Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model.

Duchenne muscular dystrophy (DMD) affecting 1 in 3500-5000 live male newborns is the frequently fatal genetic disease resulted from various mutations in DMD gene encoding dystrophin protein. About 70% of DMD-causing mutations are exon deletion leading to frameshift of open reading frame and dystrophin deficiency. To facilitate translating human DMD-targeting CRISPR therapeutics into patients, we herein establish a genetically humanized mouse model of DMD by replacing exon 50 and 51 of mouse Dmd gene with human exon 50 sequence. This humanized mouse model recapitulats patient's DMD phenotypes of dystrophin deficiency and muscle dysfunction. Furthermore, we target splicing sites in human exon 50 with adenine base editor to induce exon skipping and robustly restored dystrophin expression in heart, tibialis anterior and diaphragm muscles. Importantly, systemic delivery of base editor via adeno-associated virus in the humanized male mouse model improves the muscle function of DMD mice to the similar level of wildtype ones, indicating the therapeutic efficacy of base editing strategy in treating most of DMD types with exon deletion or point mutations via exon-skipping induction.

SimSpliceEvol2: alternative splicing-aware simulation of biological sequence evolution and transcript phylogenies.

BACKGROUND: SimSpliceEvol is a tool for simulating the evolution of eukaryotic gene sequences that integrates exon-intron structure evolution as well as the evolution of the sets of transcripts produced from genes. It takes a guide gene tree as input and generates a gene sequence with its transcripts for each node of the tree, from the root to the leaves. However, the sets of transcripts simulated at different nodes of the guide gene tree lack evolutionary connections. Consequently, SimSpliceEvol is not suitable for evaluating methods for transcript phylogeny inference or gene phylogeny inference that rely on transcript conservation. RESULTS: Here, we introduce SimSpliceEvol2, which, compared to the first version, incorporates an explicit model of transcript evolution for simulating alternative transcripts along the branches of a guide gene tree, as well as the transcript phylogenies inferred. We offer a comprehensive software with a graphical user interface and an updated version of the web server, ensuring easy and user-friendly access to the tool. CONCLUSION: SimSpliceEvol2 generates synthetic datasets that are useful for evaluating methods and tools for spliced RNA sequence analysis, such as spliced alignment methods, methods for identifying conserved transcripts, and transcript phylogeny reconstruction methods. The web server is accessible at https://simspliceevol.cobius.usherbrooke.ca , where you can also download the standalone software. Comprehensive documentation for the software is available at the same address. For developers interested in the source code, which requires the installation of all prerequisites to run, it is provided at  https://github.com/UdeS-CoBIUS/SimSpliceEvol .

eHSP90α in front-line therapy in EGFR exon 19 deletion and 21 Leu858Arg mutations in advanced lung adenocarcinoma.

PURPOSE: Extracellular heat shock protein 90 AA1(eHSP90α) is intricately linked to tumor progression and prognosis. This study aimed to investigate the difference in the value of eHSP90α in post-treatment response assessment and prognosis prediction between exon 19 deletion(19DEL) and exon 21 Leu858Arg(L858R) mutation types in lung adenocarcinoma(LUAD). METHODS: We analyzed the relationship between the expression of eHSP90α and clinicopathological features in 89 patients with L858R mutation and 196 patients with 19DEL mutation in LUAD. The Kaplan-Meier survival curve was used to determine their respective cut-off values and analyze the relationship between eHSP90α expression and the survival time of the two mutation types. The area under the curve (AUC) was used to evaluate the diagnostic performance of biomarkers. Then, the prognostic model was developed using the univariate-Cox multivariate-Cox and LASSO-multivariate logistic methods. RESULTS: In LUAD patients, eHSP90α was positively correlated with carcinoembryonic antigen(CEA), carbohydrate antigen 125(CA125), and carbohydrate antigen 153(CA153). The truncated values of eHSP90α in L858R and 19DEL patients were 44.5 ng/mL and 40.8 ng/mL, respectively. Among L858R patients, eHSP90α had the best diagnostic performance (AUC = 0.765), and higher eHSP90α and T helper cells(Th cells) expression were significantly related to shorter overall survival(OS) and worse treatment response. Also, high eHSP90a expression and short progression-free survival(PFS) were significantly correlated. Among 19DEL patients, CEA had the best diagnostic efficacy (AUC = 0.734), and CEA and Th cells were independent prognostic factors that predicted shorter OS. Furthermore, high CA125 was significantly associated with short PFS and poor curative effect. CONCLUSIONS: eHSP90α has a better prognostic value in LUAD L858R patients than 19DEL, which provides a new idea for clinical diagnosis and treatment.

