Unlocking Clinical Insights: Lymphocyte-Specific Protein Tyrosine Kinase Candidates as Promising Therapeutic Targets for Pancreatic Cancer Risk Stratification.

Background: Uncover the pivotal link between lymphocyte-specific protein tyrosine kinase (Lck)-related genes and clinical risk stratification in pancreatic cancer. Methods: This study identifies shared genes between differentially expressed genes (DEGs) and Lck-related genes in pancreatic cancer using a methodological framework rooted in The Cancer Genome Atlas database. Feature gene selection is accomplished and a signature model is constructed. Statistical significant clinical endpoints such as overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) were defined. Results: After performing random survival forest, Lasso regression, and multivariate Cox regression model, 7 trait genes out of 272 Lck-associated DEGs are selected to create a signature model that is independent of other clinical factors and can predict OS and DSS. It appears that high-risk patients have activated the TP53 signaling pathway and the cell cycle signaling pathway. LAMA3 turned out to be the hub gene of the signature with high expression in pancreatic cancer. Patients with increased expression of LAMA3 had a short OS, DSS, and PFI in comparison. The candidate competing endogenous RNA network of LAMA3 turned out to be OPI5-AS1/hsa-miR-186-5p/LAMA3 axis. Conclusions: A characteristic signature of seven Lck-related genes, especially LAMA3, has been shown to be a key factor in clinical risk stratification for pancreatic cancer.

Digenic inheritance accounts for phenotypic variability in amelogenesis imperfecta.

Amelogenesis imperfecta (AI) represents a group of clinically and genetically heterogeneous disorders that affect enamel formation and mineralization. Although AI is commonly considered a monogenic disorder, digenic inheritance is rarely reported. In this study, we recruited two nonconsanguineous Chinese families exhibiting diverse phenotypes of enamel defects among affected family members. Digenic variants were discovered in both probands. In family 1, the proband inherited a paternal frameshift variant in LAMA3 (NM_198129.4:c.3712dup) and a maternal deletion encompassing the entire AMELX gene. This resulted in a combined hypoplastic and hypomineralized AI phenotype, which was distinct from the parents' manifestations. In family 2, whole-exome sequencing analysis revealed the proband carried a maternal heterozygous splicing variant in COL17A1 (NC_000010.11 (NM_000494.3): c.4156 + 2dup) and compound heterozygous variants in RELT (paternal: NM_032871.4:c.260A > T; maternal: NM_032871.4:c.521 T > G). These genetic changes caused the abundant irregular enamel defects observed in the proband, whereas other affected family members carrying heterozygous variants in both COL17A1 and RELT displayed only horizontal grooves as their phenotype. The pathogenicity of the novel COL17A1 splice site variant was confirmed through RT-PCR and minigene assay. This study enhances our understanding by highlighting the potential association between the co-occurrence of variants in two genes and variable phenotypes observed in AI patients.

Heterozygous nonsense variants in laminin subunit 3α resulting in Ebstein's anomaly.

Ebstein's anomaly is a rare congenital heart disease characterized by tricuspid valve downward displacement and is associated with additional cardiac phenotypes such as left ventricle non-compaction. The genetic basis of Ebstein's anomaly has yet to be fully elucidated, although several genes (e.g., NKX2-5, MYH7, TPM1, and FLNA) may contribute to Ebstein's anomaly. Here, in two Ebstein's anomaly families (a three-generation family and a trio), we identified independent heterozygous nonsense variants in laminin subunit 3 α (LAMA3), cosegregated with phenotypes in families with reduced penetrance. Furthermore, knocking out Lama3 in mice revealed that haploinsufficiency of Lama3 led to Ebstein's malformation of the tricuspid valve and an abnormal basement membrane structure. In conclusion, we identified a novel gene-disease association of LAMA3 implicated in Ebstein's anomaly, and the findings extended our understanding of the role of the extracellular matrix in Ebstein's anomaly etiology.

c.151dup variant in LAMA3 in Pakistani patients affected with Shabbir Syndrome but showing mild symptoms.

