Introduction of SARS-CoV-2 C.37 (WHO VOI lambda) in the Sao Paulo State, Southeast Brazil.

The Lambda variants of interest (VOI) (C37/GR/452Q.V1/21G) was initially reported in Lima, Peru but has gained rapid dissemination through other Latin American countries. Nevertheless, the dissemination and molecular epidemiology of the Lambda VOI in Brazil is unknown apart from a single case report. In this respect, we characterized the circulation of the SARS-CoV-2 Lambda VOI (C37/GR/452Q.V1/21G) in Sao Paulo State, Brazil. From March to June 2021, we identified seven Lambda isolates in a set of approximately 8000 newly sequenced genomes of the Network for Pandemic Alert of Emerging SARS-CoV-2 variants from Sao Paulo State. Interestingly, in three of the positive patients, the Lambda VOI infection was probably related to a contact transmission. These individuals were fully vaccinated to COVID-19 and presented mild symptoms. The remaining positive for Lambda VOI individuals showed different levels of COVID-19 symptoms and one of them needed hospitalization (score 5, WHO). In our study, we present a low level of Lambda VOI circulation in the Sao Paulo State. This reinforces the essential role of molecular surveillance for the effective SARS-CoV-2 pandemic response, especially in regard to circulating variants.

Genomic monitoring unveil the early detection of the SARS-CoV-2 B.1.351 (beta) variant (20H/501Y.V2) in Brazil.

Sao Paulo State, currently experiences a second COVID-19 wave overwhelming the healthcare system. Due to the paucity of SARS-CoV-2 complete genome sequencing, we established a Network for Pandemic Alert of Emerging SARS-CoV-2 Variants to rapidly understand and monitor the spread of SARS-CoV-2 variants into the state. Through analysis of 210 SARS-CoV-2 complete genomes obtained from the largest regional health departments we identified cocirculation of multiple SARS-CoV-2 lineages such as B.1.1 (0.5%), B.1.1.28 (23.2%), B.1.1.7 (alpha variant, 6.2%), B.1.566 (1.4%), B.1.544 (0.5%), C.37 (0.5%) P.1 (gamma variant, 66.2%), and P.2 (zeta variant, 1.0%). Our analysis allowed also the detection, for the first time in Brazil, the South African B.1.351 (beta) variant of concern, B.1.351 (501Y.V2) (0.5%), characterized by the following mutations: ORF1ab: T265I, R724K, S1612L, K1655N, K3353R, SGF 3675_F3677del, P4715L, E5585D; spike: D80A, D215G, L242_L244del, A262D, K417N, E484K, N501Y, D614G, A701V, C1247F; ORF3a: Q57H, S171L, E: P71L; ORF7b: Y10F, N: T205I; ORF14: L52F. The most recent common ancestor of the identified strain was inferred to be mid-October to late December 2020. Our analysis demonstrated the P.1 lineage predominance and allowed the early detection of the South African strain for the first time in Brazil. We highlight the importance of SARS-CoV-2 active monitoring to ensure the rapid detection of potential variants for pandemic control and vaccination strategies. Highlights Identification of B.1.351 (beta) variant of concern in the Sao Paulo State. Dissemination of SARS-CoV-2 variants of concern and interest in the Sao Paulo State. Mutational Profile of the circulating variants of concern and interest.

Introduction of SARS-CoV-2 C.37 (WHO VOI lambda) in the Sao Paulo state, southeast Brazil

The Lambda variants of interest (VOI) (C37/GR/452Q.V1/21G) was initially reported in Lima, Peru but has gained rapid dissemination through other Latin American countries. Nevertheless, the dissemination and molecular epidemiology of the Lambda VOI in Brazil is unknown apart from a single case report. In this respect, we characterized the circulation of the SARS-CoV-2 Lambda VOI (C37/GR/452Q.V1/21G) in Sao Paulo State, Brazil. From March to June 2021, we identified seven Lambda isolates in a set of approximately 8000 newly sequenced genomes of the Network for Pandemic Alert of Emerging SARS-CoV-2 variants from Sao Paulo State. Interestingly, in three of the positive patients, the Lambda VOI infection was probably related to a contact transmission. These individuals were fully vaccinated to COVID-19 and presented mild symptoms. The remaining positive for Lambda VOI individuals showed different levels of COVID-19 symptoms and one of them needed hospitalization (score 5, WHO). In our study, we present a low level of Lambda VOI circulation in the Sao Paulo State. This reinforces the essential role of molecular surveillance for the effective SARS-CoV-2 pandemic response, especially in regard to circulating variants.

