简介：AbstractObjective:Biallelic mutations in the RecQ like helicase (RECQL) 4 gene, a guardian of the genome, cause Rothmund-Thomson syndrome type II (RTS-II). Two Chinese girls with mild-phenotype RTS-II mainly restricted to their skin are herein described.Methods:Blood specimens from two families with mild-phenotype RTS-II were collected. DNA isolation, RNA isolation and complementary DNA synthesis, and next-generation sequencing using a multi-gene panel were applied to verify the underlying pathogenic variants in the causative RECQL4 gene.Results:We analyzed two patients with mild phenotypes. One patient had an unreported paternal c.2885+1G>A alteration in intervening sequence 16 and the previously reported maternal exon 14 c.2272C>T (p.R758X), both resulting in premature termination codons. The other patient carried two novel alterations, c.2886-1G>A and c.2752G>T (p.E918X). Complementary DNA sequencing showed that different splice-site mutations within the same intron could lead to completely different splicing modes.Conclusion:We identified three novel pathogenic RECQL4 variants in two patients with RTS, thus expanding the mutational spectrum of RTS-II. We also explored their pathogenic effect by transcripts analysis to address genotype-phenotype correlations.

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简介：AbstractBackground:Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers without effective therapy. To explore potential molecular targets in ESCC, we quantified the mutation spectrum and explored the relationship between gene mutation and clinicopathological characteristics and programmed death-ligand 1 (PD-L1) expression.Methods:Between 2015 and 2019, 29 surgically resected ESCC tissues and adjacent normal tissues from the Fourth Hospital of Hebei Medical University were subjected to targeted next-generation sequencing. The expression levels of PD-L1 were detected by immunohistochemistry. Mutational signatures were extracted from the mutation count matrix by using non-negative matrix factorization. The relationship between detected genomic alterations and clinicopathological characteristics and PD-L1 expression was estimated by Spearman rank correlation analysis.Results:The most frequently mutated gene was TP53 (96.6%, 28/29), followed by NOTCH1 (27.6%, 8/29), EP300 (17.2%, 5/29), and KMT2C (17.2%, 5/29). The most frequently copy number amplified and deleted genes were CCND1/FGF3/FGF4/FGF19 (41.4%, 12/29) and CDKN2A/2B (10.3%, 3/29). By quantifying the contribution of the mutational signatures to the mutation spectrum, we found that the contribution of signature 1, signature 2, signature 10, signature 12, signature 13, and signature 17 was relatively high. Further analysis revealed genetic variants associated with cell cycle, chromatin modification, Notch, and Janus kinase-signal transducer and activator of transcription signaling pathways, which may be key pathways in the development and progression of ESCC. Evaluation of PD-L1 expression in samples showed that 13.8% (4/29) of samples had tumor proportion score ≥1%. 17.2% (5/29) of patients had tumor mutation burden (TMB) above 10 mut/Mb. All samples exhibited microsatellite stability. TMB was significantly associated with lymph node metastasis (r = 0.468, P = 0.010), but not significantly associated with PD-L1 expression (r = 0.246, P = 0.198). There was no significant correlation between PD-L1 expression and detected gene mutations (all P > 0.05).Conclusion:Our research initially constructed gene mutation profile related to surgically resected ESCC in high-incidence areas to explore the mechanism underlying ESCC development and potential therapeutic targets.

