Infectious vaccine-derived rubella viruses emerge, persist, and evolve in cutaneous granulomas of children with primary immunodeficiencies.

Rubella viruses (RV) have been found in an association with granulomas in children with primary immune deficiencies (PID). Here, we report the recovery and characterization of infectious immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from diagnostic skin biopsies of four patients. Sequence evolution within PID hosts was studied by comparison of the complete genomic sequences of the iVDRVs with the genome of the vaccine virus RA27/3. The degree of divergence of each iVDRV correlated with the duration of persistence indicating continuous intrahost evolution. The evolution rates for synonymous and nonsynonymous substitutions were estimated to be 5.7 x 10-3 subs/site/year and 8.9 x 10-4 subs/site/year, respectively. Mutational spectra and signatures indicated a major role for APOBEC cytidine deaminases and a secondary role for ADAR adenosine deaminases in generating diversity of iVDRVs. The distributions of mutations across the genes and 3D hotspots for amino acid substitutions in the E1 glycoprotein identified regions that may be under positive selective pressure. Quasispecies diversity was higher in granulomas than in recovered infectious iVDRVs. Growth properties of iVDRVs were assessed in WI-38 fibroblast cultures. None of the iVDRV isolates showed complete reversion to wild type phenotype but the replicative and persistence characteristics of iVDRVs were different from those of the RA27/3 vaccine strain, making predictions of iVDRV transmissibility and teratogenicity difficult. However, detection of iVDRV RNA in nasopharyngeal specimen and poor neutralization of some iVDRV strains by sera from vaccinated persons suggests possible public health risks associated with iVDRV carriers. Detection of IgM antibody to RV in sera of two out of three patients may be a marker of virus persistence, potentially useful for identifying patients with iVDRV before development of lesions. Studies of the evolutionary dynamics of iVDRV during persistence will contribute to development of infection control strategies and antiviral therapies.

Association of BRCA1/2 defects with genomic scores predictive of DNA damage repair deficiency among breast cancer subtypes.

INTRODUCTION: Homologous recombination (HR) DNA repair is of clinical relevance in breast cancer. Three DNA-based homologous recombination deficiency (HRD) scores (HRD-loss of heterozygosity score (LOH), HRD-telomeric allelic imbalance score (TAI), and HRD-large-scale state transition score (LST)) have been developed that are highly correlated with defects in BRCA1/2, and are associated with response to platinum therapy in triple negative breast and ovarian cancer. This study examines the frequency of BRCA1/2 defects among different breast cancer subtypes, and the ability of the HRD scores to identify breast tumors with defects in the homologous recombination DNA repair pathway. METHODS: 215 breast tumors representing all ER/HER2 subtypes were obtained from commercial vendors. Next-generation sequencing based assays were used to generate genome wide SNP profiles, BRCA1/2 mutation screening, and BRCA1 promoter methylation data. RESULTS: BRCA1/2 deleterious mutations were observed in all breast cancer subtypes. BRCA1 promoter methylation was observed almost exclusively in triple negative breast cancer. BRCA1/2 deficient tumors were identified with BRCA1/2 mutations, or BRCA1 promoter methylation, and loss of the second allele of the affected gene. All three HRD scores were highly associated with BRCA1/2 deficiency (HRD-LOH: P = 1.3 × 10(-17); HRD-TAI: P = 1.5 × 10(-19); HRD-LST: P = 3.5 × 10(-18)). A combined score (HRD-mean) was calculated using the arithmetic mean of the three scores. In multivariable analyses the HRD-mean score captured significant BRCA1/2 deficiency information not captured by the three individual scores, or by clinical variables (P values for HRD-Mean adjusted for HRD-LOH: P = 1.4 × 10(-8); HRD-TAI: P = 2.9 × 10(-7); HRD-LST: P = 2.8 × 10(-8); clinical variables: P = 1.2 × 10(-16)). CONCLUSIONS: The HRD scores showed strong correlation with BRCA1/2 deficiency regardless of breast cancer subtype. The frequency of elevated scores suggests that a significant proportion of all breast tumor subtypes may carry defects in the homologous recombination DNA repair pathway. The HRD scores can be combined to produce a more robust predictor of HRD. The combination of a robust score, and the FFPE compatible assay described in this study, may facilitate use of agents targeting homologous recombination DNA repair in the clinical setting.

The genome of the domesticated apple (Malus × domestica Borkh.).

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes.

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16-q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed.

Sequencing and assembly of highly heterozygous genome of Vitis vinifera L. cv Pinot Noir: problems and solutions.

A new approach to sequencing and assembling a highly heterozygous genome, that of grape, species Vitis vinifera cv Pinot Noir, is described. The combining of genome shotgun of paired reads produced by Sanger sequencing and sequencing by synthesis of unpaired reads was shown to be an efficient procedure for decoding a complex genome. About 2 million SNPs and more than a million heterozygous gaps have been identified in the 500 Mb genome of grape. More than 91% of the sequence assembled into 58,611 contigs is now anchored to the 19 linkage groups of V. vinifera.