Innovative construction of the first reliable catalogue of bovine circular RNAs.

The aim of this study was to compare the circular transcriptome of divergent tissues in order to understand: i) the presence of circular RNAs (circRNAs) that are not exonic circRNAs, i.e. originated from backsplicing involving known exons and, ii) the origin of artificial circRNA (artif_circRNA), i.e. circRNA not generated in-vivo. CircRNA identification is mostly an in-silico process, and the analysis of data from the BovReg project (https://www.bovreg.eu/) provided an opportunity to explore new ways to identify reliable circRNAs. By considering 117 tissue samples, we characterized 23,926 exonic circRNAs, 337 circRNAs from 273 introns (191 ciRNAs, 146 intron circles), 108 circRNAs from small non-coding genes and nearly 36.6K circRNAs classified as other_circRNAs. Furthermore, for 63 of those samples we analysed in parallel data from total-RNAseq (ribosomal RNAs depleted prior to library preparation) with paired mRNAseq (library prepared with poly(A)-selected RNAs). The high number of circRNAs detected in mRNAseq, and the significant number of novel circRNAs, mainly other_circRNAs, led us to consider all circRNAs detected in mRNAseq as artificial. This study provided evidence of 189 false entries in the list of exonic circRNAs: 103 artif_circRNAs identified by total RNAseq/mRNAseq comparison using two circRNA tools, 26 probable artif_circRNAs, and 65 identified by deep annotation analysis. Extensive benchmarking was performed (including analyses with CIRI2 and CIRCexplorer-2) and confirmed 94% of the 23,737 reliable exonic circRNAs. Moreover, this study demonstrates the effectiveness of a panel of highly expressed exonic circRNAs (5-8%) in analysing the tissue specificity of the bovine circular transcriptome.

The novel HLA-C*04:01:01:182 allele identified in a candidate bone marrow donor by next-generation sequencing.

HLA-C*04:01:01:182 differs from the HLA-C*04:01:01:06 allele by one nucleotide substitution in the 5'UTR.

The novel HLA-DRB1 allele, HLA-DRB1*09:54 was identified in a Chinese individual.

HLA-DRB1*09:54 shows a substitution G to A at position 449 when compared with HLA-DRB1*09:01:02:01.

The novel HLA-C*14:02:01:31 allele identified in a candidate bone marrow donor by next-generation sequencing.

Characterisation of the novel HLA-C*14:02:01:31 allele in a 21-year-old Greek bone marrow donor.

The novel HLA-A allele, HLA-A*03:478, identified in a Korean individual.

One nucleotide substitution in codon 320 of A*03:01:01:01 results in the novel allele, HLA-A*03:478.

Meet the authors: Mei-Sheng Xiao, Arun Prasath Damodaran, and Thomas Gonatopoulos-Pournatzis.

We talk to corresponding author Thomas Gonatopoulos-Pournatzis and co-first authors Arun Prasath Damodaran and Mei-Sheng Xiao about their paper "Genome-scale exon perturbation screens uncover exons critical for cell fitness" (in this issue of Molecular Cell) and get insights into their findings, career trajectories, and future directions in the pre-mRNA processing field.

Characterisation of the novel HLA-C*01:273 allele, first identified in a Brazilian individual.

The novel HLA-C*01:273 allele, first described in a potential bone marrow donor from Brazil.

Identification of the novel HLA-A*30:221 allele by next-generation sequencing.

The novel HLA-A*30:221 allele differs from HLA-A*30:01:01:01 by one nucleotide substitution in Exon 7.

Characterisation of the novel HLA-B*46:96 allele by next-generation sequencing.

