Broad-Spectrum Detection of HPV in Male Genital Samples Using Target-Enriched Whole-Genome Sequencing.

Most human papillomavirus (HPV) surveillance studies target 30-50 of the more than 200 known types. We applied our recently described enriched whole-genome sequencing (eWGS) assay to demonstrate the impact of detecting all known and novel HPV types in male genital samples (n = 50). HPV was detected in nearly all (82%) samples, (mean number of types/samples 13.6; range 1-85), and nearly all HPV-positive samples included types in multiple genera (88%). A total of 560 HPV detections (237 unique HPV types: 46 alpha, 55 beta, 135 gamma, and 1 mu types) were made. The most frequently detected HPV types were alpha (HPV90, 43, and 74), beta (HPV115, 195, and 120), and gamma (HPV134, mSD2, and HPV50). High-risk alpha types (HPV16, 18, 31, 39, 52, and 58) were not common. A novel gamma type was identified (now officially HPV229) along with 90 unclassified types. This pilot study demonstrates the utility of the eWGS assay for broad-spectrum type detection and suggests a significantly higher type diversity in males compared to females that warrants further study.

NanoString Technology for Human Papillomavirus Typing.

High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter® platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 104 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 104 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.

Bioinformatics Pipeline for Human Papillomavirus Short Read Genomic Sequences Classification Using Support Vector Machine.

We recently developed a test based on the Agilent SureSelect target enrichment system capturing genomic fragments from 191 human papillomaviruses (HPV) types for Illumina sequencing. This enriched whole genome sequencing (eWGS) assay provides an approach to identify all HPV types in a sample. Here we present a machine learning algorithm that calls HPV types based on the eWGS output. The algorithm based on the support vector machine (SVM) technique was trained on eWGS data from 122 control samples with known HPV types. The new algorithm demonstrated good performance in HPV type detection for designed samples with 25 or greater HPV plasmid copies per sample. We compared the results of HPV typing made by the new algorithm for 261 residual epidemiologic samples with the results of the typing delivered by the standard HPV Linear Array (LA). The agreement between methods (97.4%) was substantial (kappa= 0.783). However, the new algorithm identified additionally 428 instances of HPV types not detectable by the LA assay by design. Overall, we have demonstrated that the bioinformatics pipeline is an accurate tool for calling HPV types by analyzing data generated by eWGS processing of DNA fragments extracted from control and epidemiological samples.

Universal human papillomavirus typing by whole genome sequencing following target enrichment: evaluation of assay reproducibility and limit of detection.

BACKGROUND: We recently described a method for unbiased detection of all known human papillomaviruses (HPV) types with the potential for the determination of their variant and integration from the resulting whole genome sequence data. Considering the complex workflow for target-enriched next generation sequencing (NGS), we focused on the reproducibility and limit of detection (LOD) of this new universal HPV typing assay in this study. RESULTS: We evaluated the reproducibility and LOD for HPV genotyping based on our recently published method that used RNA-baits targeting whole genomes of 191 HPV types, Agilent SureSelect protocol for target enrichment and Illumina HiSeq 2500 for sequencing (eWGS, enriched whole genome sequencing). Two libraries, prepared from pooled plasmids representing 9 vaccine HPV types at varying input (1-625 copies/reaction), were sequenced twice giving four replicates for evaluating reproducibility and LOD. eWGS showed high correlation in the number of reads mapped to HPV reference genomes between the two flow-cell lanes within (R2 = 1) and between experiments (R2 = 0.99). The number of mapped reads was positively correlated to copy number (ß = 13.9, p < 0.0001). The limit of blank (LOB) could be calculated based on mapped reads to HPV types not included in each sample. HPV genotyping was reproducible for all 9 types at 625 copies using multiple cut-off criteria but LOD was 25 copies based on number of reads above LOB even when multiple types were present. eWGS showed no bias for HPV genotyping under single or multiple infection (p = 0.16-0.99). CONCLUSIONS: The universal eWGS method for HPV genotyping has sensitivity, competitive with widely used consensus PCR methods with reduced type competition, and with the potential for determination of variant and integration status. The protocol used in this study, using defined samples varying in complexity and copy number, analyzed in replicate and duplicate assays, is applicable to most WGS methods.

Precision Medicine Requires Precision Laboratories.

This commentary highlights the validation study by Lih et al that supports the use of precision medicine for improved clinical trials.

Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment.

We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology.

Females with FVIII and FIX deficiency have reduced joint range of motion.