Concerted evolution of vertebrate CCR2 and CCR5 genes and the origin of a recombinant equine CCR5/2 gene.

Chemokine receptors (CCRs) play an essential role in the initiation of an innate immune host response. Several of these receptors have been shown to modulate the outcome of viral infections. The recent availability of complete genome sequences from a number of species provides a unique opportunity to analyze the evolution of the CCR genes. A phylogenetic analysis revealed that the CCR2 gene evolved in concert with the paralogous CCR5 gene, but not with another paralogous gene, CCR3, in the opossum, platypus, rabbit, guinea pig, cat, and rodent lineages. In addition, evidence of concerted evolution of the CCR2 and CCR5 genes was observed in chicken and lizard genomes. A unique CCR5/2 gene that originated by unequal crossing over between the CCR2 and CCR5 genes was detected in the domestic horse. The CCR2, CCR5, and CCR5/2 genes were mapped to ECA16q21 using fluorescent in situ hybridization (FISH). Single-nucleotide polymorphisms identified in the equine CCR5 gene and characterized within 5 horse breeds provide haplotype markers for future case/control studies investigating the genetic bases of horse susceptibility to infectious diseases.

A reference integrated map for cultivated grapevine (Vitis vinifera L.) from three crosses, based on 283 SSR and 501 SNP-based markers.

We have developed an integrated map from five elite cultivars of Vitis vinifera L.; Syrah, Pinot Noir, Grenache, Cabernet Sauvignon and Riesling which are parents of three segregating populations. A new source of markers, SNPs, identified in ESTs and unique BAC-end sequences was added to the available IGGP reference set of SSRs. The complete integrated map comprises 1,134 markers (350 AFLP, 332 BESs, 169 ESTs, 283 SSRs) spanning 1,443 cM over 19 linkage groups and shows a mean distance between neighbouring loci of 1.27 cM. Marker order was mainly conserved between the integrated map and the highly dense SyrahxPinot Noir consensus map except for few inversions. Moreover, the marker order has been validated through the assembled genome sequence of Pinot Noir. We have also assessed the transferability of SNP-based markers among five V. vinifera varieties, enabling marker validation across different genotypes. This integrated map can serve as a fundamental tool for molecular breeding in V. vinifera and related species and provide a basis for studies of genome organization and evolution in grapevines.

SNP high-throughput screening in grapevine using the SNPlex genotyping system.

BACKGROUND: Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods. RESULTS: Herein we report the first application of the SNPlexgenotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah x Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay. CONCLUSION: Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety.

BACKGROUND: Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. PRINCIPAL FINDINGS: We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). CONCLUSIONS: Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

Characterization of the equine 2'-5' oligoadenylate synthetase 1 (OAS1) and ribonuclease L (RNASEL) innate immunity genes.

BACKGROUND: The mammalian OAS/RNASEL pathway plays an important role in antiviral host defense. A premature stop-codon within the murine Oas1b gene results in the increased susceptibility of mice to a number of flaviviruses, including West Nile virus (WNV). Mutations in either the OAS1 or RNASEL genes may also modulate the outcome of WNV-induced disease or other viral infections in horses. Polymorphisms in the human OAS gene cluster have been previously utilized for case-control analysis of virus-induced disease in humans. No polymorphisms have yet been identified in either the equine OAS1 or RNASEL genes for use in similar case-control studies. RESULTS: Genomic sequence for equine OAS1 was obtained from a contig assembly generated from a shotgun subclone library of CHORI-241 BAC 100I10. Specific amplification of regions of the OAS1 gene from 13 horses of various breeds identified 33 single nucleotide polymorphisms (SNP) and two microsatellites. RNASEL cDNA sequences were determined for 8 mammals and utilized in a phylogenetic analysis. The chromosomal location of the RNASEL gene was assigned by FISH to ECA5p17-p16 using two selected CHORI-241 BAC clones. The horse genomic RNASEL sequence was assembled. Specific amplification of regions of the RNASEL gene from 13 horses identified 31 SNPs. CONCLUSION: In this report, two dinucleotide microsatellites and 64 single nucleotide polymorphisms within the equine OAS1 and RNASEL genes were identified. These polymorphisms are the first to be reported for these genes and will facilitate future case-control studies of horse susceptibility to infectious diseases.

The mammalian 2'-5' oligoadenylate synthetase gene family: evidence for concerted evolution of paralogous Oas1 genes in Rodentia and Artiodactyla.

