Is cytology/HPV co-testing for cervical cancer screening useful in Japan?

OBJECTIVE: In Japan, no region has introduced primary HPV testing for cervical cancer screening. We assessed the diagnostic value and possible harm of HPV testing in Japan. METHODS: This cross-sectional study with historical controls used cytology-based screening and co-testing data in Japan. As surrogate indicators of possible harm, colposcopy referral rate and cervical intraepithelial neoplasm (CIN) 1 detection rates were calculated. As surrogate indicators with diagnostic values, the detection rates of CIN2 or greater (CIN2+) and CIN3+ were calculated. RESULTS: The data of 297 970 women (182 697 for cytology-based, 115 273 for co-testing) were examined. The detection rates of CIN1, CIN2+, and CIN3+ were significantly higher in the co-testing group than in the cytology-based group (P < 0.001, P < 0.0001, P < 0.01, respectively). Between ages 25-49, CIN2+ detection rates were significantly higher in the co-testing group than in the cytology-based group (P < 0.05 for each 5-year age group). Between ages 30-49, CIN3+ detection rates were significantly higher in the co-testing group than in the cytology-based group (P < 0.05 for each 5-year age group). CONCLUSION: Limiting the target age group may minimize the possible harm of screening. Cytology/HPV co-testing may be useful in Japanese populations if balance is maintained between benefit and harm.

Implementation of primary HPV testing in Japan.

Cervical cancer screening has been shifting from primary cytology to primary HPV testing worldwide as primary HPV testing is more sensitive than primary cytology. To the best of our knowledge, the current study is the first in Japan to examine the feasibility of primary HPV testing. One of the disadvantages of this shift is that hrHPV-/≥LSIL/CIN2+ (high-risk HPV negative cancers or pre-cancerous lesions with abnormal cytology results) can be missed. The objectives of the present study are to clarify in detail CIN2+ missed by this shift and to evaluate the feasibility of primary HPV testing in Japan. Data from 115,273 women who underwent co-testing with cytology and HPV testing in cancer screening were used in the current study. The cases with hrHPV-/≥LSIL ('hrHPV-/≥L-SIL' include CIN2-, in contrast, 'hrHPV-/≥L-SIL/CIN2+' doesn't include CIN2-) were analysed in detail. Women with hrHPV-/≥LSIL comprised 0.3% of the total. The prevalence of CIN2, CIN3, SCC or cervical adenocarcinomas in the lesions with HPV-/≥LSIL was 0.03% in the cancer screening group. Only one case of 14 cervical adenocarcinomas in ≥LSIL was hrHPV-. The prevalence of cancer missed by the shift in patients >50 years of age was significantly higher compared with patients younger than 49 years. In conclusion, the prevalence of CIN2+, which might be missed by the shift from primary cytology to primary HPV testing, was remarkably low in this Japanese cancer screening. The data indicated that primary HPV testing, which was more sensitive for CIN2+ than primary cytology, was a feasible method that can be used in Japan. In particular, primary HPV testing should be introduced for women <50 years old.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.

Construction of pseudomolecule sequences of the aus rice cultivar Kasalath for comparative genomics of Asian cultivated rice.

Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone.

A BAC physical map of aus rice cultivar 'Kasalath', and the map-based genomic sequence of 'Kasalath' chromosome 1.

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice (Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome (BAC) physical map for the aus rice cultivar 'Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar 'Nipponbare'. Examination of physical distances between computational and experimental measurements of 'Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of 'Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317,843 single-nucleotide polymorphisms (SNPs) and 66,331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome.

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis.

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene-enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous-nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome-wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri-nucleotide repeats were most common among the SSRs and were overrepresented in the protein-coding sequences, we found that selection against indels of tri-nucleotide repeats was relatively weak in both African and Asian rice. Our genome-wide sequencing of O. glaberrima and in-depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.

Comparative analysis of complete orthologous centromeres from two subspecies of rice reveals rapid variation of centromere organization and structure.

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. This function is conserved across species, but the DNA components that are involved in kinetochore formation differ greatly, even between closely related species. To shed light on the nature, evolutionary timing and evolutionary dynamics of rice centromeres, we decoded a 2.25-Mb DNA sequence covering the centromeric region of chromosome 8 of an indica rice variety, 'Kasalath' (Kas-Cen8). Analysis of repetitive sequences in Kas-Cen8 led to the identification of 222 long terminal repeat (LTR)-retrotransposon elements and 584 CentO satellite monomers, which account for 59.2% of the region. A comparison of the Kas-Cen8 sequence with that of japonica rice 'Nipponbare' (Nip-Cen8) revealed that about 66.8% of the Kas-Cen8 sequence was collinear with that of Nip-Cen8. Although the 27 putative genes are conserved between the two subspecies, only 55.4% of the total LTR-retrotransposon elements in 'Kasalath' had orthologs in 'Nipponbare', thus reflecting recent proliferation of a considerable number of LTR-retrotransposons since the divergence of two rice subspecies of indica and japonica within Oryza sativa. Comparative analysis of the subfamilies, time of insertion, and organization patterns of inserted LTR-retrotransposons between the two Cen8 regions revealed variations between 'Kasalath' and 'Nipponbare' in the preferential accumulation of CRR elements, and the expansion of CentO satellite repeats within the core domain of Cen8. Together, the results provide insights into the recent proliferation of LTR-retrotransposons, and the rapid expansion of CentO satellite repeats, underlying the dynamic variation and plasticity of plant centromeres.

Physical maps and recombination frequency of six rice chromosomes.

We constructed physical maps of rice chromosomes 1, 2, and 6-9 with P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones. These maps, with only 20 gaps, cover more than 97% of the predicted length of the six chromosomes. We submitted a total of 193 Mbp of non-overlapping sequences to public databases. We analyzed the DNA sequences of 1316 genetic markers and six centromere-specific repeats to facilitate characterization of chromosomal recombination frequency and of the genomic composition and structure of the centromeric regions. We found marked changes in the relative recombination rate along the length of each chromosome. Chromosomal recombination at the centromere core and surrounding regions on the six chromosomes was completely suppressed. These regions have a total physical length of about 23 Mbp, corresponding to 11.4% of the entire size of the six chromosomes. Chromosome 6 has the longest quiescent region, with about 5.6 Mbp, followed by chromosome 8, with quiescent region about half this size. Repetitive sequences accounted for at least 40% of the total genomic sequence on the partly sequenced centromeric region of chromosome 1. Rice CentO satellite DNA is arrayed in clusters and is closely associated with the presence of Centromeric Retrotransposon of Rice (CRR)- and RIce RetroElement 7 (RIRE7)-like retroelement sequences. We also detected relatively small coldspot regions outside the centromeric region; their repetitive content and gene density were similar to those of regions with normal recombination rates. Sequence analysis of these regions suggests that either the amount or the organization patterns of repetitive sequences may play a role in the inactivation of recombination.

The genome sequence and structure of rice chromosome 1.

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.