database from UCLA, the antisense transcripts data from Yelin et al., 2003, and the H-invitational full-length cDNA database from JBIRC, Japan. These data gives new insight into the complexity of genomic organization and provide candidate loci for experimentalists to study antisense mediated regulation of alternative initiation, splicing and termination. Our result contains 468 clusters with this characteristic genomic organization and can be found at http://aistar.bii.a-star.edu.sg/.

The Drosophila Minutes are haploinsufficient mutations that are defective in ribosomal protein (rp) production, resulting in short, thin bristles, delayed development and recessive lethality. In a Minute fly, the amount of rp gene messenger RNA (mRNA) is reduced to >or=50% of the normal amount of gene product, and becomes rate limiting for ribosome biogenesis, cell proliferation and growth. Haploinsufficiency increases the vulnerability to complete loss of gene function (homozygous null state) if hit by a second mutation. Because of the homozygous lethality, it has only been possible to study the effects of Minute mutations in heterozygous animals. To be able to study the consequences of a loss-of-function of an rp gene (0%>mRNA<50%) in developing and differentiated cells we used heritable RNA interference (RNAi) in combination with the yeast GAL4/UAS binary system to spatiotemporally knock down the ribosomal protein L14 (RpL14) gene. We show, at the RNA and phenotypic levels, that RNAi efficiently reduces RpL14 gene expression throughout development, causing lethality and distinct and dramatic somatic anomalies in both developing and differentiated cells.

NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation.

The ribosomal protein RpL14 gene has been characterized in several species, including, human, rat and fruit fly. Haploinsufficiency for the gene causes the Minute phenotype in Drosophila, and it has been proposed as a regulator in the tumorigenic pathway in human. Several features concerning the gene structure have been studied, and some of these differ between human/rat and Drosophila. To address functional and evolutionary questions about these differences we have isolated and sequenced a cDNA and a genomic clone covering the RpL14 gene from the pufferfish Takifugu rubripes (Fugu). The Fugu RpL14 gene is approximately 2 Kb, with 5 introns, and encodes a protein of 137 amino acids. The protein contains a KOW-motif and a nuclear localization signal, which are conserved among a wide range of RPL14 proteins. On the other hand, a variable amino acid (alanine) repeat observed in human is missing in Takifugu rubripes, and the protein is shorter than its mammalian counterparts. Compared with human, the RpL14 gene in Fugu contains introns localized at identical positions in the gene, and most of them are shorter. A comparison of the RpL14 gene structure from a broad range of organisms indicates that both loss and gain of introns have occurred during the evolution of the gene.

Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modifications (2'-O-ribose methylation and pseudouridylation). Their genomic organization show great flexibility: some are individually or polycistronically transcribed, while others are encoded within introns of other genes. Here, we present an evolutionary analysis of the U49 gene in seven species. In all species analyzed, U49 contains the typical hallmarks of C and D box motifs, and a conserved 12-15 nt sequence complementary to rRNA that define them as homologs. In mouse, human, and Drosophila U49 is found encoded within introns of different genes, and in plants it is transcribed polycistronically from four different locations. In addition, U49 has two copies in two different introns of the RpL14 gene in Drosophila. The results indicate a substantial degree of duplication and translocation of the U49 gene in evolution. In light of its variable organization we discuss which of the two proposed mechanisms of rearrangement has acted upon the U49 snoRNA gene: chromosomal duplication or transposition through an RNA intermediate.

Ribosomal protein S3 (RPS3) is a multifunctional ribosomal protein: it is a structural and functional component of the ribosome, and also a DNA repair enzyme involved in the DNA base excision repair pathway. Here we cloned and characterized the genomic organization of the ribosomal protein S3 gene (RpS3) homolog in Drosophila virilis. We then compared gene structure and protein sequences of RpS3 from vertebrates, invertebrates, and plants. These comparisons revealed that RpS3 genes from plants to mammals have highly conserved coding and amino acid sequences, and also protein size. Further comparisons of the protein sequences show that important domains are well conserved in both localization and sequence. In contrast, comparison of gene size and organization reveals differing patterns and levels of conservation. Whereas invertebrate RpS3 genes are small in size and gene organization is variable (from zero to four introns), vertebrates have a considerably larger (but variable) gene size and a uniform gene organization. The larger gene size in vertebrates is due to increased number and expansion of introns. Although the plant RpS3 genes are relatively small ( approximately 1.8 kb), their organization resembles that seen in vertebrates. The high conservation through different phyla may suggest that RPS3 might be under great functional constraints, both in its capacity as a component of the ribosome and as a component of a DNA repair system. Finally, electrophoretic mobility shift assays indicate that an upstream element binds a nuclear protein(s).

In vertebrate genomes the dinucleotide CpG is heavily methylated, except in CpG islands, which are normally unmethylated. It is not clear why the CpG islands are such poor substrates for DNA methyltransferase. Plant genomes display methylation, but otherwise the genomes of plants and animals represent two very divergent evolutionary lines. To gain a further understanding of the resistance of CpG islands to methylation, we introduced a human CpG island from the proteasome-like subunit I gene into the genome of the plant Arabidopsis thaliana. Our results show that prevention of methylation is an intrinsic property of CpG islands, recognized even if a human CpG island is transferred to a plant genome. Two different parts of the human CpG island - the promoter region/ first exon and exon 2-4 - both displayed resistance against methylation, but the promoter/ exon1 construct seemed to be most resistant. In contrast, certain sites in a plant CpG-rich region used as a control transgene were always methylated. The frequency of silencing of the adjacent nptII (KmR) gene in the human CpG constructs was lower than observed for the plant CpG-rich region. These results have implications for understanding DNA methylation, and for construction of vectors that will reduce transgene silencing.

In Drosophila melanogaster, the apparently unrelated genes anon-66Da, RpL14, and anon-66Db (from telomere to centromere) are located on a 5547 bp genomic fragment on chromosome arm 3L at cytological position 66D8. The three genes are tightly linked, and flanked by two relatively large genes with unknown function. We have taken a comparative genomic approach to investigate the evolutionary history of the three genes. To this end we isolated a Drosophila virilis 7.3 kb genomic fragment which is homologous to a 5.5 kb genomic region of D. melanogaster. Both fragments map to Muller's element D, namely to section 66D in D. melanogaster and to section 32E in D. virilis, and harbor the genes anon-66Da, RpL14, and anon-66Db. We demonstrate that the three genes exhibit a high conservation of gene topography in general and in detail. While most introns and intergenic regions reveal sequence divergences, there are, however, a number of interspersed conserved sequence motifs. In particular, two introns of the RpL14 gene contain a short, highly conserved 60 nt long sequence located at corresponding positions. This sequence represents a novel Drosophila small nucleolar RNA, which is homologous to human U49. Whereas DNA flanking the three genes shows no significant interspecies homologies, the 3'-flanking region in D. virilis contains sequences from the transposable element Penelope. The Penelope family of transposable elements has been shown to promote chromosomal rearrangements in the D. virilis species group. The presence of Penelope sequences in the D. virilis 7.3 kb genomic fragment may be indicative for a transposon-induced event of transposition which did not yet scramble the order of the three genes but led to the breakdown of sequence identity of the flanking DNA.