Objective: To identify genetic causes of Shabbir syndrome in two patients of Pakistani origin. Methods: In the present study, we have investigated a Pakistani family with two affected members segregating Laryngo-onycho-cutaneous (LOC) syndrome. The patients were diagnosed as suspected cases of LOC based on phenotypes including abnormal larynx, nails, and hyperpigmentation in patients' eyes. Genetic investigation was done by performing whole exome sequencing (WES) using DNA of the patients. Sanger sequencing was performed to validate WES findings and segregation analysis in the family. Results: Data analysis of exomes and Sanger sequencing of patients revealed a homozygous one base pair duplication (NM_000227.6; LAMA3; c.151dup; p.Val51GlyfsTer4) in LAMA3 in the patients. Parents of the patients were heterozygous for the identified variant. Conclusion: Previously, the same variant has been found in most of the Pakistani Punjabi patients affected with LOC. Therefore, Pakistani Punjabi families affected with Shabbir Syndrome may be screened for c.151dup variant in LAMA3 using targeted sequencing. Sanger sequencing is a cost-effective and time-saving technique as compared to whole exome/genome sequencing. Hence, developing ethnicity-specific LAMA3 targeted molecular diagnostic test would be cost-effective. Further, the study would assist in carrier testing and prenatal diagnosis of the affected families.

LINC00936/microRNA-221-3p Regulates Tumor Progression in Ovarian Cancer by Interacting with LAMA3.

BACKGROUND: Ovarian cancer remains a leading cause of mortality in women. It is known that long non-coding RNA (lncRNA) controls various biological processes and pathogenesis of many diseases, including cancers. This study aimed to determine whether LINC00936 and microRNA-221-3p (miR-221-3p) influence the laminin alpha 3 chain gene (LAMA3) in the development of ovarian cancer. METHODS: The expressions of LINC00936, miR-221-3p, and LAMA3 in ovarian cancer and adjacent tissues were assessed. Furthermore, ovarian cancer cells were transfected with vectors with overexpressed LINC00936, miR-221-3p mimic, miR-221-3p inhibitor, and si-LAMA3 to elucidate their functions in ovarian cancer cell proliferation, migration, invasion, angiogenesis, and tumorigenesis. The binding relationship between LINC00936 and miR-221-3p and the relationship between miR-221-3p and LAMA3 were verified to explore the mechanism of action of LINC00936 in ovarian cancer. LINC00936 binds to miR-221-3p as a ceRNA and regulates the expression of LAMA3. RESULTS: LINC00936 and LAMA3 were poorly expressed, while miR-221-3p was highly expressed in ovarian cancer tissues. Over-expression of LINC00936 contributed to decreasing miR- 221-3p expression and increasing LAMA3 expression. LINC00936 overexpression or miR-221- 3p silencing downregulated the levels of PCNA, MMP-2, MMP-9, and VEGF and decreased cell proliferation, migration, invasion, angiogenesis, and ovarian cancer tumorigenesis. CONCLUSION: Collectively, overexpression of LINC00936 suppressed the development of ovarian cancer by competitively binding to miR-221-3p and controlling LAMA3 expression. These results could serve as a novel theoretical base for the treatment of ovarian cancer.

Novel variants in LAMA3 and COL7A1 and recurrent variant in KRT5 underlying epidermolysis bullosa in five Chinese families.

Epidermolysis bullosa (EB) is a group of clinically and genetically heterogeneous diseases characterized by trauma-induced mucocutaneous fragility and blister formation. Here, we investigated five Chinese families with EB, and eight variants including a novel nonsense variant (c.47G>A, p.W16*) in LAMA3, a known recurrent variant (c.74C>T, p.P25L) in KRT5, 2 novel (c.2531T>A, p.V844E; c.6811_6814del, p.R2271fs) and 4 known (c.6187C>T, p.R2063W; c.7097G>A, p.G2366D; c.8569G>T, p.E2857*; c.3625_3635del, p.S1209fs) variants in COL7A1 were detected. Notably, this study identified a nonsense variant in LAMA3 that causes EB within the Chinese population and revealed that this variant resulted in a reduction in LAMA3 mRNA and protein expression levels by nonsense-mediated mRNA decay. Our study expands the mutation spectra of Chinese patients with EB.

[A pedigree study of laryngo-onycho-cutaneous syndrome with a novel mutation on LAMA3 gene].

Objective:To detect genetic mutations in a case of laryngo-onycho-cutaneous syndrome, and to explore the possible molecular biological pathogenic causes. Methods:With informed consent, the family clinical data of the child with laryngo-onycho-cutaneous syndrome were collected, peripheral blood of the protester and his parents was collected and DNA was extracted, and gene detection was performed by high-throughput sequencing method. Sanger sequencing was used to verify and analyze the mutation sites of the probs and their families. Results:Genetic testing of the proband revealed homozygous mutation of LAMA3 gene c.171+1G>A site, which is splicing mutation. There was no report in the literature, which was a new mutation site. The parents of the proband had normal phenotype and heterozygous mutation at this locus was detected. Conclusion:Homozygous mutation of LAMA3 c.171+1G>A is the likely pathogenic of the proband, and this study expands the mutant spectrum of LAMA3. The clinical phenotype of laryngo-onycho-cutaneous syndrome is highly variable, and the multidisciplinary diagnosis and treatment can effectively avoid missed diagnosis and misdiagnosis.