Multiple 2'-5' oligoadenylate (2-5A) synthetases are important components of innate immunity in mammals. Gene families encoding these proteins have previously been studied mainly in humans and mice. To reconstruct the evolution of this gene family in mammals, a search for additional 2-5A synthetase genes was performed in rat, cattle, pig, and dog. Twelve 2'-5' oligoadenylate synthetase (Oas) genes were identified in the rat genome, including eight Oas1 genes, two Oas1 pseudogenes, single copies of Oas2 and Oas3, and two Oas-like genes, Oasl1 and Oasl2. Four OAS genes were detected in the pig genome and five OAS genes were found in both the cattle and dog genomes. An OAS3 gene was not found in either the cattle or the pig genome. While two tandemly duplicated OAS-like (OASL) genes were identified in the dog genome, only a single OASL orthologue was found in both the cattle and the pig genomes. The bovine and porcine OASL genes contain premature stop codons and encode truncated proteins, which lack the typical C-terminal double ubiquitin domains. The cDNA sequences of the rat, cattle, pig, and dog OAS genes were amplified, sequenced and compared with each other and with those in the human, mouse, horse, and chicken genomes. Evidence of concerted evolution of paralogous 2'-5' oligoadenylate synthetase 1 genes was obtained in rodents (Rodentia) and even-toed ungulates (Artiodactyla). Calculations using the nonparametric Kolmogorov-Smirnov test suggested that the homogenization of paralogous OAS1 sequences was due to gene conversion rather than stabilizing selection.

Comparative analysis of vertebrate EIF2AK2 (PKR) genes and assignment of the equine gene to ECA15q24-q25 and the bovine gene to BTA11q12-q15.

The structures of the canine, rabbit, bovine and equine EIF2AK2 genes were determined. Each of these genes has a 5' non-coding exon as well as 15 coding exons. All of the canine, bovine and equine EIF2AK2 introns have consensus donor and acceptor splice sites. In the equine EIF2AK2 gene, a unique single nucleotide polymorphism that encoded a Tyr329Cys substitution was detected. Regulatory elements predicted in the promoter region were conserved in ungulates, primates, rodents, Afrotheria (elephant) and Insectifora (shrew). Western clawed frog and fugu EIF2AK2 gene sequences were detected in the USCS Genome Browser and compared to those of other vertebrate EIF2AK2 genes. A comparison of EIF2AK2 protein domains in vertebrates indicates that the kinase catalytic domains were evolutionarily more conserved than the nucleic acid-binding motifs. Nucleotide substitution rates were uniform among the vertebrate sequences with the exception of the zebrafish and goldfish EIF2AK2 genes, which showed substitution rates about 20% higher than those of other vertebrates. FISH was used to physically assign the horse and cattle genes to chromosome locations, ECA15q24-q25 and BTA11q12-15, respectively. Comparative mapping data confirmed conservation of synteny between ungulates, humans and rodents.

Seasonal differences and protection by creatine or arginine pretreatment in ischemia of mammalian and molluscan neurons in vitro.

We investigated the dose-response relationship of protection by creatine against ischemic damage, and we asked whether or not such protection may be observed in invertebrate neurons that might provide a simpler experimental model. Rat isolated pyramidal neurons from the CA3 region of hippocampus subjected to ischemia ("in vitro ischemia") showed anoxic depolarization (AD) after 3-7 min of incubation in anoxic medium. Membrane potential (MP) was reduced 25-78% from preanoxic value. Inward current was decreased by an average 49%. Supplementation with creatine protected against these changes, with 1 mM being the minimal effective concentration, 2 mM providing a near-maximal protection, a maximal effect being obtained with 5 mM creatine. No additional protection was provided by up to 20 mM creatine. Isolated giant neurons of Lymnaea stagnalis showed a similar response to in vitro ischemia. However, a clear seasonal dependence of sensitivity of these cells was detected. In cells obtained during summertime (May-August), AD latency ranged from 3 to 10 min; during wintertime (December-March), this response did not occur even after 25-50 min. The addition of creatine to the medium did not cause changes in AD latency, probably because these neurons rely on a phosphoarginine/arginine energy system. However, treatment of the cells, harvested during summertime, with 2 mM arginine did provide clear protection against anoxic-aglycaemic changes. Summing up, besides confirming previous findings on creatine protection in mammalian neurons, we (1) better characterized their dose-response relationship and extended the findings to the CA3 region and to isolated neurons, (2) found that invertebrate neurons are not protected by creatine but by arginine supplementation and (3) reported a novel mechanism of seasonal dependence in sensitivity of in vitro ischemia by invertebrate neurons.

Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes.

The compact genome of Fugu rubripes has been sequenced to over 95% coverage, and more than 80% of the assembly is in multigene-sized scaffolds. In this 365-megabase vertebrate genome, repetitive DNA accounts for less than one-sixth of the sequence, and gene loci occupy about one-third of the genome. As with the human genome, gene loci are not evenly distributed, but are clustered into sparse and dense regions. Some "giant" genes were observed that had average coding sequence sizes but were spread over genomic lengths significantly larger than those of their human orthologs. Although three-quarters of predicted human proteins have a strong match to Fugu, approximately a quarter of the human proteins had highly diverged from or had no pufferfish homologs, highlighting the extent of protein evolution in the 450 million years since teleosts and mammals diverged. Conserved linkages between Fugu and human genes indicate the preservation of chromosomal segments from the common vertebrate ancestor, but with considerable scrambling of gene order.

A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.