Novel Pathogenic Mutation of P209L in TRPC6 Gene Causes Adult Focal Segmental Glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a leading kidney disease, clinically associated with proteinuria and progressive renal failure. The occurrence of this disease is partly related to gene mutations. We describe a single affected family member who presented with FSGS. We used high-throughput sequencing, sanger sequencing to identify the pathogenic mutations, and a systems genetics analysis in the BXD mice was conducted to explore the genetic regulatory mechanisms of pathogenic genes in the development of FSGS. We identified high urinary protein (++++) and creatinine levels (149 µmol/L) in a 29-year-old male diagnosed with a 5-year history of grade 2 hypertension. Histopathology of the kidney biopsy showed stromal hyperplasia at the glomerular segmental sclerosis and endothelial cell vacuolation degeneration. Whole-exome sequencing followed by Sanger sequencing revealed a heterozygous missense mutation (c.643C > T) in exon 2 of TRPC6, leading to the substitution of arginine with tryptophan at position 215 (p.Arg215Trp). Systems genetics analysis of the 53 BXD mice kidney transcriptomes identified Pygm as the upstream regulator of Trpc6. Those two genes are jointly involved in the regulation of FSGS mainly via Wnt and Hippo signaling pathways. We present a novel variant in the TRPC6 gene that causes FSGS. Moreover, our data suggested TRPC6 works with PYGM, as well as Wnt and Hippo signaling pathways to regulate renal function, which could guide future clinical prevention and targeted treatment for FSGS outcomes.

Joint analysis of WES and RNA-Seq identify signature genes related to metastasis in prostate cancer.

Prostate cancer (PCa) has a certain degree of heritability, and metastasis occurs as cancer progresses. However, its underlying mechanism remains largely unknown. We sequenced four cases of cancer without metastasis, four metastatic cancer, and four benign hyperplasia tissues as controls. A total of 1839 damaging mutations were identified. Pathway analysis, gene clustering, and weighted gene co-expression network analysis were employed to find characteristics associated with metastasis. Chr19 had the most mutation density and 1p36 had the highest mutation frequency across the genome. These mutations occurred in 1630 genes, including the most frequently mutated genes TTN and PLEC, and dozens of metastasis-related genes, such as FOXA1, NCOA1, CD34, and BRCA2. Ras signalling and arachidonic acid metabolism were uniquely enriched in metastatic cancer. Gene programmes 10 and 11 showed the signatures indicating the occurrence of metastasis better. A module (135 genes) was specifically associated with metastasis. Of them, 67.41% reoccurred in program 10, with 26 genes further retained as the signature genes related to PCa metastasis, including AGR3, RAPH1, SOX14, DPEP1, and UBL4A. Our study provides new molecular perspectives on PCa metastasis. The signature genes and pathways could be served as potential therapeutic targets for metastasis or cancer progression.

Preliminary investigation of ixodid ticks in Jiangxi Province of Eastern China.

