Identifying Circulating MicroRNA in Kawasaki Disease by Next-Generation Sequencing Approach

Kawasaki disease (KD) typically occurs in children aged under 5 years and can cause coronary artery lesions (CALs). Early diagnosis and treatment with intravenous immunoglobulin can reduce the occurrence of CALs; therefore, identifying a good biomarker for diagnosing KD is essential. Here, using next-generation sequencing in patients with recurrent KD, those with viral infection, and healthy controls, we identified dysregulated circulating microRNAs as diagnostic biomarkers for KD. Pathway enrichment analysis illustrated the putative role of these miRNAs in KD progression. Their expression levels were validated using real-time polymerase chain reaction (qPCR). Fifteen dysregulated circulating miRNAs (fold changes >2 and <0.5) were differentially expressed in the recurrent KD group compared with the viral infection and control groups. These miRNAs were significantly involved in the transforming growth factor-β, epithelial–mesenchymal transition, and cell apoptosis signaling pathways. Notably, their expression levels were frequently restored after intravenous immunoglobulin treatment. Among the candidates, miR-24-3p expression level was significantly higher in patients with recurrent KD compared with healthy controls or viral infection controls (p < 0.001). Receiver operating characteristic analysis revealed that high miR-24-3p expression levels may be a potential biomarker for KD diagnosis. In conclusion, we identified miR-24-3p significantly higher in KD patients, which may be a potential diagnostic biomarker for KD.     

A Novel Predicted Calcium-Regulated Kinase Family Implicated in Neurological Disorders

The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca²⁺-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca²⁺-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1) and CXORF36 (DIA1R), are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins.     

Whole-Genome Sequencing Analysis from the Chikungunya Virus Caribbean Outbreak Reveals Novel Evolutionary Genomic Elements

Background

Chikungunya virus (CHIKV), an alphavirus and member of the Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naïve populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak.

Methodology/Principle Findings

We used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3’ untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells.

Conclusions/Significance

Here we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3’UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naïve population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV.     

Targeted Next-Generation Sequencing Reveals Novel USH2A Mutations Associated with Diverse Disease Phenotypes: Implications for Clinical and Molecular Diagnosis

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also demonstrated that a targeted NGS approach is a valuable tool for the genetic diagnosis of USH2 and RP.     

Targeted Next-Generation Sequencing Revealed Novel Mutations in Chinese Ataxia Telangiectasia Patients: A Precision Medicine Perspective

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia and immunodeficiency due to mutations in the ATM gene. We performed targeted next-generation sequencing (NGS) on three unrelated patients and identified five disease-causing variants in three probands, including two pairs of heterozygous variants (FAT–1:c.4396C>T/p.R1466X, c.1608-2A>G; FAT–2:c.4412_4413insT/p.L1472Ffs*19, c.8824C>T/p.Q2942X) and one pair of homozygous variants (FAT–3: c.8110T>G/p.C2704G, Hom). With regard to precision medicine for rare genetic diseases, targeted NGS currently enables the rapid and cost-effective identification of causative mutations and is an updated molecular diagnostic tool that merits further optimization. This high-throughput data-based strategy would propel the development of precision diagnostic methods and establish a foundation for precision medicine.     

一种快速简便构建DNA病毒测序文库的方法

为了对1株中国棉铃虫核型多角体缺失病毒HZ-9进行基因组测序,采用了一种新的方法,通过超声波振断HaBacHZ9细菌人工染色体质粒（bacterial artificial chromosome plasmid,Bacmid）基因组DNA,用Taq酶在DNA片段两端加腺噤呤A,胶回收后得到预期的1—2kb的DNA片段,然后与pGEM-Teasy载体连接,构建了中国棉铃虫缺失病毒HaBacHZ9的亚克隆文库。结果随机挑选10个克隆子酶切分析,显示9个克隆子有1500bp左右的插入片断,并对HaBaeHZ9进行了全基因组测序。结论成功构建了HaBaeHZ9的DNA测序文库,为HZ-9功能基因组学研究奠定了基础,这是一种简单快速的构建DNA病毒测序文库的方法。     

A Novel DNA Polymerase Family Found in Archaea

One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeon Pyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes in M. jannaschii encoding the proteins that are homologous to the DNA polymerase II of P. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia coli had both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.     

Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC.     

Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer

Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by de novo sequencing using a next-generation DNA sequencer on total RNAs extracted from an autopsy lung of a patient who died of viral pneumonia with A/H1N1/2009. Among a total of 9.4×10⁶ 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than ×200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly₁₇₂Glu and Gly₂₃₉Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.     

Structural,Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.Plants produce a vast array of secondary metabolites, many of them being restricted to specific groups of plant species. This extraordinary chemical diversity is believed to have evolved from a limited number of ubiquitous biosynthetic pathways through gene duplication followed by functional divergence (Pichersky and Gang, 2000). The phenylpropanoid pathway, derived from Phe, illustrates perfectly this phenomenon, as it gives rise to a large diversity of phenolic compounds playing key roles in plants, including participation in structural polymers, defense against herbivores and pathogens, protection from abiotic stress, and important functions in plant-pollinator interactions. Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including dicotyledon angiosperms such as grapevine (Vitis vinifera), peanut (Arachis hypogaea), and Japanese knotweed (Fallopia japonica, formerly Polygonum cuspidatum), monocotyledons like sorghum (Sorghum bicolor), and gymnosperms such as several Pinus and Picea species. In addition to their participation in both constitutive and inducible defense mechanisms in plants, several stilbenes display important pharmacological properties. Since resveratrol (3,5,4′-trihydroxy-trans-stilbene) was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Renaud and de Lorgeril, 1992), plant stilbenes have received considerable interest. Nowadays, resveratrol ranks among the most extensively studied natural products, and hundreds of studies have shown that it can slow the progression of a wide variety of illnesses, including cancer and cardiovascular disease, as well as extend the life spans of various organisms (Baur and Sinclair, 2006). Stilbene synthases (STSs) are characteristic of stilbene-producing plants and catalyze the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative. STSs are members of the type III polyketide synthases family, chalcone synthases (CHSs), which catalyze the first step of flavonoid biosynthesis, being the most ubiquitous polyketide synthase in plants. Both CHS and STS use p-coumaroyl-CoA and malonyl-CoA as substrates and synthesize the same linear tetraketide intermediate. However, STS uses a specific cyclization mechanism involving a decarboxylation to form the stilbene backbone. STS proteins share extensive amino acid sequence identity with CHS, and phylogenetic analysis of the STS and CHS gene families has shown that STS genes may have evolved from CHS genes several times independently (Tropf et al., 1994). In most stilbene-producing plants, STS genes form small families of closely related paralogs. For example, two STS cDNAs have been cloned from peanut (Schröder et al., 1988), the genome of Scots pine (Pinus sylvestris) has been shown to contain a small family of four STS genes (Preisig-Müller et al., 1999), and three STS genes have been characterized in Japanese red pine (Pinus densiflora; Kodan et al., 2002). Only one STS gene has been isolated from Japanese knotweed to date (Liu et al., 2011), and the sequencing of sorghum genome has shown that SbSTS1 was the only STS gene in this plant species (Yu et al., 2005; Paterson et al., 2009). Grapevine is a noteworthy exception among stilbene-producing plants, as its genome has been shown to contain a large family of putative STS genes. Early Southern-blot experiments suggested that the grapevine genome contained more than 20 STS genes (Sparvoli et al., 1994). Analyses of the first drafts of the grapevine genome sequence confirmed the large size of this multigene family, with an estimated number of STS genes ranging from 21 to 43 (Jaillon et al., 2007; Velasco et al., 2007). However, these relatively low-coverage sequence drafts did not allow a precise analysis of large families of highly similar genes. The more recently released 12× genome sequence of grapevine inbred Pinot Noir cultivar PN40024 offered an improved sequence quality, allowing an accurate analysis of the STS gene family. In this work, we take advantage of the improved 12× sequence of the grapevine ‘PN40024’ genome to analyze the grapevine STS gene family. Furthermore, we combine molecular evolution to structural and functional analyses to gain more insight into the significance of the remarkable amplification of the STS family in grapevine.     