Little is known about rates of joint bleeding among females with FVIII/FIX deficiency or hemophilia carriers. In a cross-sectional study, we tested the hypothesis that females with FVIII or FIX deficiency enrolled in the Universal Data Collection (UDC) project had a reduced mean overall joint range of motion (ROM) compared with historic controls from the Normal Joint Study. Demographics, clinical characteristics, and joint ROM measurements on 303 females without a bleeding disorder and 148 females with FVIII and FIX deficiency, respectively, between the ages of 2-69 years and a body mass index (BMI) ≤ 35 were compared. Multivariate linear regression was performed with the overall joint ROM (sum of the right and left ROM measurements of five joints) as the dependent variable and FVIII or FIX activity as the independent variable adjusting for age, race, BMI, and number of joint bleeds reported over the last 6 months. As FVIII and FIX activity decreased, the mean overall joint ROM became reduced and in most cases was significantly lower than that of the controls regardless of age and clinical hemophilia severity. Further investigation of reduced joint ROM as evidence of subclinical joint bleeding in females with FVIII and FIX deficiency is warranted.

A role for WDR5 in TRA-1/Gli mediated transcriptional control of the sperm/oocyte switch in C. elegans.

The hermaphrodite germline of Caenorhabditis elegans initially engages in spermatogenesis and then switches to oogenesis during late stages of larval development. TRA-1, a member of the Ci/Gli family of transcriptional repressors, plays an essential role in this switch by repressing genes that promote spermatogenesis. WDR5 proteins are conserved components of histone methyltransferase complexes normally associated with gene activation. However, two C. elegans WDR5 homologs, wdr-5.1 and wdr-5.2 are redundantly required for normal TRA-1 dependent repression, and this function is independent of their roles in histone methylation. Animals lacking wdr-5.1/wdr-5.2 function fail to switch to oogenesis at 25°C, resulting in a masculinization of germline (Mog) phenotype. The Mog phenotype is caused by ectopic expression of fog-3, a direct target of TRA-1 repression. WDR-5.1 associates with the fog-3 promoter and is required for TRA-1 to bind to fog-3 promoter. Other direct targets of TRA-1 are similarly derepressed in the double mutant. These results show that WDR5 plays a novel and important role in stabilizing transcriptional repression during C. elegans sex determination, and provide evidence that this important protein may operate independently of its established role in histone methyltransferase complexes.

Mutation analysis of a cohort of US patients with hemophilia B.

Hemophilia B (HB) is a disorder resulting from genetic mutations in the Factor 9 gene (F9). Genotyping of HB patients is important for genetic counseling and patient management. Here we report a study of mutations identified in a large sample of HB patients in the US. Patients were enrolled through an inhibitor surveillance study at 17 hemophilia treatment centers. A total of 87 unique mutations were identified from 225 of the 226 patients, including deletions, insertions, and point mutations. Point mutations were distributed throughout the F9 gene and were found in 86% of the patients. Of these mutations, 24 were recurrent in the population, and 3 of them (c.316G>A, c.1025C>T, and c.1328T>A) accounted for 84 patients (37.1%). Haplotype analysis revealed that the high recurrence arose from a founder effect. The severity of HB was found to correlate with the type of mutation. Inhibitors developed only in severe cases with large deletions and nonsense mutations. None of the mild or moderate patients developed inhibitors. Our results provide a resource describing F9 mutations in US HB patients and confirm previous findings that patients bearing large deletions and nonsense mutations are at high risk of developing inhibitors.

The CDC Hemophilia B mutation project mutation list: a new online resource.

Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

A role for Set1/MLL-related components in epigenetic regulation of the Caenorhabditis elegans germ line.

The methylation of lysine 4 of Histone H3 (H3K4me) is an important component of epigenetic regulation. H3K4 methylation is a consequence of transcriptional activity, but also has been shown to contribute to "epigenetic memory"; i.e., it can provide a heritable landmark of previous transcriptional activity that may help promote or maintain such activity in subsequent cell descendants or lineages. A number of multi-protein complexes that control the addition of H3K4me have been described in several organisms. These Set1/MLL or COMPASS complexes often share a common subset of conserved proteins, with other components potentially contributing to tissue-specific or developmental regulation of the methyltransferase activity. Here we show that the normal maintenance of H3K4 di- and tri-methylation in the germ line of Caenorhabditis elegans is dependent on homologs of the Set1/MLL complex components WDR-5.1 and RBBP-5. Different methylation states that are each dependent on wdr-5.1 and rbbp-5 require different methyltransferases. In addition, different subsets of conserved Set1/MLL-like complex components appear to be required for H3K4 methylation in germ cells and somatic lineages at different developmental stages. In adult germ cells, mutations in wdr-5.1 or rbbp-5 dramatically affect both germ line stem cell (GSC) population size and proper germ cell development. RNAi knockdown of RNA Polymerase II does not significantly affect the wdr-5.1-dependent maintenance of H3K4 methylation in either early embryos or adult GSCs, suggesting that the mechanism is not obligately coupled to transcription in these cells. A separate, wdr-5.1-independent mode of H3K4 methylation correlates more directly with transcription in the adult germ line and in embryos. Our results indicate that H3K4 methylation in the germline is regulated by a combination of Set1/MLL component-dependent and -independent modes of epigenetic establishment and maintenance.

