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Objective: To explore through big data analysis whether aberrant alternative splicing (AS) events precede tau P301S -induced neurodegenerative phenotype in 6-month-old PS19 mice., Methods: The original sequencing files of the GSE182170 dataset was downloaded from the European Nucleotide Archive (ENA) database with axel, aligned to the reference genome of the ENSEMBL database by using STAR software, and common AS event analysis and visualization were performed with rMATS and rmats2sashimiplot R packages. RSEM software was utilized for gene transcript quantification, Deseq2, edgeR, and limma R packages were used for differential expression analysis, and clusterProfiler R package was applied for GO enrichment analysis. String and Cytoscape were used for protein-protein interaction (PPI) analysis. Gene expression correlation analysis was performed with ggcorrplot R package. AS events were validated using PCR followed by agarose electrophoresis., Results: A total of 8 079 AS events were identified with rMATS and 117 significant AS events (ΔPSI>0.1, sequencing coverage >1) were selected eventually. The most frequent type of AS event was skipped exon (SE) (50.43%), followed by alternative 3' splice site (A3SS) and mutually exclusive exons (MXE). GO enrichment analysis revealed that synapse organization genes were aberrantly spliced in SE events and spliceosome genes were spliced in A3SS events. PPI and correlation analyses showed that the splicing factor Snrpn was significantly associated with the largest number of transcripts. Agarose electrophoresis confirmed the aberrant AS event of the Lrp8 gene in PS19 mice., Conclusion: Dysregulated splicing factors may contribute to tau P301S -induced aberrant AS changes. The study also increases the understanding of the cycling of tau protein and splicing factors in tauopathies., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective: To identify the pathogenic variants from a patient with suspected congenital contractural arachnodactyly, and to explore the possible molecular genetic pathogenesis, so as to provide evidence for clinical diagnosis., Methods: Whole exome sequencing was performed for the patient. The splicing site variation of candidate pathogenic genes was verified by Sanger sequencing, and the new transcript sequence was determined by RT-PCR and TA-cloning sequencing., Results: The patient carried a heterozygous c.533-1G>C variant of FBN2 gene, which was not reported. The sequencing of mRNA showed that the variant leaded to the disappearance of the canonical splice acceptor site of FBN2 gene and the activation of a cryptic splice acceptor site at c.533-71, resulting in the insertion of 70 bp sequence in the new transcript. It was speculated that the polypeptide encoded by the new transcript changed from valine (Val) to serine (Ser) at amino acid 179, and prematurely terminated after 26 aminoacids. According to the guidelines of American College of Medical Genetics and Genomics, the variant of FBN2 gene c. 533-1G>C was determined as pathogenic (PVS1+PM2+PP3 )., Conclusion: A novel splicing variant of FBN2 gene (c.533-1G>C) was identified, which can lead to congenital contractural arachnodactyly.

Objective: To detect mutations of the XPC (XPC complex subunit, DNA damage recognition and repair factor) gene in a family affected with xeroderma pigmentosum group C (XP-C)., Methods: The patient was subjected to next-generation sequencing and Sanger sequencing. Suspected mutations were validated by Sanger sequencing. Effect of splicing mutation was confirmed by reverse transcription-PCR (RT-PCR)., Results: Compound heterozygous mutations of c.2098G to T and c.2034-7_2040del were found in the XPC gene in the proband. Among these, c.2098G to T (p.G700X) is a nonsense mutation resulting in a truncated XPC protein. C.2034-7_2040del involves the -1 position, which may alter the splice donor site of the intron 11 of XPC and result in a truncated XPC protein with loss of amino acids from 940 to 679 positions. The two mutations were not detected among 100 unrelated healthy controls., Conclusion: Mutations of c.2098 G to T and c.2034-7_2040del of the XPC gene may lead to abnormal XPC expression and reduction or elimination of normal XPC functions, which may underlie the disease in this family.

Objective: To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism., Methods: The coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis. Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing, with its abundance compared with that of healthy controls., Results: DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to A) at the 5'splice sites of intron 3 of the EXT1 gene. The same mutation was not found in the healthy controls. Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing. TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (< 0.05) compared with the controls., Conclusion: The c.1164+1G to A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts. As the result, the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.