Genomic monitoring unveil the early detection of the SARS-CoV-2 B.1.351 (beta) variant (20H/501Y.V2) in Brazil

Sao Paulo State, currently experiences a second COVID-19 wave overwhelming the healthcare system. Due to the paucity of SARS-CoV-2 complete genome sequencing, we established a Network for Pandemic Alert of Emerging SARS-CoV-2 Variants to rapidly understand and monitor the spread of SARS-CoV-2 variants into the state. Through analysis of 210 SARS-CoV-2 complete genomes obtained from the largest regional health departments we identified cocirculation of multiple SARS-CoV-2 lineages such as B.1.1 (0.5%), B.1.1.28 (23.2%), B.1.1.7 (alpha variant, 6.2%), B.1.566 (1.4%), B.1.544 (0.5%), C.37 (0.5%) P.1 (gamma variant, 66.2%), and P.2 (zeta variant, 1.0%). Our analysis allowed also the detection, for the first time in Brazil, the South African B.1.351 (beta) variant of concern, B.1.351 (501Y.V2) 0.5%, characterized by the following mutations: ORF1ab: T265I, R724K, S1612L, K1655N, K3353R, SGF 3675_F3677del, P4715L, E5585D; spike: D80A, D215G, L242_L244del, A262D, K417N, E484K, N501Y, D614G, A701V, C1247F; ORF3a: Q57H, S171L, E: P71L; ORF7b: Y10F, N: T205I; ORF14: L52F. The most recent common ancestor of the identified strain was inferred to be mid-October to late December 2020. Our analysis demonstrated the P.1 lineage predominance and allowed the early detection of the South African strain for the first time in Brazil. We highlight the importance of SARS-CoV-2 active monitoring to ensure the rapid detection of potential variants for pandemic control and vaccination strategies.

Application of Whole-Exome Sequencing in Detecting Copy Number Variants in Patients with Developmental Delay and/or Multiple Congenital Malformations.

Overcoming challenges for the unambiguous detection of copy number variations is essential to broaden our understanding of the role of genomic variants in the clinical phenotype. With the improvement of software and databases, whole-exome sequencing quickly can become an excellent strategy in the routine diagnosis of patients with a developmental delay and/or multiple congenital malformations. However, even after a detailed analysis of pathogenic single-nucleotide variants and indels in known disease genes, using whole-exome sequencing, some patients with suspected syndromic conditions are left without a conclusive diagnosis. These negative results could be the result of different factors including nongenetic etiologies, lack of knowledge about the genes that cause different disease phenotypes, or, in some cases, a deletion or duplication of genomic information not routinely detectable by whole-exome sequencing variant calling. Although copy number variant detection is possible using whole-exome sequencing data, such analysis presents significant challenges and cannot yet be used to replace chromosomal arrays for identification of deletions or duplications.

A rare genomic duplication in 2p14 underlies autosomal dominant hearing loss DFNA58.

Here we define a ~200 Kb genomic duplication in 2p14 as the genetic signature that segregates with postlingual progressive sensorineural autosomal dominant hearing loss (HL) in 20 affected individuals from the DFNA58 family, first reported in 2009. The duplication includes two entire genes, PLEK and CNRIP1, and the first exon of PPP3R1 (protein coding), in addition to four uncharacterized long non-coding (lnc) RNA genes and part of a novel protein-coding gene. Quantitative analysis of mRNA expression in blood samples revealed selective overexpression of CNRIP1 and of two lncRNA genes (LOC107985892 and LOC102724389) in all affected members tested, but not in unaffected ones. Qualitative analysis of mRNA expression identified also fusion transcripts involving parts of PPP3R1, CNRIP1 and an intergenic region between PLEK and CNRIP1, in the blood of all carriers of the duplication, but were heterogeneous in nature. By in situ hybridization and immunofluorescence, we showed that Cnrip1, Plek and Ppp3r1 genes are all expressed in the adult mouse cochlea including the spiral ganglion neurons, suggesting changes in expression levels of these genes in the hearing organ could underlie the DFNA58 form of deafness. Our study highlights the value of studying rare genomic events leading to HL, such as copy number variations. Further studies will be required to determine which of these genes, either coding proteins or non-coding RNAs, is or are responsible for DFNA58 HL.

Loss of TNR causes a nonprogressive neurodevelopmental disorder with spasticity and transient opisthotonus.

