Clonal overgrowth of esophageal smooth muscle cells in diffuse leiomyomatosis-Alport syndrome caused by partial deletion in COL4A5 and COL4A6 genes

This is a study of a patient who manifests all of the features of a diffuse leiomyomatosis-Alport syndrome (DL-ATS), and her two-year-old son who has already been diagnosed with Alport syndrome. Fourteen years ago, the patient underwent a partial esophageal resection followed by a replacement with jejunum. Recently, she underwent a surgical resection of the esophagus due to esophageal dysfunction. Genetic analyses of COL4A5 and COL4A6 on the X-chromosome were efficiently performed using the genomic DNA of her son. We have identified a novel deletion of 194-kb in length, encompassing COL4A5-COL4A6 promoters as well as nearly the entire large intron 1 of COL4A5 and intron 2 of COL4A6. To uncover the relationship of the esophagus-specific occurrence of the tumor and the expression of those genes, immunohistochemical analyses of type IV collagen α chains were conducted in the non-affected individuals. The esophageal smooth muscle-specific expression of α5(IV) and α6(IV) chains in the gastrointestinal tract was observed. Moreover, CAG repeat analysis of the androgen receptor gene and an immunohistochemical analysis in the leiomyoma revealed clonal overgrowth of the cells which received X-inactivation on the non-affected allele. These results may suggest that the dominant effect was caused by the partial deletion of the esophageal smooth muscle-specific genes, COL4A5 and COL4A6.     

A novel heterozygous COL4A4 missense mutation in a Chinese family with focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the most common glomerular histological lesion associated with high‐grade proteinuria and end‐stage renal disease. Histologically, FSGS is characterized by focal segmental sclerosis with foot process effacement. The aim of this study was to identify the disease‐causing mutation in a four‐generation Chinese family with FSGS. A novel missense mutation, c.1856G>A (p.Gly619Asp), in the collagen type IV alpha‐4 gene (COL4A4) was identified in six patients and it co‐segregated with the disease in this family. The variant is predicted to be disease‐causing and results in collagen IV abnormalities. Our finding broadens mutation spectrum of the COL4A4 gene and extends the phenotypic spectrum of collagen IV nephropathies. Our study suggests that exome sequencing is a cost‐effective and efficient approach for identification of disease‐causing mutations in phenotypically complex or equivocal disorders. Timely screening for COL4A3/COL4A4 mutations in patients with familial FSGS may help both accurately diagnose and treat these patients.     

The COL5A1 genotype is associated with range of motion

[Purpose]

The aim of our study was to investigate the association between COL5A1 genotype and Range of Motion as measured by the passive straight leg raise (SLR) and whole body join laxity (WBJL) in Asian population.

[Methods]

One hundred and seventy seven participants including Korean and Japanese college students (male = 109, female = 68) participated in the study. Each subject performed the passive straight leg raise and whole body join laxity test. Genotyping for the COL5A1 (rs 12722) polymorphism was performed using the TaqMan approach. The COL5A1 genotype exhibited a Hardy-Weinberg equilibrium distribution in our population.

[Results]

The physical parameters including height, weight, and BMI were higher in < 90° group than > 90° group. The SLR exhibited significant difference among the COL5A1 group. However, the WBJL did not differ significantly among the COL5A1 genotype, but significant difference was seen in CC genotype when compared to CT (2.99 ± 1.72) or TT (2.70 ± 1.52) genotype.

[Conclusion]

We concluded that COL5A1 gene polymorphism is associated with increased SLR ROM in Asian population.     

High expression of COL5A2, a member of COL5 family,indicates the poor survival and facilitates cell migration in gastric cancer