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简介：AbstractBackground:The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is pandemic. However, the origins and global transmission pattern of SARS-CoV-2 remain largely unknown. We aimed to characterize the origination and transmission of SARS-CoV-2 based on evolutionary dynamics.Methods:Using the full-length sequences of SARS-CoV-2 with intact geographic, demographic, and temporal information worldwide from the GISAID database during 26 December 2019 and 30 November 2020, we constructed the transmission tree to depict the evolutionary process by the R package "outbreaker" . The affinity of the mutated receptor-binding region of the spike protein to angiotensin-converting enzyme 2 (ACE2) was predicted using mCSM-PPI2 software. Viral infectivity and antigenicity were tested in ACE2-transfected HEK293T cells by pseudovirus transfection and neutralizing antibody test.Results:From 26 December 2019 to 8 March 2020, early stage of the COVID-19 pandemic, SARS-CoV-2 strains identified worldwide were mainly composed of three clusters: the Europe-based cluster including two USA-based subclusters; the Asia-based cluster including isolates in China, Japan, the USA, Singapore, Australia, Malaysia, and Italy; and the USA-based cluster. The SARS-CoV-2 strains identified in the USA formed four independent clades while those identified in China formed one clade. After 8 March 2020, the clusters of SARS-CoV-2 strains tended to be independent and became "pure" in each of the major countries. Twenty-two of 60 mutations in the receptor-binding domain of the spike protein were predicted to increase the binding affinity of SARS-CoV-2 to ACE2. Of all predicted mutants, the number of E484K was the largest one with 86 585 sequences, followed by S477N with 55 442 sequences worldwide. In more than ten countries, the frequencies of the isolates with E484K and S477N increased significantly. V367F and N354D mutations increased the infectivity of SARS-CoV-2 pseudoviruses (P < 0.001). SARS-CoV-2 with V367F was more sensitive to the S1-targeting neutralizing antibody than the wild-type counterpart (P < 0.001).Conclusions:SARS-CoV-2 strains might have originated in several countries simultaneously under certain evolutionary pressure. Travel restrictions might cause location-specific SARS-CoV-2 clustering. The SARS-CoV-2 evolution appears to facilitate its transmission via altering the affinity to ACE2 or immune evasion.

简介：AbstractImportance:First branchial cleft anomalies (FBCAs) are rare congenital malformations, accounting for < 8% of all branchial cleft anomalies. However, little is currently known about the cause of FBCAs at the molecular level.Objective:To identify genomic alterations related to the genetic etiology of FBCAs in Chinese children.Methods:We performed whole-exome sequencing of samples from 10 pediatric patients with FBCAs. Data analysis was carried out using the Burrow-Wheeler Alignment software package, and the dbSNP database for comparisons. Rare variants were further validated by Sanger sequencing. Insertion/deletions (indels) were examined using the Genome Analysis Toolkit.Results:We identified 14 non-synonymous mutations in seven potential FBCA-susceptibility genes (TRAPPC12, NRP2, NPNT, SH3RF2, RHPN1, TENM4, and ARMCX4). We also detected 133 shared small indels in 125 genes. Gene Ontology analysis indicated that most of the identified genes played critical roles in development and differentiation pathways involved in regulating organ development.Interpretation:We characterized the mutational landscape in pathways involved in development and differentiation in Chinese children with FBCA. The results identified potential pathogenic genes and mutations related to FBCA, and provide molecular-level support for the branchial theory of FBCA pathogenesis.

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简介：AbstractBackground:Investigations of the pathogenic mechanisms in motor neurons (MNs) derived from amyotrophic lateral sclerosis (ALS) disease-specific induced pluripotent stem (iPS) cell lines could improve understanding of the issues affecting MNs. Therefore, in this study we explored mutant superoxide dismutase 1 (SOD1) protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations.Methods:We generated induced pluripotent stem cell (iPSC) lines from two familial ALS (FALS) patients with SOD1-V14M and SOD1-C111Y mutations, and then differentiated them into MNs. We investigated levels of the SOD1 protein in iPSCs and MNs, the intracellular Ca2+ levels in MNs, and the lactate dehydrogenase (LDH) activity in the process of differentiation into the MNs derived from the controls and ALS patients’ iPSCs.Results:The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers. We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the two SOD1 mutant patients. However, at no time did we observe stronger LDH activity in the patient lines compared with the control lines.Conclusions:MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis, providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy. Incremental mutant expressions of SOD1 in MNs may have disrupted MN function, either causing or contributing to the intracellular calcium disturbances, which could lead to the occurrence and development of the disease.