Induction of Cross-Reactive Antibodies to Novel H7N9 Influenza Virus by Recombinant Newcastle Disease Virus Expressing a North American Lineage H7 Subtype Hemagglutinin

Severe human disease caused by the emerging H7N9 influenza virus in China warrants a rapid response. Here, we present a recombinant Newcastle disease virus expressing a North American lineage H7 influenza virus hemagglutinin. Sera from immunized mice were cross-reactive to a broad range of H7 subtype viruses and inhibited hemagglutination by the novel H7 hemagglutinin. Immunized mice were protected against a heterologous H7 subtype challenge, and genetic analysis suggested that cross-protective antibodies recognize conserved antigenic sites.     

Isolation and Identification of a Novel Rabies Virus Lineage in China with Natural Recombinant Nucleoprotein Gene

Rabies virus (RABV) causes severe neurological disease and death. As an important mechanism for generating genetic diversity in viruses, homologous recombination can lead to the emergence of novel virus strains with increased virulence and changed host tropism. However, it is still unclear whether recombination plays a role in the evolution of RABV. In this study, we isolated and sequenced four circulating RABV strains in China. Phylogenetic analyses identified a novel lineage of hybrid origin that comprises two different strains, J and CQ92. Analyses revealed that the virus 3′ untranslated region (UTR) and part of the N gene (approximate 500 nt in length) were likely derived from Chinese lineage I while the other part of the genomic sequence was homologous to Chinese lineage II. Our findings reveal that homologous recombination can occur naturally in the field and shape the genetic structure of RABV populations.     

基于新一代测序技术的mtDNA突变检测方法学的建立

目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法.     

Identification and Clinical Implications of Novel MYO15A Mutations in a Non-consanguineous Korean Family by Targeted Exome Sequencing

Mutations of MYO15A are generally known to cause severe to profound hearing loss throughout all frequencies. Here, we found two novel MYO15A mutations, c.3871C>T (p.L1291F) and c.5835T>G (p.Y1945X) in an affected individual carrying congenital profound sensorineural hearing loss (SNHL) through targeted resequencing of 134 known deafness genes. The variant, p.L1291F and p.Y1945X, resided in the myosin motor and IQ2 domains, respectively. The p.L1291F variant was predicted to affect the structure of the actin-binding site from three-dimensional protein modeling, thereby interfering with the correct interaction between actin and myosin. From the literature analysis, mutations in the N-terminal domain were more frequently associated with residual hearing at low frequencies than mutations in the other regions of this gene. Therefore we suggest a hypothetical genotype-phenotype correlation whereby MYO15A mutations that affect domains other than the N-terminal domain, lead to profound SNHL throughout all frequencies and mutations that affect the N-terminal domain, result in residual hearing at low frequencies. This genotype-phenotype correlation suggests that preservation of residual hearing during auditory rehabilitation like cochlear implantation should be intended for those who carry mutations in the N-terminal domain and that individuals with mutations elsewhere in MYO15A require early cochlear implantation to timely initiate speech development.     

DNA Configuration in the Core of Marek''s Disease Virus

Marek's disease virus DNA appears to be wound around a central structure connecting the two inner poles of the capsid. Based on electron micrographs of Marek's disease virions, a diagram of spatial configuration of virus DNA is presented. This diagram may explain the pleomorphic character of the herpesvirus core observed with the electron microscope.     

A Novel Virus Causes Scale Drop Disease in Lates calcarifer

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.     

A Novel Evolutionary Strategy Revealed in the Phaeoviruses

Phaeoviruses infect the brown algae, which are major contributors to primary production of coastal waters and estuaries. They exploit a Persistent evolutionary strategy akin to a K- selected life strategy via genome integration and are the only known representatives to do so within the giant algal viruses that are typified by r- selected Acute lytic viruses. In screening the genomes of five species within the filamentous brown algal lineage, here we show an unprecedented diversity of viral gene sequence variants especially amongst the smaller phaeoviral genomes. Moreover, one variant shares features from both the two major sub-groups within the phaeoviruses. These phaeoviruses have exploited the reduction of their giant dsDNA genomes and accompanying loss of DNA proofreading capability, typical of an Acute life strategist, but uniquely retain a Persistent life strategy.