Background: Gastric cancer (GC) metastasis determines the prognosis of patients, and exploring the molecular mechanism of GC metastasis is expected to provide a theoretical basis for clinical treatment. Recent studies have shown that extracellular matrix protein is closely related to GC metastasis. The present study aimed to explore the expression profile and role of COL5A2, as an extracellular matrix protein, in GC.Methods: The expression, overall survival, and progression-free survival data of COL5 family members were extracted from The Cancer Genome Atlas (TCGA) database, respectively. Weighted gene co-expression network analysis of the {"type":"entrez-geo","attrs":{"text":"GSE62229","term_id":"62229"}}GSE62229 database was performed out to identify modules and associated genes.Results: COL5A2 was selected as our research target in the TCGA database, and was also verified in the {"type":"entrez-geo","attrs":{"text":"GSE62229","term_id":"62229"}}GSE62229 and {"type":"entrez-geo","attrs":{"text":"GSE15459","term_id":"15459"}}GSE15459 datasets. COL5A2 was up-regulated in GC tissues by paraffin immunohistochemistry and RT-qPCR. The prognosis of patients with low COL5A2 expression was better than that of patients with high COL5A2 expression. Scratch and migration experiments showed that knockdown of COL5A2 decreased the migration ability of gastric cancer cells compared with the control group. In vivo, mice with tail vein injection COL5A2 knockdown had fewer and smaller metastatic nodules in liver. GSEA results showed that the TCGA and {"type":"entrez-geo","attrs":{"text":"GSE62229","term_id":"62229"}}GSE62229 samples were significantly enriched in several well-known cancer-related pathways, such as the TGF-β, MAPK, and JAK2 signaling pathways.Conclusion: COL5A2 was most closely related to advanced GC among COL5 family members. High COL5A2 expression is associated with a poor prognosis, and may be a novel therapeutic target for GC.     

Silencing of CA1 mRNA in tumour cells does not change the gene expression of the extracellular matrix proteins

We report the silencing of CA1 mRNA in PC3 and MDA cells. The levels of mRNA coding CA1 protein in the knock‐down mRNA (CA1 siRNA) cells have been measured by RT‐PCR and were approximately 5% (PC3) and 20% (MDA‐MB‐231), respectively, of the level of control (Mock siRNA) used during silencing. In PC3 and MDA‐MB‐231 cells, the mRNAs for COL1A1 and COL4A4 were up‐regulated. The mRNAs for CTHRC1, LAMC2, and WNT7B were not changed when compared to the control. The morphology of the cells during the treatments remained the same. On the Western blots, the lysate from the silenced cells showed lower levels of CA I as well.     

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type Ⅰ collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (Ⅰ) and pro-α2 (Ⅰ) chains of type Ⅰ collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A＞G and c.3305G＞C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G＞C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A＞G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.     

Mutation c.359_363delGTATTinsATAC in the COL4A5 Causes alport syndrome in a Chinese family

The X-linked form of Alport syndrome is associated with mutations in the COL4A5 gene, which is located at Xq22.3 and encodes the α5 chain of type IV collagen. Here we clinically characterized a Chinese family with Alport Syndrome, but no ocular or hearing abnormalities have been observed in any patient in the family. Through Linkage analysis and direct DNA sequencing, a novel complex deletion/insertion mutation c.359_363delGTATTinsATAC in the COL4A5 gene was identified in the family. The mutation was found in all affected family members, but was not present in the unaffected family individuals or the 200 controls. The predicted mutant protein in the family is a truncated protein consisting of only 153 residues. Our report for the first time revealed that the frameshift mutation in the type IV collagen chain α5 causes only renal disease, without extrarenal lesion. Our study broadens genotypic and phenotypic spectrum of COL4A5 mutations associated with Alport syndrome.     

LncRNA COL1A2-AS1 inhibits the scar fibroblasts proliferation via regulating miR-21/Smad7 pathway

lncRNA COL1A2-AS1 (COL1A2 antisense RNA 1), a lncRNA overexpressed in hypertrophic scar, has been demonstrated to be involved in the hypertrophic scar formation. However, the mechanisms of lncRNA COL1A2-AS1 inhibiting the scar fibroblasts proliferation remains not well understood. In this study, we demonstrated that lncRNA COL1A2-AS1 was upregulated in hypertrophic scar tissue and fibroblasts, and suppressed fibroblasts proliferation by promoting Smad7 expression. Furthermore, we found that miR-21 was involved in lncRNA COL1A2-AS1-induced expression of Smad7, by which COL1A2-AS1 acted as endogenous sponge to adsorb miR-21 and in turn regulated Smad7 and a cascade of molecular to play a protective role in hypertrophic scar. In addition, overexpression of miR-21 attenuated COL1A2-AS1-mediated proliferation suppression of hypertrophic scar fibroblasts. In conclusion, our study demonstrated that COL1A2-AS1/miR-21/Smad pathway plays an important role in inhibiting hypertrophic scar formation, and suggested this novel pathway may be a new target for hypertrophic scar treatment.     