HLA-B*46:96 differs from HLA-B*46:01:01:01 by a single nucleotide substitution at position 479 C>T.

Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro.

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.

Skeletal muscle vulnerability in a child with Pitt-Hopkins syndrome.

BACKGROUND: TCF4 acts as a transcription factor that binds to the immunoglobulin enhancer Mu-E5/KE5 motif. Dominant variants in TCF4 are associated with the manifestation of Pitt-Hopkins syndrome, a rare disease characterized by severe mental retardation, certain features of facial dysmorphism and, in many cases, with abnormalities in respiratory rhythm (episodes of paroxysmal tachypnea and hyperventilation, followed by apnea and cyanosis). Frequently, patients also develop epilepsy, microcephaly, and postnatal short stature. Although TCF4 is expressed in skeletal muscle and TCF4 seems to play a role in myogenesis as demonstrated in mice, potential myopathological findings taking place upon the presence of dominant TCF4 variants are thus far not described in human skeletal muscle. METHOD: To address the pathological effect of a novel deletion affecting exons 15 and 16 of TCF4 on skeletal muscle, histological and immunofluorescence studies were carried out on a quadriceps biopsy in addition to targeted transcript studies and global proteomic profiling. RESULTS: We report on muscle biopsy findings from a Pitt-Hopkins patient with a novel heterozygous deletion spanning exon 15 and 16 presenting with neuromuscular symptoms. Microscopic characterization of the muscle biopsy revealed moderate fiber type I predominance, imbalance in the proportion of fibroblasts co-expressing Vimentin and CD90, and indicate activation of the complement cascade in TCF4-mutant muscle. Protein dysregulations were unraveled by proteomic profiling. Transcript studies confirmed a mitochondrial vulnerability in muscle and confirmed reduced TCF4 expression. CONCLUSION: Our combined findings, for the first time, unveil myopathological changes as phenotypical association of Pitt-Hopkins syndrome and thus expand the current clinical knowledge of the disease as well as support data obtained on skeletal muscle of a mouse model.

Primary malignant melanoma of the gastroesophageal junction with KIT gene exon 11 mutation.

Mucosal melanoma (MM) represents an uncommon form of melanoma. Primary gastrointestinal tract (GIT) melanoma is even rarer. A 70-year-old male visited the Liaoning Cancer Hospital and Institute, China, due to upper abdominal discomfort for the past two months. His endoscopy revealed a prominent, 6-cm ulcerated neoplasm in the gastroesophageal junction (GEJ). Lesion endoscopic biopsy showed diffusely distributed tumour cells. He underwent subtotal gastrectomy with lymph node dissection (LND). Postoperative histopathology revealed a diffuse distribution of tumour cells with numerous tumourinfiltrating lymphocytes (TILs) and pigment granules. Immunohistochemical (IHC) results were positive for both S-100 and HMB-45. Molecular analysis showed KIT gene exon 11 mutations. Although the clinicians emphasised the necessity of systemic chemotherapy and immunotherapy with the patient and his family, the patient did not receive any adjuvant therapy and died 36 months after surgery. Primary malignant melanoma of GEJ should be considered in a differential diagnosis for gastrointestinal malignancies, especially after excluding the source of metastasis through a systemic examination.

HLA-B*51:197: A composite product of interlocus recombination of B*51:01:01:01 and C*01:02:01:01.

The HLA-B*51:197 allele is generated from the interlocus recombination of both HLA-B*51:01:01:01 and HLA-C*01:02:01:01 alleles.

Identification of the novel HLA-A*11:01:117 allele by next-generation sequencing.

HLA-A*11:01:117 differs from HLA-A*11:01:01:01 by a single nucleotide substitution at position 780 A>G.

Identification of the novel allele HLA-A*02:1144 in a Chinese platelet donor.

The novel HLA-A*02:1144 allele differs from HLA-A*02:03:01:01 by 3 nucleotides in exon 7.

Identification of the new allele HLA-A*24:632 in a Greek individual using next generation sequencing.

The HLA-Α*24:632 allele differs from HLA-A*24:02:01:01 by a single nucleotide substitution in exon 2.