This study was aimed to analyze the β-globin gene mutations in a patient with β-thalassemia minor. Genomic DNA was extracted from peripheral blood cells of the patient. The full-length DNA sequence coding for β-globin was amplified by polymerase chain reaction, and the gene mutation was determined by DNA sequencing. The results indicated that a heterogeneous A→G mutation was found at position 129 in intron 1 of the β-thalassemia minor patient. It is concluded that the IVS-I-129(A→G) mutation is a splicing site mutation leading to a splicing error in immature messenger RNA and a protein translation error for the β-globin gene. Thus, the IVS-I-129(A→G) is a novel mutation.

Objective: To study the expression of plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in spiral ganglion cell (SGC) from inner ear of newborn rats and further check PMCA2 splice variants at site A and C., Methods: Spiral ganglion tissues isolated from cochlea of newborn rats (P3-P4) were cultured and identified in vitro. The cochlea of newborn rats (P3-P4) were isolated and cut into frozen sections. The expression of PMCA2 was detected by immunofluorescence analyses. The SGC cultured in 4 wells of the 6-well culture plate were collected and the total RNA was extracted by Trizol and reverse transcribed to cDNA. The site A and C splice variants of PMCA2 were respectively checked by nested PCR and common PCR., Results: The SGC grew well with good refraction and showed positive immunoreactivity for neuronal marker NF-200. Strong green fluorescence could be seen in cytomembrane, cytoplasm and neuritis, as well showing SGC immunoreactivity for PMCA2 antibody. In the cochlear sections, the spiral ganglion tissues were strongly stained by PMCA2. PMCA2z was present at splice site A, but PMCA2b and PMCA2c were present at splice site C., Conclusion: SGC from newborn rats strongly expresses PMCA2 and different splice variants are present at PMCA2 splice site A and C.

Objective: To investigate the existence of alternatively spliced variants of constitutive androstane receptor (CAR) in liver of mouse., Methods: The nucleotide from liver of mouse was purified and the CAR cDNA was amplified by PCR. The fragments of CAR cDNA were cloned to T vector and sequence analysis was performed., Result: Various spliced variants of CAR in liver mouse were confirmed by DNA sequencing., Conclusion: There are alternatively spliced variants in CAR, which are located in the ligand binding sequence of CAR.

Objective: To search for possible novel mutations in palmitoyl-protein thioesterase 1 (PPT1) gene in two Chinese babies with infantile neuronal ceroid lipofuscinosis (INCL)., Methods: Two probands with INCL, confirmed clinically and pathologically, were used for mutation search in PPT1 gene. Onset of the disease occurred before the age of 1 year and they mainly showed progressive mental and motor retardation. The 9 coding exons and their flanking intron sequences of palmitoyl-protein thioesterase 1 (PPT1) gene were amplified by using PCR and sequenced. The parents of proband 1 were also examined., Results: One splicing mutation and two missense mutations were identified in the two probands: the proband 1 carrying a compound heterozygous mutation of a IVS1 + 1G-->A mutation in intron 1 and a c550G-->A mutation in exon 6 leading to the amino acid substitution of E184K. Additionally, the parents of the proband 1 also harbored one of the mutations of the patient, respectively. The proband 2 carrying a homozygous mutation of c272A-->C in exon 3, which resulted in the amino acid substitutions of Q91P., Conclusions: The IVS1 + 1G-->A mutation and Q91P mutation are novel mutations, which lead to INCL. The genetic abnormalities of PPT1 in Chinese patients may not be completely the same as those in the patients of other regions of the world.

Objective: To explore the molecular mechanism involved in patient with congenital FV deficiency., Methods: Activity of FV was determined by biochemical method. The PCR products of FV gene was analyzed by DNA sequencing directly or cloned into T-vector prior to DNA analysis. The mutation of FV gene in proband and his family numbers was analysed by restriction enzyme analysis. Its occurrence was investigated in the control group. DNA was extracted from the peripheral blood mono1nuclear cells of the proband, male, 18 years old, and his parents. The PCR products were analyzed by direct sequencing or cloned into T-vector prior to DNA analysis. One hundred patients with different kind of hemotopathy were used as controls., Results: A single point mutation, AG-->GG was found at position 3' splice site of intron 8 of the proband. This mutation was confirmed by family screening., Conclusion: A single point mutation, AG-->GG at position 3' splice site of intron 8 mutation of FV gene is related to the pathogenesis of congenital FV deficiency.