A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family

Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes.     

一性连锁Alport综合征中国家系COL4A5基因新突变位点的检测

通过PCR和直接测序的方法,对一性连锁Alport综合征家系17个受检个体的COL4A5基因所有51个外显子及其相邻内含子的DNA序列进行检测。结果发现,在第26外显子2240位点,男患者存在C碱基缺失(2240delc),女患者存在杂合缺失,同时对女患者相应的PCR产物进行克隆和测序以验证PCR测序结果的可靠性,而在正常家系成员和80例对照中均未发现此位点异常,说明2240delc为引起该家系临床病变的突变位点,不是多态性位点。在性连锁Alport综合征中,COL4A5基因的这个单碱基缺失突变位点为首次报道。     

Molecular prognostic of nine parthanatos death-related genes in glioma,particularly in COL8A1 identification

Post-operative progression and chemotherapy resistance are the main causes of treatment failure in glioma patients. There is a lack of ideal prediction models for post-operative glioma patient progression and drug sensitivity. We aimed to develop a prognostic model of parthanatos mRNA biomarkers for glioma outcomes. A total of 11 parthanatos genes were obtained from ParthanatosCluster database. ConsensusClusterPlus and R “Limma” package were used to cluster The Cancer Genome Atlas (TCGA)-glioma cohort and analyze the differential mRNAs. Univariate Cox regression analysis, random survival forest model, and least absolute shrinkage and selection operator (LASSO) regression analysis were used to determine the nine ParthanatosScore prognostic genes combination. ParthanatosScore was verified by 656 patients and 979 patients in TCGA and CGCA-LGG/GBM datasets. Differences in genomic mutations, tumor microenvironments, and functional pathways were assessed. Drug response prediction was performed using pRRophetic. Kaplan–Meier survival analysis was analyzed. Finally, COL8A1 was selected to evaluate its potential biological function and drug sensitivity of temozolomide and AZD3759 in glioma cells. ParthanatosScore obtained a combination of nine glioma prognostic genes, including CD58, H19, TNFAIP6, FTLP3, TNFRSF11B, SFRP2, LOXL1, COL8A1, and FABP5P7. In the TCGA-LGG/GBM dataset, glioma prognosis was poor in high ParthanatosScore. Low-score glioma patients were sensitive to AZD3759_1915, AZD5582_1617, AZD8186_1918, Dasatinib_1079, and Temozolomide_1375, while high-score patients were less sensitive to these drugs. Compared with HA cells, COL8A1 was significantly over-expressed in LN229 and U251 cells. Silencing COL8A1 inhibited the malignant characterization of LN229 and U251 cells. Temozolomide and AZD3759 also promoted parthanatos gene expression in glioma cells. Temozolomide and AZD3759 inhibited COL8A1 expression and cell viability and promoted apoptosis in glioma cells and PGM cells. ParthanatosScore can accurately predict clinical prognosis and drug sensitivity after glioma surgery. Silencing COL8A1 inhibited the malignant characterization. Temozolomide and AZD3759 inhibited COL8A1 expression and cell viability and promoted apoptosis and parthanatos gene expression, which is a target to improve glioma.     

THE +1245G/T POLYMORPHISMS IN THE COLLAGEN TYPE I ALPHA 1 (COL1A1) GENE IN POLISH SKIERS WITH ANTERIOR CRUCIATE LIGAMENT INJURY

Objectives

The aim of this study was to examine the association of +1245G/T polymorphisms in the COL1A1 gene with ACL ruptures in Polish male recreational skiers in a case-control study.

Methods

A total of 138 male recreational skiers with surgically diagnosed primary ACL ruptures, all of whom qualified for ligament reconstruction, were recruited for this study. The control group comprised 183 apparently healthy male skiers with a comparable level of exposure to ACL injury, none of whom had any self-reported history of ligament or tendon injury. DNA samples extracted from the oral epithelial cells were genotyped for the +1245G/T polymorphisms using real-time PCR method.