PDigenic Mutations in Junctional Epidermolysis Bullosa in An Iranian Family.

In this study, we describe one Iranian patient who was diagnosed with Epidermolysis Bullosa (EB) because of mutations in three candidate genes, including 3 mutations. Two missense mutations in the LAMA3 (D3134H) and LAMB3 (Y339H) genes and also, a synonymous mutation in the ITGB4 (H422H) gene were identified that leads to the Junctional-EBHerlitz (JEB-Herlitz) clinical phenotype. The patient had a heterozygous LAMA3 mutation combined with a heterozygous mutation in LAMB3. Our results propose that these mutations produce novel protein-coding transcripts which explain the JEB-Herlitz phenotype in the patient. Interestingly, this is the first report indicating that a digenic inheritance in the LAMA3 and LAMB3 which is responsible for JEB-Herlitz. Also, this is the first digenic inheritance recognized in the JEB-Herlitz family. This study provides a new way to clarify the molecular mechanisms of LAMA3 and LAMB3 genes in JEB-Herlitz.

LAMA3 DNA methylation and transcriptome changes associated with chemotherapy resistance in ovarian cancer.

OBJECTIVE: LAMA3 is a widely studied methylated gene in multiple tumors, but the relationship between chemotherapy resistance in ovarian cancer is unclear. In this study, LAMA3 methylation was predicted by bioinformatics, and the ability of LAMA3 methylation to predict the chemotherapy resistance and prognosis of ovarian cancer was confirmed in experiments. METHODS: Multiple databases have performed the bioinformatics analysis of methylation and transcription factor binding site (TFBS) on the promoter region of LAMA3 gene. Pyrosequencing detected the methylation of LAMA3. QRT-PCR and immunohistochemistry detected the expression of LAMA3. Real Time Cell Analyzer (RTCA) detects changes in cell proliferation, migration and invasion ability. Flow cytometry was used to detect apoptosis. RESULTS: CPG islands of 176 bp, 134 bp, 125 bp and 531 bp were predicted in the promoter region of LAMA3 gene. The 4 prediction results are basically overlapped. 7 transcription factor binding sites were predicted, and the one with the highest score was on the predicted CpG island located in the proximal promoter region. LAMA3 hypermethylation and low expression are both associated with chemotherapy resistance and poor prognosis in ovarian cancer. LAMA3 methylation was negatively correlated with expression. After upregulation of LAMA3, the proliferation ability of chemoresistant ovarian cancer cell decreased, while the ability of apoptosis, invasion and migration increased. CONCLUSION: LAMA3 hypermethylation is associated with chemotherapy resistance and poor prognosis. As a typical CpG island gene, LAMA3(cg20937934) and LAMA3(cg13270625) hypermethylation is negatively correlated with low expression. LAMA3 promotes the invasion, migration and apoptosis of SKOV3DDP. In the future, the mechanism of LAMA3 methylation in ovarian cancer will need to be further studied.

Surfce Functionalized via AdLAMA3 Multilayer Coating for Re-epithelization Around Titanium Implants.

The peri-implant epithelium (PIE) forms a crucial seal between the oral environment and the implant surface. Compared with the junctional epithelium (JE), the biological sealing of PIE is fragile, which lacks hemidesmosomes (HDs) and internal basal lamina (extracellular matrix containing laminin332, IBL) on the upper part of the interface. In the study, we aim to prepare a coating with good biocompatibility and ability to immobilize the recombinant adenovirus vector of LAMA3 (AdLAMA3) for promoting the re-epithelization of PIE. The titanium surface functionalized with AdLAMA3 was established via layer-by-layer assembly technique and antibody-antigen specific binding. The biological evaluations including cell adhesion and the re-epithelization of PIE were investigated. The results in vitro demonstrated that the AdLAMA3 coating could improve epithelial cell attachment and cell spreading in the early stage. In vivo experiments indicated that the AdLAMA3 coating on the implant surface has the potential to accelerate the healing of the PIE, and could promote the expression of laminin α3 and the formation of hemidesmosomes. This study might provide a novel approach and experimental evidence for the precise attachment of LAMA3 to titanium surfaces. The process could improve the re-epithelization of PIE.

