Aberrant RNA splicing as the molecular basis of some pathogenic variants.

Identification and correct classification of disease-associated mutations are essential for molecular diagnosis and clinical management of many genetic disorders. Although next-generation sequencing has greatly accelerated the detection of nucleotide changes, the biological interpretation of most variants has become a real challenge. Moreover, attention is typically paid to protein-coding changes and the potential impact of exonic variants on RNA splicing is often ignored. There is increasing evidence showing that disease-causing aberrant RNA splicing is more widespread than currently appreciated. Here, we review the major types of the variants involved in RNA splicing and the approaches used to identify and characterize these variants. We hope to provide a reference for evaluation of the effects of mutations on diseases.

Aberrant promoter hypermethylation inhibits RGMA expression and contributes to tumor progression in breast cancer.

Breast cancer (BC) is the most common cancer in women worldwide, and the exploration of aberrantly expressed genes might clarify tumorigenesis and help uncover new therapeutic strategies for BC. Although RGMA was recently recognized as a tumor suppressor gene, its detailed biological function and regulation in BC remain unclear. Herein, we found that RGMA was downregulated in BC tissues compared with non-tumorous breast tissues, particularly in metastatic BC samples, and that patients with low RGMA expression manifested a poorer prognosis. Furthermore, DNMT1 and DNMT3A were found to be recruited to the RGMA promoter and induced aberrant hypermethylation, resulting in downregulation of RGMA expression in BC. In contrast, RGMA overexpression suppressed BC cell proliferation and colony-formation capabilities and increased BC cell apoptosis. Furthermore, RGMA knockdown accelerated BC cell proliferation and suppressed cellular apoptosis in vitro and in vivo. Reversal of RGMA promoter methylation with 5-Aza-CdR restored RGMA expression and blocked tumor growth. Overall, DNMT1- and DNMT3A-mediated RGMA promoter hypermethylation led to downregulation of RGMA expression, and low RGMA expression contributed to BC growth via activation of the FAK/Src/PI3K/AKT-signaling pathway. Our data thus suggested that RGMA might be a promising therapeutic target in BC.

lncRNA THAP7-AS1, transcriptionally activated by SP1 and post-transcriptionally stabilized by METTL3-mediated m6A modification, exerts oncogenic properties by improving CUL4B entry into the nucleus.

Long noncoding RNAs (lncRNAs) are dysregulated in different cancer types, and thus have emerged as important regulators of the initiation and progression of human cancers. However, the biological functions and the underlying mechanisms responsible for their functions in gastric cancer (GC) remain poorly understood. Here, by lncRNA microarray, we identified 1414 differentially expressed lncRNAs, among which THAP7-AS1 was significantly upregulated in GC tissues compared with non-tumorous gastric tissues. High expression of THAP7-AS1 was correlated with positive lymph node metastasis and poorer prognosis. SP1, a transcription factor, could bind directly to the THAP7-AS1 promoter region and activate its transcription. Moreover, the m6A modification of THAP7-AS1 by METTL3 enhanced its expression depending on the "reader" protein IGF2BP1-dependent pathway. THAP7-AS1 promoted GC cell progression. Mechanistically, THAP7-AS1 interacted with the 1-50 Amino Acid Region (nuclear localization signal) of CUL4B through its 1-442 nt Sequence, and it promoted interaction between nuclear localization signal (NLS) and importin α1, and improved the CUL4B protein entry into the nucleus, repressing miR-22-3p and miR-320a expression by CUL4B-catalyzed H2AK119ub1 and the EZH2-mediated H3K27me3, subsequently activating PI3K/AKT signaling pathway to promote GC progression. Moreover, LV-sh-THAP7-AS1 treatment could suppress GC growth, invasion and metastasis, indicating that THAP7-AS1 may act as a promising molecular target for GC therapies. Taken together, our results show that THAP7-AS1, transcriptionally activated by SP1 and then modified by METTL3-mediated m6A, exerts oncogenic functions, by promoting interaction between NLS and importin α1 and then improving the CUL4B protein entry into the nucleus to repress the transcription of miR-22-3p and miR-320a.

[Two novel mutations of GLA gene in Chinese patients with Fabry disease].

OBJECTIVE: To observe the disease-causing GLA gene mutations in Chinese patients with Fabry disease and the correlation between the genotype and phenotype. METHODS: DNA from 2 Chinese patients with Fabry disease and their relatives were collected. The seven exons and nonjunctional regions of GLA gene were amplified with polymerase chain reaction and the products were sequenced. The correlation between the genotype and phenotype was analyzed. RESULTS: Two mutations, G1168A and G1170A, located in 5' untranslated regions (5'UTR) were identified in the two probands and the two mutations were absent in normal controls. Three patients with the same genotype were found in the pedigree with G1168A mutation and there was no gene mutation carrier in the pedigree with G1170A mutation. Symptoms of the disease are less in female patients than that in male patients. CONCLUSION: GLA gene mutation in 5'UTR may also be involved in the disease process of patients with Fabry disease and the phenotype is partly affected by gender.

GSK-3ß-regulated N-acetyltransferase 10 is involved in colorectal cancer invasion.

PURPOSE: NAT10 (N-acetyltransferase 10) is a nucleolar protein, but may show subcellular redistribution in colorectal carcinoma. In this study, we evaluated membranous staining of NAT10 in colorectal carcinoma and its clinical implications, and explored the mechanism of regulation of NAT10 redistribution. EXPERIMENTAL DESIGN: The expression and subcellular redistribution of NAT10, ß-catenin, E-cadherin, and GSK-3ß were evaluated by immunohistochemistry in 222 cases of colorectal carcinoma. Regulation of NAT10 and its influence on cell motility were analyzed with inhibitors of GSK-3ß, transfection of wild-type or kinase-inactivated GSK-3ß, or expression of various domains of NAT10, and evaluated with immunofluorescence, Western blotting, and Transwell assays. RESULTS: NAT10 localized mainly in the nucleoli of normal tissues, and was redistributed to the membrane in cancer cells, particularly at the invasive "leading edge" of the tumor. This correlated well with nuclear accumulation of ß-catenin (P<0.001; χ2=68.213). In addition, NAT10 membrane staining reflected the depth of invasion and tendency to metastasize (all P values<0.001), and was associated with a poorer prognosis (P=0.023; χ2=5.161). Evaluation of the mechanism involved demonstrated that subcellular redistribution of NAT10 may result from its increased stability and nuclear export, which is brought about by inhibition of GSK-3ß. Moreover, redistribution of NAT10 induces alteration of cytoskeletal dynamics and increases cancer cell motility. CONCLUSION: The subcellular redistribution of NAT10 can be induced by decreases in GSK-3ß activity. This redistribution increases cancer cell motility, and is, thus, correlated with invasive potential and poorer clinical outcome. This finding suggests that NAT10 may be a useful prognostic marker and potential therapeutic target in colorectal carcinoma.