Results

Genotype distributions among cases and controls conformed to Hardy-Weinberg equilibrium (p = 0.2469 and p = 0.33, respectively). There was a significant difference in the genotype distribution between skiers and controls (p = 0.045, Fisher''s exact test). There was no statistical difference in allele distribution: OR 1.43 (0.91-2.25), p = 0.101 (two-sided Fisher''s exact test).

Conclusions

The risk of ACL ruptures was around 1.43 times lower in carriers of a minor allele G as compared to carriers of the allele T.     

Overrepresentation of the COL3A1 AA genotype in Polish skiers with anterior cruciate ligament injury

Although various intrinsic and extrinsic risk factors for anterior cruciate ligament (ACL) rupture have been identified, the exact aetiology of the injury is not yet fully understood. Type III collagen is an important factor in the repair of connective tissue, and certain gene polymorphisms may impair the tensile strength. The aim of this study was to examine the association of the COL3A1 rs1800255 polymorphism with ACL rupture in Polish male recreational skiers. A total of 321 male Polish recreational skiers were recruited for this study; 138 had surgically diagnosed primary ACL ruptures (ACL-injured group) and 183 were apparently healthy male skiers (control group – CON) who had no self-reported history of ligament or tendon injury. Both groups had a comparable level of exposure to ACL injury. Genomic DNA was extracted from the oral epithelial cells. All samples were genotyped on a real-time polymerase chain reaction instrument. The genotype distribution in the ACL-injured group was significantly different than in CON (respectively: AA=10.1 vs 2.2%, AG=22.5 vs 36.1, GG=67.4 vs 61.8%; p=0.0087). The AA vs AG+GG genotype of COL3A1 (odds ratio (OR)=5.05; 95% confidence interval (CI), 1.62-15.71, p=0.003) was significantly overrepresented in the ACL-injured group compared with CON. The frequency of the A allele was higher in the ACL-injured group (21.4%) compared with CON (20.2%), but the difference was not statistically significant (p=0.72). This study revealed an association between the COL3A1 rs1800255 polymorphism and ACL ruptures in Polish skiers.     

Identification of potential biomarkers associated with immune infiltration in the esophageal carcinoma tumor microenvironment

Tumor immune cell infiltration was significantly correlated with the progression and the effect of immunotherapy in cancers including esophageal carcinoma (ESCA). However, no biomarkers were identified which were associated with immune infiltration in ESCA. In the present study, a total of 128 common differentially expressed genes (DEGs) were identified between esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC). The results of gene ontology (GO) enrichment and Reactome pathway analysis displayed that the up-regulated DEGs were mainly involved in the regulation of extracellular matrix (ECM), while the down-regulated DEGs were mainly involved in the regulation of cornification and keratinocyte differentiation. The most significant module of up-regulated DEGs was selected by Molecular Complex Detection (MCODE). Top ten similar genes of COL1A2 were explored, then validation and the prognostic analysis of these genes displayed that COL1A2, COL1A1, COL3A1, ZNF469 and Periostin (POSTN) had the prognostic value which were up-regulated in ESCA. The expressions of COL1A2 and its four similar genes were mainly correlated with infiltrating levels of macrophages and dendritic cells (DCs) and showed strong correlations with diverse immune marker sets in ESCA. To summarize, COL1A2 and its four similar genes were identified as the potential biomarkers associated with immune infiltration in ESCA. These genes might be applied to immunotherapy for ESCA.     

Novel cytisine derivatives exert anti-liver fibrosis effect via PI3K/Akt/Smad pathway

A series of new cytisine derivatives with a unique endocyclic scaffold were synthesized and evaluated for their inhibitory effect on collagen α1 (I) (COL1A1) promotor in human LX2 cells, taking cytisine as the lead. Structure-activity relationship (SAR) revealed that introducing a 12N-benzyl substitution might significantly enhance the activity. Compound 5f exhibited a promising inhibitory potency against COL1A1 with an IC₅₀ value of 12.8 μM in human LX2 cells, and an inspiring inhibition activity against COL1A1 on both mRNA and protein levels. It also effectively inhibited the expression of α smooth muscle actin (α-SMA), connective tissue growth factor (CTGA), matrix metalloprotein 2 (MMP-2), and transforming growth factor β1 (TGFβ1), indicating an extensive inhibitory effect against fibrogenetic proteins. In addition, compound 5f displayed reasonable PK and safety profiles. The primary mechanism study indicated that it might repress the hepatic fibrogenesis via PI3K/Akt/Smad signaling pathway. The results provided powerful information for further structure optimization, and compound 5f was selected as a novel anti-liver fibrosis agent for further investigation.     