PURPOSE: TNR, encoding Tenascin-R, is an extracellular matrix glycoprotein involved in neurite outgrowth and neural cell adhesion, proliferation and migration, axonal guidance, myelination, and synaptic plasticity. Tenascin-R is exclusively expressed in the central nervous system with highest expression after birth. The protein is crucial in the formation of perineuronal nets that ensheath interneurons. However, the role of Tenascin-R in human pathology is largely unknown. We aimed to establish TNR as a human disease gene and unravel the associated clinical spectrum. METHODS: Exome sequencing and an online matchmaking tool were used to identify patients with biallelic variants in TNR. RESULTS: We identified 13 individuals from 8 unrelated families with biallelic variants in TNR sharing a phenotype consisting of spastic para- or tetraparesis, axial muscular hypotonia, developmental delay, and transient opisthotonus. Four homozygous loss-of-function and four different missense variants were identified. CONCLUSION: We establish TNR as a disease gene for an autosomal recessive nonprogressive neurodevelopmental disorder with spasticity and transient opisthotonus and highlight the role of central nervous system extracellular matrix proteins in the pathogenicity of spastic disorders.

Heterozygous loss of function of NR4A2 is associated with intellectual deficiency, rolandic epilepsy, and language impairment.

Recognition of a de novo mutation in NR4A2 associated with a neurodevelopmental phenotype reinforces its role in 2q23q24 microdeletion syndrome. Using the proband WES data and the probability of loss-of-function intolerance index (pLi) set at 1.0 (highest intolerance constraint), we could target NR4A2 as the candidate gene in this patient.

RNAseq Transcriptional Profiling following Whip Development in Sugarcane Smut Disease.

Sugarcane smut disease is caused by the biotrophic fungus Sporisorium scitamineum. The disease is characterized by the development of a whip-like structure from the primary meristems, where billions of teliospores are produced. Sugarcane smut also causes tillering and low sucrose and high fiber contents, reducing cane productivity. We investigated the biological events contributing to disease symptoms in a smut intermediate-resistant sugarcane genotype by examining the transcriptional profiles (RNAseq) shortly after inoculating the plants and immediately after whip emission. The overall picture of disease progression suggests that premature transcriptional reprogramming of the shoot meristem functions continues until the emergence of the whip. The guidance of this altered pattern is potentially primarily related to auxin mobilization in addition to the involvement of other hormonal imbalances. The consequences associated with whip emission are the modulation of typical meristematic functions toward reproductive organ differentiation, requiring strong changes in carbon partitioning and energy production. These changes include the overexpression of genes coding for invertases and trehalose-6P synthase, as well as other enzymes from key metabolic pathways, such as from lignin biosynthesis. This is the first report describing changes in the transcriptional profiles following whip development, providing a hypothetical model and candidate genes to further study sugarcane smut disease progression.

Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome.

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patient's fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology.

Complete Genome Sequence of Sporisorium scitamineum and Biotrophic Interaction Transcriptome with Sugarcane.

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

Combinatory microarray and SuperSAGE analyses identify pairing-dependently transcribed genes in Schistosoma mansoni males, including follistatin.

BACKGROUND: Schistosomiasis is a disease of world-wide importance and is caused by parasitic flatworms of the genus Schistosoma. These parasites exhibit a unique reproduction biology as the female's sexual maturation depends on a constant pairing-contact to the male. Pairing leads to gonad differentiation in the female, and even gene expression of some gonad-associated genes is controlled by pairing. In contrast, no morphological changes have been observed in males, although first data indicated an effect of pairing also on gene transcription in males. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the influence of pairing on males, we performed a combinatory approach applying SuperSAGE and microarray hybridization, generating the most comprehensive data-set on differential transcription available to date. Of 6,326 sense transcripts detected by both analyses, 29 were significantly differentially transcribed. Besides mutual confirmation, the two methods complemented each other as shown by data comparison and real-time PCR, which revealed a number of genes with consistent regulation across all methods. One of the candidate genes, follistatin of S. mansoni (SmFst) was characterized in more detail by in situ hybridization and yeast two-hybrid (Y2H) interaction analyses with potential binding partners. CONCLUSIONS/SIGNIFICANCE: Beyond confirming previously hypothesized differences in metabolic processes between pairing-experienced (EM) and pairing-unexperienced males (UM), our data indicate that neuronal processes are involved in male-female interaction but also TGFß-signaling. One candidate revealing significant down-regulation in EM was the TGFß-pathway controlling molecule follistatin (SmFst). First functional analyses demonstrated SmFst interaction with the S. mansoni TGFß-receptor agonists inhibin/activin (SmInAct) and bone morphogenic protein (SmBMP), and all molecules colocalized in the testes. This indicates a yet unknown role of the TGFß-pathway for schistosome biology leading to male competence and a possible influence of pairing on the male gonad.

Non-coding RNAs in schistosomes: an unexplored world.