In recent years, a large effort has been made for tick surveys for public health importance around China, especially after outbreaks of severe fever with thrombocytopenia syndrome (SFTS) occurred in humans in 2009. In this paper, the preliminary species composition and population distribution of ticks in Jiangxi Province of Eastern China is reported. Ticks were collected in three habitats (grassland, shrubs and woodland) and from nine host groups in 12 sampling sites throughout Jiangxi Province between 2011 and 2018. Six tick species including Haemaphysalis longicornis, Rhipicephalus sanguineus sensu lato, Haemaphysalis yeni, Haemaphysalis kitaoka, Ixodes sinensis and Dermacentor auratus were collected from the vegetation. Haemaphysalis longicornis was most abundant tick species, accounting for 90.6% of the total ticks. Haemaphysalis yeni and H. kitaoka were newly recorded tick species in Jiangxi Province. Tick presence was remarkably greater in grassland (89.4%) than in woodland (9.4%) and shrubs (1.2%), and nymphs (68.2%) and larvae (19.1%) were more frequently found than adult females (6.6%) and males (6.0%). On hosts, a total of 1513 ticks, from 13 species and four genera, were collected. These were H. longicornis, Haemaphysalis campanulata, Haemaphysalis flava, Haemaphysalis phasiana, H. yeni, H. kitaoka, Haemaphysalis hystricis, R. sanguineus (s.l.), Rhipicephalus haemaphysaloides, Rhipicephalus microplus, Ixodes granulatus, I. sinensis and Amblyomma testudinarium. Amblyomma testudinarium was a newly recorded tick species in Jiangxi Province. Based on this investigation, H. longicornis was the most frequently collected species (30.5%) and widely distributed tick species of the total collection ticks (in 11 sampling sites). Haemaphysalislongicornis had a broad host range and its presence (hosts with at least one tick) was significantly greater on Lepus sinensis (33.3%) than on Canis familiaris (2.3%) (χ2 = 23.68, p = 0.0013). In addition, the number of H. longicornis collected on L. sinensis (64.0%) was higher than on other host groups. Of all ticks collected on hosts, different developmental stages were obtained, which included 347 larvae (22.9%), 249 nymphs (16.5%), 404 adult males (26.7%) and 513 females (33.9%) and sex distribution was relatively uniform. These data indicate that a broad range of tick species is widely distributed throughout Jiangxi Province in Eastern China.

Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing.

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.

Extrachromosomal circular DNA orchestrates genome heterogeneity in urothelial bladder carcinoma.

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients. Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data. Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome. Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.

Haemaphysalis longicornis calreticulin is not an effective molecular tool for tick bite diagnosis and disruption of tick infestations.

BACKGROUND: Tick calreticulin (CRT) is a calcium-binding protein secreted into the host during blood feeding. It has been used as a biomarker of tick exposure and has potential as an anti-tick vaccine, but there is no information about these uses for Haemaphysalis longicornis CRT (HlCRT). We synthesized recombinant H. longicornis CRT (rHlCRT) and evaluated its potential for tick bite diagnosis and for disrupting tick infestations. METHODS: The responses of mice and rabbits exposed to H. longicornis ticks were measured with ELISA to determine the antibody level against rHlCRT. To evaluate the effects of rHlCRT-induced anti-tick immunity, engorgement weight, tick engorgement index (TEI), feeding duration, ecdysis rate, and egg weight per engorged tick were compared between ticks fed on immunized and normal mice. RESULTS: Mean anti-tick CRT antibody levels in sera collected from mice at 1 and 15 days after primary tick exposure were not significantly different from the mean antibody levels in negative control mice that were not bitten by ticks (all P values > 0.05). No significant anti-HlCRT IgG responses developed in mice after second exposure to tick bites compared with the level of anti-HlCRT antibody response in negative control mice (all P values > 0.25). For rabbits, no significant differences in the antibody levels were observed in animals before challenge infestation and after tick exposures, and in animals after two tick exposures (all P values > 0.10). There were no significant differences in the body weight of ticks fed on immunized and normal mice (all P values > 0.15). No significant differences in TEI were observed between ticks fed on immunized mice and normal control mice (all P values > 0.50). There were no significant differences in feeding duration for female ticks, and feeding duration and ecdysis rate for nymphs in the experimental and control groups (all P values > 0.10 for feeding duration and P value = 0.19 for ecdysis rate). We did not observe a significant difference in egg weight per tick in the rHlCRT-immunized and the control groups (P = 0.88). CONCLUSIONS: HlCRT in H. longicornis tick saliva proteins appears to be nonimmunogenic to mammalian hosts like mice and rabbits. Vaccination with rHlCRT did not generate effective immunity against parthenogenetic and bisexual H. longicornis nymphs or female ticks. These results indicate that HlCRT is not a suitable molecular candidate for H. longicornis tick bite diagnosis and not effective for the disruption of tick infestations.

Molecular differentiation and species composition of genus Culicoides biting midges (Diptera: Ceratopogonidae) in different habitats in southern China.

