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HPV16 causes half of cervical cancers worldwide; for unknown reasons, most infections resolve within two years. Here, we analyze the viral genomes of 5,328 HPV16-positive case-control samples to investigate mutational signatures and the role of human APOBEC3-induced mutations in viral clearance and cervical carcinogenesis. We identify four de novo mutational signatures, one of which matches the COSMIC APOBEC-associated signature 2. The viral genomes of the precancer/cancer cases are less likely to contain within-host somatic HPV16 APOBEC3-induced mutations (Fisher’s exact test, P = 6.2 x 10 −14 ), and have a 30% lower nonsynonymous APOBEC3 mutation burden compared to controls. We replicate the low prevalence of HPV16 APOBEC3-induced mutations in 1,749 additional cases. APOBEC3 mutations also historically contribute to the evolution of HPV16 lineages. We demonstrate that cervical infections with a greater burden of somatic HPV16 APOBEC3-induced mutations are more likely to be benign or subsequently clear, suggesting they may reduce persistence, and thus progression, within the host. The APOBEC mutational signature is prevalent in different tumour types. Here, using HPV16- positive cervical samples, the authors show that the signature is more prevalent in the viral genome of benign or clearing HPV16 infections compared to the viral genomes of the more advanced precancerous lesions or cervical cancer.

Invasive cervical cancers (ICC), caused by HPV infections, have a heterogeneous molecular landscape. We investigate the detection, timing, and HPV type specificity of somatic mutations in 3929 HPV-positive exfoliated cervical cell samples from individuals undergoing cervical screening in the U.S. using deep targeted sequencing in ICC cases, precancers, and HPV-positive controls. We discover a subset of hotspot mutations rare in controls (2.6%) but significantly more prevalent in precancers, particularly glandular precancer lesions (10.2%), and cancers (25.7%), supporting their involvement in ICC carcinogenesis. Hotspot mutations differ by HPV type, and HPV18/45-positive ICC are more likely to have multiple hotspot mutations compared to HPV16-positive ICC. The proportion of cells containing hotspot mutations is higher (i.e., higher variant allele fraction) in ICC and mutations are detectable up to 6 years prior to cancer diagnosis. Our findings demonstrate the feasibility of using exfoliated cervical cells for detection of somatic mutations as potential diagnostic biomarkers. Invasive cervical cancer is caused by HPV infection, but the disease itself is highly variable. Here, the authors use deep targeted sequencing to identify hotspot mutations in routine screening samples prior to diagnosis, which differed depending on HPV type.

Of the ~60 human papillomavirus (HPV) genotypes that infect the cervicovaginal epithelium, only 12–13 “high-risk” types are well-established as causing cervical cancer, with HPV16 accounting for over half of all cases worldwide. While HPV16 is the most important carcinogenic type, variants of HPV16 can differ in their carcinogenicity by 10-fold or more in epidemiologic studies. Strong genotype-phenotype associations embedded in the small 8-kb HPV16 genome motivate molecular studies to understand the underlying molecular mechanisms. Understanding the mechanisms of HPV genomic findings is complicated by the linkage of HPV genome variants. A panel of experts in various disciplines gathered on 21 November 2016 to discuss the interdisciplinary science of HPV oncogenesis. Here, we summarize the discussion of the complexity of the viral–host interaction and highlight important next steps for selected applied basic laboratory studies guided by epidemiological genomic findings.

APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0–29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0–5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (p = 0.028), and APOBEC-associated RNA editing (p = 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation.

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17–2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings' history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ∼ 3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463-65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of ≤ 1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.

Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count.

Background A low cost and accurate method for detecting high-risk (HR) human papillomavirus (HPV) is important to permit HPV testing for cervical cancer prevention. We used a commercially available HPV method (H13, Hybribio) which was documented to function accurately in a reduced volume of cervical specimen to determine the most prevalent HPV types and the distribution of HPV infections in over 1795 cancer-free women in Guatemala undergoing primary screening for cervical cancer by cytology. Methods HR-HPV detection was attempted in cervical samples from 1795 cancer-free women receiving Pap smears using the Hybribio™ real-time PCR assay of 13 HR types. The test includes a globin gene internal control. HPV positive samples were sequenced to determine viral type. Age-specific prevalence of HPV was also assessed in the study population. Results A total of 13% (226/1717) of women tested HPV+, with 78 samples (4.3%) failing to amplify the internal control. The highest prevalence was found in younger women (< 30 years, 22%) and older ones (≥60 years, 15%). The six most common HR-HPV types among the 148 HPV+ typed were HPV16 (22%), HPV18 (11%), HPV39 (11%), HPV58 (10%), HPV52 (8%), and HPV45 (8%). Conclusions In this sample of cancer free women in Guatemala, HPV16 was the most prevalent HR type in Guatemala and the age-specific prevalence curve peaked in younger ages. Women in the 30-59-year age groups had a prevalence of HR-HPV of 8%, however, larger studies to better describe the epidemiology of HPV in Guatemala are needed.

Following publication of the original article [1], an error was reported in the tagging of Joël Fokom Domgue in the author group. The tagging in this correction article has been fixed.Following publication of the original article [1], an error was reported in the tagging of Joël Fokom Domgue in the author group. The tagging in this correction article has been fixed.