TGF-β1 induces type I collagen deposition in granulosa cells via the AKT/GSK-3β signaling pathway-mediated MMP1 down-regulation

Type I collagen is the most abundant extracellular matrix (ECM) protein in the mammalian ovary, and comprises two COL1A1 subunits and one COL1A2 subunit. Matrix metalloproteinase 1 (MMP1) is a typical collagenase of type I collagen, that can be detected in ovarian follicles and early corpus luteum. Previous studies demonstrated that MMP1-mediated degradation of type I collagen plays a functional role in regulating corpus luteum formation, and transforming growth factor β1 (TGF-β1) inhibits luteinization and progesterone production in granulosa cells (GCs). Whether TGF-β1 regulates the expression of MMP1, COL1A1, or the deposition of type I collagen during corpus luteum formation remains to be elucidated. This study aimed to investigate the molecular mechanisms through which TGF-β1 regulates MMP1 expression and type I collagen deposition in GCs. Our results show that TGF-β1 upregulates COL1A1 expressions and downregulates MMP1 expression. Inhibition approaches, including pharmacological inhibitors such as p38 inhibitor (SB203580), ERK1/2 inhibitor (U0126), AKT inhibitor (LY294002), and GSK-3β inhibitor (LiCl), as well as knockdown using siRNA specific to these genes, were used. Our results suggest that TGF-β1 decreases MMP1 production via an ALK5-mediated AKT/GSK-3β-dependent signaling pathway, and a decrease in MMP1 levels and an increase in COL1A1 levels synergistically promote type I collagen deposition in GCs. Collectively, these findings provide novel insights into the underlying molecular mechanisms by which TGF-β1 upregulates type I collagen deposition in GCs.     

中国人COL2A1基因座的扩增片段长度多态性

用聚合酶链式反应、高分辨聚丙烯酰胺凝胶水平电泳及银染技术对位于人类Ⅱ型胶原基因终止密码下游非转录区１．３ｋｂ处的可主数目串联重复育列进行了研究。制备了由人类不同基因型ＤＮＡ混合而成的人类等位基因型参考物，根据实验结果进行了命名。     

Insertion/deletion polymorphism in the 3′ untranslated region of COL1A2 disrupts its interaction with microRNA-382 and leads to decreased susceptibility to osteoporotic fracture

A growing body of evidence has proved that the expression of COL1A2 is associated with a reduced risk of osteoporotic fracture. One single-nucleotide polymorphism (rs3917) located within the 3′-untranslated region of COL1A2 may “alter” binding site of miR-382 and thereby associated with the risk of osteoporotic fracture. Bioinformatic analysis, luciferase reporter assay, site-directed mutagenesis, Western blot and real-time PCR were performed in this study. In this study, we validated COL1A2 as a target of miR-382 in osteoblast. In addition, bone tissue samples were genotyped as wild-type rs3917, heterozygous rs3917, and homozygous rs3917. The expression of miR-382 was comparable between the genotype groups, whereas the expression of COL1A2 mRNA and protein was much higher in heterozygous rs3917 and homozygous rs3917 than the wild-type rs3917 group. Furthermore, we transfected the wild-type rs3917 and heterozygous rs3917 cells with miR-382 mimics or inhibitors and found that the transfection with miR-382 mimics significantly increased the level of the miR-382 in the cells of both genotypes, and the introduction of miR-382 inhibitors substantially suppressed the level of miR-382 in both cells. In wild-type rs3917 cells, transfection of miR-382 mimics and COL1A2 small interfering RNA (siRNA) similarly and substantially downregulated the expression of COL1A2, while in heterozygous rs3917 cells, only COL1A2 siRNA notably reduced the expression of COL1A2, whereas introduction of miR-382 mimics left expression of COL1A2 intact. The findings showed rs3917 polymorphism interfered with the interaction between COL1A2 mRNA and miR-382, and minor allele is associated with a reduced risk of osteoporotic fracture.