Long Non-coding RNA LINC00628 Interacts Epigenetically with the LAMA3 Promoter and Contributes to Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular progress in lung adenocarcinoma. In this study, to identify cancer-related lncRNAs and genes, we screened for those lncRNAs that were differentially expressed in lung adenocarcinoma, which revealed LINC00628 overexpression and low expression of laminin subunit alpha 3 (LAMA3). This was further validated in the cancerous tissues from patients diagnosed with lung adenocarcinoma. Thereafter, we explored the functional relevance of LINC00628 and LAMA3 in lung adenocarcinoma by analyzing the recruitment of DNA methyltransferase (DNMT) and the cellular processes of lung adenocarcinoma cells following treatments that induced LINC00628 overexpression or LINC00628 silencing or with 5-azacytidine (5-Aza, a DNMT inhibitor). The results showed that LINC00628 silencing decreased cell proliferation, migration, and invasion as well as the drug resistance of lung adenocarcinoma cells to vincristine (VCR). The results were opposite in the cells with LAMA3 demethylation induced by 5-Aza treatment. Further research indicated that LINC00628 recruited DNMT1, DNMT3A, and DNMT3B to promote the methylation of LAMA3 promoter, thereby decreasing its expression. Moreover, an in vivo experiment was performed in nude mice to assess the tumor growth ability and drug resistance of human lung adenocarcinoma cells. It was observed that LINC00628 silencing or 5-Aza treatment inhibited the in vivo tumor growth ability of the human lung adenocarcinoma cells and reduced their resistance to VCR. Altogether, our results provide evidence of a mechanism by which LINC00628 silencing exerts an inhibitory role in lung adenocarcinoma by modulating the DNA methylation of LAMA3, indicative of a novel molecular target for treatment of lung adenocarcinoma patients showing resistance to VCR.

Correlation of LAMA3 with onset and prognosis of ovarian cancer.

Correlation of laminin subunit α3 (LAMA3) gene with onset and prognosis of ovarian cancer was investigated. In total, 210 ovarian cancer patients who received surgical resection in West China Second Hospital from March 2011 to March 2013 were randomly selected, and another 160 non-ovarian cancer patients who needed ovariectomy were also selected. The relative expression of LAMA3 gene was compared via quantitative polymerase chain reaction (qPCR) in carcinoma tissues, para-carcinoma tissues and non-carcinoma normal tissues in ovarian cancer patients. The methylation level was compared among the above three tissue types. The correlation between the mutation site rs12373237 in LAMA3 gene and onset was analyzed. The expression of laminin in ovarian cancer was detected using immunohistochemistry. Moreover, the 5-year survival rate after operation was recorded and the survival curve was plotted. The expression level of LAMA3 was lower in carcinoma tissues than those in normal tissues and para-carcinoma tissues (P<0.05). The methylation degree was lower in para-carcinoma tissues and normal tissues than that in carcinoma tissues (P<0.05). The CC homozygous mutation of rs12373237 was highly correlated with the onset of ovarian cancer (OR=4.333, P=0.028). The expression of LAMA3 was classified via immunohistochemistry, and the number in high-expression group (63.8%) was larger than that in low-expression group (36.2%) (P<0.05). According to the analysis of 5-year survival rate, the recurrence-free survival rate and overall survival rate in LAMA3 high-expression group were significantly higher than those in LAMA3 low-expression group (P<0.05). The expression level and base mutation of LAMA3 gene can change the level of laminin, which have a certain influence on the onset and prognosis of ovarian cancer.

[Induction of specific CD8(+) T cells against hepatocellular carcinoma-associated neoantigens].

Objective: To predict the tumor neoantigen peptides in hepatocellular carcinoma (HCC), and examine their specific immune effects against the tumor cells without injury to normal cells. Methods: The data of whole-genome sequencing and exome sequencing of HCC tumor and matched non-tumor liver tissues were analyzed to confirm the HCC-associated somatic mutations. Based on the HLA phenotype of the patients, we used NetMHC software to predict the neoantigen epitopes with high binding affinity to their MHC-I molecules. The predicted peptides with mutation sites included were synthesized. GPL10687 platform was applied to examine the gene expression difference between tumor and normal tissues of the selected genes in GSE25097, one of the GEO databases. The quantitative real-time PCR (qRT-qPCR) and immunohistochemistry were used to confirm the expressions in tumors and normal tissues of the selected genes. By using the predicted peptides, we induced the generation of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) and examined their specific effects against tumor cells. Results: The mutation frequency of TP53 (tumor protein p53) was 40%, and LAMA3 (Laminin Subunit Alpha 3) was 8% in the analyzed HCC tissues. In GSE25097 database, TP53 and LAMA3 mRNA levels in tumors were 1.57±0.02 and 1.37±0.10, which were significantly increased than those in matched no-tumor tissue (0.54±0.01 and 0.36±0.01, P<0.05). The differences of expression levels of TP53 and LAMA3 in tumor and no-tumor tissues were validated by using qRT-qPCR and immunohistochemistry in 10 HCC tissues. The mRNA levels of TP53 and LAMA3 in tumors were 0.24±0.03 and 0.13±0.06, which were significantly elevated than those in matched no-tumor tissue (0.11±0.01 and 0.01±0.01, P<0.05). Among the Chinese population, HLA-A2 and HLA-A11 and HLA-A24 accounted for 70%, representing the major MHC-I molecules. The CTLs induced by predicted peptides showed cytotoxicity to the targets pulsed with mutated peptide, with no effect on the target pulsed with normal peptide and on normal cells. Conclusions: TP53 and LAMA3 existed relative higher mutation frequency in HCC, and expressed higher in tumor tissues. The induced CTLs by predicted peptides derived from mutation-associated protein could specific kill the target cells without injury to normal cells.