Trans-generational epigenetic regulation of C. elegans primordial germ cells.

BACKGROUND: The processes through which the germline maintains its continuity across generations has long been the focus of biological research. Recent studies have suggested that germline continuity can involve epigenetic regulation, including regulation of histone modifications. However, it is not clear how histone modifications generated in one generation can influence the transcription program and development of germ cells of the next. RESULTS: We show that the histone H3K36 methyltransferase maternal effect sterile (MES)-4 is an epigenetic modifier that prevents aberrant transcription activity in Caenorhabditis elegans primordial germ cells (PGCs). In mes-4 mutant PGCs, RNA Pol II activation is abnormally regulated and the PGCs degenerate. Genetic and genomewide analyses of MES-4-mediated H3K36 methylation suggest that MES-4 activity can operate independently of ongoing transcription, and may be predominantly responsible for maintenance methylation of H3K36 in germline-expressed loci. CONCLUSIONS: Our data suggest a model in which MES-4 helps to maintain an 'epigenetic memory' of transcription that occurred in germ cells of previous generations, and that MES-4 and its epigenetic product are essential for normal germ cell development.

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species.

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.

WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells rely on growth factors provided by feeder cells or exogenously to maintain their pluripotency. In order to identify such factors, we have established sub-lines of STO feeder cells which exhibit variable ability in supporting ES cell self-renewal. Functional screening identifies WNT5A and WNT6 as STO cell-produced factors that potently inhibit ES cell differentiation in a serum-dependent manner. Furthermore, direct activation of beta-catenin without disturbing the upstream components of the WNT/beta-catenin pathway fully recapitulates the effect of WNTs on ES cells. Importantly, the WNT/beta-catenin pathway up-regulates the mRNA for Stat3, a known regulator of ES cell self-renewal in the mouse. Finally, LIF is able to mimic the serum effect to act synergistically with WNT proteins to inhibit ES cell differentiation. Therefore, our study reveals part of the molecular mechanisms by which the WNT/beta-catenin pathway acts to prevent ES cell differentiation through convergence on the LIF/JAK-STAT pathway at the level of STAT3.

Developmental expression and function of Bmp4 in spermatogenesis and in maintaining epididymal integrity.

Bone morphogenetic proteins (BMPs) play essential roles in many aspects of developmental biology. We have previously shown that Bmp7, Bmp8a, and Bmp8b of the 60A class of Bmp genes have additive effects in spermatogenesis and in maintaining the epididymal integrity of the caput and caudal regions. Here we report that Bmp4 of the Dpp class has a unique expression pattern in the developing testis and epididymis. Bmp4 heterozygous males on a largely C57BL/6 background show compromised fertility due to degeneration of germ cells, reduced sperm counts, and decreased sperm motility. More interestingly, some of these males show extensive degeneration of the epididymal epithelium in the corpus region, rather than in the caput and cauda regions as for Bmp7 and Bmp8 mutants. Thus, these genetic data reveal a region-specific requirement of different classes of BMPs for epididymal epithelium to survive and have significant implications on male reproductive health and perhaps birth control.

BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways.

The fate of pluripotent stem cells is tightly controlled during early embryonic development. Both the derivation and the maintenance of embryonic stem cells (ES cells) in vitro depend on feeder cell-derived growth factors that are largely unidentified. To dissect the mechanisms governing pluripotency, we conducted a screen to identify factors that are produced by mouse embryonic fibroblast STO cells and are required to maintain the pluripotency of ES cells. One of the factors is bone morphogenetic protein 4 (BMP4). Unexpectedly, the major effect of BMP4 on the self-renewal of ES cells is accomplished by means of the inhibition of both extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways, and inhibitors of ERK and p38 MAPKs mimic the effect of BMP4 on ES cells. Importantly, inhibition of the p38 MAPK pathway by SB203580 overcomes the block in deriving ES cells from blastocysts lacking a functional Alk3, the BMP type IA receptor. These results uncover a paradigm for BMP signaling in the biology of pluripotent stem cells.