Distinct genetic patterns of shared and unique genes across four neurodevelopmental disorders.

Neurodevelopmental disorders, including autism spectrum disorder (ASD), intellectual disability (ID), developmental disorders (DD) and epileptic encephalopathy (EE), have a strong clinical comorbidity, which indicates a common genetic etiology across various disorders. However, the underlying genetic mechanisms of comorbidity and specificity remain unknown across neurodevelopmental disorders. Based on de novo mutations, we compared systematically the functional characteristics between shared and unique genes under these disorders, as well as the spatiotemporal trajectory of development in brain and common molecular pathways of all shared genes. We observed that shared genes present more constrained against functional rare genetic variation, and harbor more pathogenic rare variants than do unique genes in each disorder. Furthermore, 71 shared genes formed two clusters related to synaptic transmission, transcription regulation and chromatin regulator. Particularly, we also found that two core genes STXBP1 and SCN2A, that were shared by the four neurodevelopmental disorders showed prominent pleiotropy. Our findings shed light on the shared and specific patterns across neurodevelopmental disorders and will enable us to further comprehend the etiology and provide valuable information for the diagnosis of neurodevelopmental disorders.

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling.

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC ß-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism of resistance of K. pneumoniae during carbapenem treatment. RESULTS: Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 was found to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored blaSHV-187 and blaTEM-116. The blaSHV-187 and blaTEM-116 genes were not detected in FK-2723 and FK-2820. blaDHA-1, qnrB4, aac (6')-IIc, and blaSHV-12, EreA2, CatA2, SulI, and tetD, were identified in both FK-2723 and FK-2820. Moreover, the genes blaDHA-1, qnrB4, aac (6')-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate. CONCLUSION: This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.

Circular RNA sequencing indicates circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers of primary Sjögren's syndrome.

OBJECTIVES: This study aims to characterize the expression profiles of circRNAs in primary Sjogren's Syndrome (pSS) and examine the potential of noninvasive circular RNAs (circRNAs) as biomarkers of pSS. METHODS: We performed RNA sequencing of minor salivary gland (MSG) biopsies from four pSS and four non-pSS individuals (subjects undergoing MSG biopsies but not meeting 2012 or 2016 ACR classification criteria for SS). Differentially expressed circRNAs were identified by DESeq2, and confirmed by quantitative real-time PCR in the MSGs as well as in plasma exosomes in 37 pSS and 14 non-pSS subjects. Discriminatory capacity testing using receiver operating characteristic analysis was used to evaluate the performance of circRNAs as diagnostic biomarkers for pSS. RESULTS: Circ-IQGAP2 and circ-ZC3H6 had significantly upregulated expression in the MSGs of pSS patients, and this elevated expression was confirmed by quantitative real-time PCR of plasma exosome RNA. The expression of these circRNAs also showed significant correlation with both clinical features, serum IgG level and MSG focus scores. Receiver operating characteristic analysis showed that the indices comprised of both the two circRNAs and clinical features were better able to distinguish pSS from non-pSS subjects with high mean areas under the curve of 0.93 in the MSGs and 0.92 in the plasma exosomes. CONCLUSION: This study indicated the potential roles of circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers for the diagnosis of pSS.

Elevated plasma phage load as a marker for intestinal permeability in leukemic patients.

Microbial translocation (MT) and altered gut microbiota have been described in acute leukemic patients and contribute to immune activation and inflammation. However, phage translocation has not been investigated in leukemia patients yet. We recruited 44 leukemic patients and 52 healthy adults and quantified the levels of 3 phages in peripheral blood, which were the most positive phages screened from fecal samples. The content of 16S rRNA in plasma was detected by qPCR to assess the intestinal mucosa of these patients. Spearman's rank correlation was used to analyze the relationship between phage load and the relevant clinical data. We found the most prevalent phages in fecal samples were λ phage, Wphi phage, and P22 phage, and λ phage had the highest detection rate in plasma (68%). Phage content was affected by chemotherapy and course of disease and correlated with the levels of CRP (r = 0.43, p = 0.003), sCD14 (r = 0.37, p = 0.014), and sCD163 (r = 0.44, p = 0.003). Our data indicate that plasma phage load is a promising marker for gut barrier damage and that gut phage translocation correlates with monocyte/macrophage activation and systemic inflammatory response in leukemic patients.

Revision of serum ALT upper limits of normal facilitates assessment of mild liver injury in obese children with non-alcoholic fatty liver disease.