Culicoides biting midges (Diptera:Ceratopogonidae) cause a significant biting nuisance to humans, livestock, which are the biological vectors of a range of risky pathogens. Accurate illustration of vector play a key role in arthropod borne diseases surveillance. However, few studies have focused on the Culicoides, which caused bluetongue disease in 29 provinces of China since 1979. In this study, we assessed cytochrome oxidase subunit I (COI) mtDNA molecular marker for identification of ten major vector species and analyzed the Culicoides species community and diversity in different habitats. A total of 20,795 Culicoides samples collected from 11 sample sites were identified as 23 species belonging to 7 subgenera. Sequences of COI gene worked well as barcodes for identifying all the determined specimen in this study and were comparable with the existing sequence data from GenBank. We first reported COI barcode sequences of C. morisitai, C. insignipennis and C. homotomus. Morphological identification of Culicoides spp. samples within southern China appears relatively robust and some unidentified species were required further study. Our study shows that the COI sequence data can be used as a tool to identify species of Culicoides in Jiangxi Province. In our sampled area, the most abundant species was C. arakawae (61.89%), followed by C. oxystoma (13.77%), C. punctatus (10.10%), C. nipponensis (8.82%), C. homotomus (3.19%) and C.morisitai (1.17%) in this study. C. punctatus was the dominant species of Park habitat (62.22%), C. arakawae was the predominant species of Chicken habitat (96.66%), Vegetable plot habitat (92.0%), and Peasant household habitat (83.21%), respectively. C. oxystoma was the abundant species of Residential area habitat (40.11%), Hospital habitat (56.65%), and Pig & Cow habitat (48.77%), respectively. Results also show that the potential Bluetongue virus vectors belong to the Obsoletus and Pulicaris groups, are also included and relatively abundant, notably: Culicoides punctatus. These findings expand the current knowledge of Culicoides population composition in the southern part of China.

First Molecular Evidence of Anaplasma phagocytophilum in Rodent Populations of Nanchang, China.

In this study, systematic surveillance of rodent populations in Nanchang of China and determination of Anaplasma phagocytophilum infection in rodents were performed. Between 2011 and 2015, 110,084 rodent snap traps were set in 4 counties and in the city center of Nanchang, China. Finally, 942 rodents were captured, with a relative density of 0.86%. The densities varied considerably by geographical area with Anyi being the most rodent-infested County. Frequently captured rodents were sewer rats (Rattus norvegicus), house mice (Mus musculus), and Rattus flavipectus. The Anaplasma genera were investigated by PCR in 19 live rodents trapped by welded cages in Anyi, 6 rodents were assessed as positive based on amplification of 16S rRNA. Sequence analysis revealed 3 variants of A. phagocytophilum in Nanchang. PCR analysis of the gltA (citrate synthase) gene found 1 sample that was positive for A. phagocytophilum infection. The sequence of A. phagocytophilum gltA gene formed a clade with and showed 99% identity to A. phagocytophilum that has been previously described in rodents from South-Eastern China. Taken together, our research indicated that commensal rodents are potential hosts for A. phagocytophilum and controlling the rodent population may facilitate subsequent prevention of human granulocytic anaplasmosis in Nanchang, China, in the future.

First molecular detection of tick-borne pathogens in dogs from Jiangxi, China.

In this study, blood samples obtained from 162 dogs in Jiangxi, China, were employed in molecular screening of canine tick-borne pathogens by PCR and sequencing. Babesia spp. gene fragment was detected in 12 (7.41%) dogs. All samples were negative for Hepatozoon spp., Ehrlichia canis, Coxiella spp., Borrelia spp., Rickettsia spp. and Anaplasma platys. Species-specific PCR analysis further confirmed that 8 (4.94%) and 4 (2.47%) dogs were infected by Babesia canis vogeli and Babesia gibsoni, respectively. Based on our analyses, Babesia spp. infection in Jiangxi appeared not related to age, gender, breed, usage, activity and health status or tick infestation history of the dogs. This is the first molecular report of Babesia canis vogeli and Babesia gibsoni in dogs from Jiangxi, China.