Non-coding RNAs (ncRNAs) were recently given much higher attention due to technical advances in sequencing which expanded the characterization of transcriptomes in different organisms. ncRNAs have different lengths (22 nt to >1,000 nt) and mechanisms of action that essentially comprise a sophisticated gene expression regulation network. Recent publication of schistosome genomes and transcriptomes has increased the description and characterization of a large number of parasite genes. Here we review the number of predicted genes and the coverage of genomic bases in face of the public ESTs dataset available, including a critical appraisal of the evidence and characterization of ncRNAs in schistosomes. We show expression data for ncRNAs in Schistosoma mansoni. We analyze three different microarray experiment datasets: (1) adult worms' large-scale expression measurements; (2) differentially expressed S. mansoni genes regulated by a human cytokine (TNF-α) in a parasite culture; and (3) a stage-specific expression of ncRNAs. All these data point to ncRNAs involved in different biological processes and physiological responses that suggest functionality of these new players in the parasite's biology. Exploring this world is a challenge for the scientists under a new molecular perspective of host-parasite interactions and parasite development.

Non-coding RNAs in schistosomes: an unexplored world

Non-coding RNAs (ncRNAs) were recently given much higher attention due to technical advances in sequencing which expanded the characterization of transcriptomes in different organisms. ncRNAs have different lengths (22 nt to >1, 000 nt) and mechanisms of action that essentially comprise a sophisticated gene expression regulation network. Recent publication of schistosome genomes and transcriptomes has increased the description and characterization of a large number of parasite genes. Here we review the number of predicted genes and the coverage of genomic bases in face of the public ESTs dataset available, including a critical appraisal of the evidence and characterization of ncRNAs in schistosomes. We show expression data for ncRNAs in Schistosoma mansoni. We analyze three different microarray experiment datasets: (1) adult worms' large-scale expression measurements; (2) differentially expressed S. mansoni genes regulated by a human cytokine (TNF-α) in a parasite culture; and (3) a stage-specific expression of ncRNAs. All these data point to ncRNAs involved in different biological processes and physiological responses that suggest functionality of these new players in the parasite's biology. Exploring this world is a challenge for the scientists under a new molecular perspective of host-parasite interactions and parasite development.

RNAs não codificadores (ncRNAs) têm sido recentemente objeto de atenção muito maior devido aos avanços técnicos no sequenciamento que expandiram a caracterização dos transcritomas em diferentes organismos. ncRNAs possuem diferentes comprimentos (22 nt a >1.000 nt) e mecanismos de ação que essencialmente compreendem uma sofisticada rede de regulação de expressão gênica. A publicação recente dos genomas e transcritomas dos esquistossomos aumentou a descrição e caracterização de um grande número de genes do parasita. Aqui nós revisamos o número de genes preditos e a cobertura das bases do genoma em face dos ESTs públicos disponíveis, incluindo uma avaliação crítica da evidência e caracterização de ncRNAs em esquistossomos. Nós mostramos dados de expressão de ncRNAs em Schistosoma mansoni. Nós analisamos três conjuntos diferentes de dados de experimentos com microarranjos: (1) medidas de expressão em larga escala de vermes adultos; (2) genes diferencialmente expressos de S. mansoni regulados por uma citocina humana (TNF-α) no parasita em cultura; e (3) expressão estágio-especifica de ncRNAs. Todos estes dados apontam para ncRNAs envolvidos em diferentes processos biológicos e respostas fisiológicas que sugerem funcionalidade destes novos personagens na biologia do parasita. Explorar este mundo é um desafio para os cientistas sob uma nova perspectiva molecular da interação parasita-hospedeiro e do desenvolvimento do parasita.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli.

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Comparative analysis of the complete plastid genome sequence of the red alga Gracilaria tenuistipitata var. liui provides insights into the evolution of rhodoplasts and their relationship to other plastids.

We sequenced to completion the circular plastid genome of the red alga Gracilaria tenuistipitata var. liui. This is the first plastid genome sequence from the subclass Florideophycidae (Rhodophyta). The genome is composed of 183,883 bp and contains 238 predicted genes, including a single copy of the ribosomal RNA operon. Comparisons with the plastid genome of Porphyra pupurea reveal strong conservation of gene content and order, but we found major genomic rearrangements and the presence of coding regions that are specific to Gracilaria. Phylogenetic analysis of a data set of 41 concatenated proteins from 23 plastid and two cyanobacterial genomes support red algal plastid monophyly and a specific evolutionary relationship between the Florideophycidae and the Bangiales. Gracilaria maintains a surprisingly ancient gene content in its plastid genome and, together with other Rhodophyta, contains the most complete repertoire of plastid genes known in photosynthetic eukaryotes.

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.