BACKGROUND: The serum alanine aminotransferase (ALT) level is a critical parameter for evaluating liver injury in non-alcoholic fatty liver disease (NAFLD). However, the currently accepted upper limits of normal (ULN) for serum ALT (ULN-ALT) are debated, as they may be excessively high. METHODS: A total of 1638 children aged 6-16 years, comprising 507 children with normal BMI (500 healthy children and 7 children with NAFLD), 199 overweight children, and 932 obese children, were included in the analysis. We re-evaluated the ULN-ALT in 500 healthy Chinese children using the 95th percentiles of serum ALT levels as revised ULN-ALT. Fatty liver was identified by ultrasound examination. RESULTS: Significant positive correlations between serum ALT levels and body mass index (BMI) were detected in overweight boys (r = .399, P < .001), obese boys (r = .398, P < .001), and obese girls (r = .392, P < .001). The prevalence percentages of NAFLD were 93.6%, 75.8%, and 37.9% in obese boys with serum ALT levels of >50, 25-50, and ≤25 U/L and were 81.6%, 67.9%, and 20.6% in obese girls with serum ALT levels of >40, 20-40, and ≤20 U/L, respectively. CONCLUSION: Serum ALT levels significantly correlated with abnormal BMI values in children, suggesting a rigorous BMI threshold is needed to establish the cutoffs for serum ULN-ALT in children. Besides, the revised serum ULN-ALT can uncover mild liver injury in obese children with NAFLD.

A de novo pathogenic CSNK1E mutation identified by exome sequencing in family trios with epileptic encephalopathy.

Recent whole-exome sequencing (WES) studies have demonstrated the contribution of de novo mutations (DNMs) to epileptic encephalopathies (EEs). Here, we performed WES on four trios with West syndrome and identified three loss-of-function DNMs in both CSNK1E (c.885+1G>A) and STXBP1 (splicing, c.1111-2A>G; nonsense, p.(Y519X)). The splicing mutation in CSNK1E creates insertion of 116 new amino acids at position 246 followed by a premature stop codon. Both CSNK1E and STXBP1 showed a closer coexpression relationship with epilepsy candidate genes beyond that expected by chance. In addition, genes coexpressed with CSNK1E were enriched in early prenatal stages across multiple brain regions. We also found that 60 CSNK1E-interacting genes share an association with multiple neuropsychiatric disorders, and these genes formed a significant interconnected interaction network with roles in the midbrain development. Our study supported the potential role of CSNK1E variants in EE susceptibility and expanded the phenotypic spectrum associated with CSNK1E variation.

Signatures of B-cell receptor diversity in B lymphocytes following Epstein-Barr virus transformation.

Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.

Differential circRNA expression profiles in latent human cytomegalovirus infection and validation using clinical samples.

Human cytomegalovirus (HCMV) is an opportunistic prototypic beta-herpesvirus that can cause severe and even fatal diseases in immune-naive newborns and immunocompromised adults. Host-virus interactions occurring at the transcriptional and posttranscriptional levels are critical for establishing an HCMV latent or lytic infection, but the mechanisms remain poorly understood. Herein, we investigated the expression of circRNAs in human leukemia monocytes (THP-1 cells) latently infected with HCMV and explored the diagnostic value of circRNAs in children with HCMV infection. A total of 2,110 and 1,912 circRNAs were identified in mock-infected and HCMV latent-infected THP-1 cells, respectively. Of these, we identified 1,421 differently expressed circRNAs, of which 650 were upregulated and 771 were downregulated. The host genes corresponding to the differentially expressed circRNAs were mainly involved in the regulation of host cell secretion pathways, cell cycle, and cell apoptosis. The differentially expressed circRNAs had binding sites for microRNAs, suggesting an important role in the mechanism of HCMV latent infection. Furthermore, a clinical analysis showed that the expression levels of hsa_circ_0001445 and hsa_circ_0001206 were statistically significantly different in HCMV-infected patients vs. normal controls, suggesting that these circRNAs could potentially serve as biomarkers of HCMV-infection.

High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis.

Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testing (pDST) is time-consuming, and PCR-based assays are limited to hotspot mutations. In this study, we developed and validated a convenient and efficient approach based on high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). The analytical performance of NTS was evaluated, and 99 clinical samples were collected to assess its clinical performance. The NTS results showed that MTB and its drug resistance were successfully identified in approximately 7.5 h. Furthermore, compared to the pDST and Xpert MTB/RIF assays, NTS provided much more drug resistance information, covering 14 anti-TB drugs, and it identified 20 clinical cases of drug-resistant MTB. The mutations underlying these drug-resistant cases were all verified using Sanger sequencing. Our approach for this TB drug resistance assay offers several advantages, including being culture-free, efficient, high-throughput, and highly accurate, which would be very helpful for clinical patient management and TB infection control.

Human cytomegalovirus-induced immune regulation is correlated with poor prognosis in patients with colorectal cancer.

Evidence suggests that human cytomegalovirus (HCMV) infection may be implicated in the progression of colorectal cancer (CRC). However, the correlation between HCMV infection and survival outcomes in patients with CRC remains unclear. Here, we constructed a flow algorithm to identify HCMV sequences based on the RNA-seq data of patients with CRC derived from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The patients' clinical information matrix was used to calculate the Euclidean distance to filter out suitable patients not infected with HCMV, combined with patients' survival outcomes, to reveal how HCMV infection is involved in CRC progression. HCMV infection is widespread in patients with CRC, and the prevalence of HCMV infection ranges from 10 to 36% in four independent CRC datasets, with infection being concentrated in carcinoma tissue rather than in normal tissue. In addition, HCMV-positive patients had a poor survival prognosis, with three HCMV genes, UL82, UL42, and UL117, associated with poor patient survival outcomes. Most importantly, we suppose that the regulation of immune function by HCMV may be key to the poor prognosis of patients with CRC. We found that HCMV infection was associated with poor prognosis in CRC patients and identified three prognosis-associated HCMV genes. The regulation of immune function caused by HCMV infection was the key factor, while HCMV-positive patients with CRC mostly presented with a state of immunosuppression. This may provide new ideas for the personalized treatment of patients with CRC, especially with respect to immunotherapy.

The Mitochondrial tRNAPhe 625G>A Mutation in Three Han Chinese Families With Cholecystolithiasis.

In this study, we assessed three Chinese families with inherited cholecystolithiasis and conducted the clinical, genetic, and molecular characterization of these subjects. Eight of eighteen matrilineal relatives had a clinical phenotype in these three families. Sequence analysis of complete mitochondrial genomes in these probands identified the homoplasmic tRNAPhe 625 G > A mutation and distinct sets of mtDNA polymorphisms belonging to haplogroups H2, F4b, and M10a. The 625G > A mutation disturbed the classic G-C base-pairings at a highly conserved position 49 in the T-stem of mitochondrial tRNAs. Molecular dynamics simulation showed that the structure of tRNAphe with 625 G > A mutation was noticeably remodeled while compared with the isoform of the wild type. The occurrence of tRNAPhe 625 G > A mutation in these various genetically unrelated subjects strongly indicates that this mutation is involved in the pathogenesis of cholecystolithiasis. This is the first evidence that tRNA mutations are associated with cholecystolithiasis, and it provided more insights into the genetic mechanism of cholecystolithiasis.

De Novo Germline Mutations in SEMA5A Associated With Infantile Spasms.

Infantile spasm (IS) is an early-onset epileptic encephalopathy that usually presents with hypsarrhythmia on an electroencephalogram with developmental impairment or regression. In this study, whole-exome sequencing was performed to detect potential pathogenic de novo mutations, and finally we identified a novel damaging de novo mutation in SEMA5A and a compound heterozygous mutation in CLTCL1 in three sporadic trios with IS. The expression profiling of SEMA5A in the human brain showed that it was mainly highly expressed in the cerebral cortex, during the early brain development stage (8 to 9 post-conception weeks and 0 to 5 months after birth). In addition, we identified a close protein-protein interaction network between SEMA5A and candidate genes associated with epilepsy, autism spectrum disorder (ASD) or intellectual disability. Gene enrichment and function analysis demonstrated that genes interacting with SEMA5A were significantly enriched in several brain regions across early fetal development, including the cortex, cerebellum, striatum and thalamus (q < 0.05), and were involved in axonal, neuronal and synapse-associated processes. Furthermore, SEMA5A and its interacting genes were associated with ASD, epilepsy syndrome and developmental disorders of mental health. Our results provide insightful information indicating that SEMA5A may contribute to the development of the brain and is associated with IS. However, further genetic studies are still needed to evaluate the role of SEMA5A in IS to definitively establish the role of SEMA5A in this disorder.