Induction of specific CD8+ T cells against hepatocellular carcinoma-associated neoantigens / 中华肿瘤杂志

Objective@#To predict the tumor neoantigen peptides in hepatocellular carcinoma (HCC), and examine their specific immune effects against the tumor cells without injury to normal cells.@*Methods@#The data of whole-genome sequencing and exome sequencing of HCC tumor and matched non-tumor liver tissues were analyzed to confirm the HCC-associated somatic mutations. Based on the HLA phenotype of the patients, we used NetMHC software to predict the neoantigen epitopes with high binding affinity to their MHC-I molecules. The predicted peptides with mutation sites included were synthesized. GPL10687 platform was applied to examine the gene expression difference between tumor and normal tissues of the selected genes in GSE25097, one of the GEO databases. The quantitative real-time PCR (qRT-qPCR) and immunohistochemistry were used to confirm the expressions in tumors and normal tissues of the selected genes. By using the predicted peptides, we induced the generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) and examined their specific effects against tumor cells.@*Results@#The mutation frequency of TP53 (tumor protein p53) was 40%, and LAMA3 (Laminin Subunit Alpha 3) was 8% in the analyzed HCC tissues. In GSE25097 database, TP53 and LAMA3 mRNA levels in tumors were 1.57±0.02 and 1.37±0.10, which were significantly increased than those in matched no-tumor tissue (0.54±0.01 and 0.36±0.01, P<0.05). The differences of expression levels of TP53 and LAMA3 in tumor and no-tumor tissues were validated by using qRT-qPCR and immunohistochemistry in 10 HCC tissues. The mRNA levels of TP53 and LAMA3 in tumors were 0.24±0.03 and 0.13±0.06, which were significantly elevated than those in matched no-tumor tissue (0.11±0.01 and 0.01±0.01, P<0.05). Among the Chinese population, HLA-A2 and HLA-A11 and HLA-A24 accounted for 70%, representing the major MHC-I molecules. The CTLs induced by predicted peptides showed cytotoxicity to the targets pulsed with mutated peptide, with no effect on the target pulsed with normal peptide and on normal cells.@*Conclusions@#TP53 and LAMA3 existed relative higher mutation frequency in HCC, and expressed higher in tumor tissues. The induced CTLs by predicted peptides derived from mutation-associated protein could specific kill the target cells without injury to normal cells.

A stop-gain in the laminin, alpha 3 gene causes recessive junctional epidermolysis bullosa in Belgian Blue cattle.

Four newborn purebred Belgian Blue calves presenting a severe form of epidermolysis bullosa were recently referred to our heredo-surveillance platform. SNP array genotyping followed by autozygosity mapping located the causative gene in a 8.3-Mb interval on bovine chromosome 24. Combining information from (i) whole-genome sequencing of an affected calf, (ii) transcriptomic data from a panel of tissues and (iii) a list of functionally ranked positional candidates pinpointed a private G to A nucleotide substitution in the LAMA3 gene that creates a premature stop codon (p.Arg2609*) in exon 60, truncating 22% of the corresponding protein. The LAMA3 gene encodes the alpha 3 subunit of the heterotrimeric laminin-332, a key constituent of the lamina lucida that is part of the skin basement membrane connecting epidermis and dermis layers. Homozygous loss-of-function mutations in this gene are known to cause severe junctional epidermolysis bullosa in human, mice, horse, sheep and dog. Overall, our data strongly support the causality of the identified gene and mutation.

MicroRNA-17/20a functions to inhibit cell migration and can be used a prognostic marker in oral squamous cell carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) accounts for>90% oral cancer which is a leading cause of cancer death worldwide. Early diagnosis may well offer an opportunity to increase survival to this neoplasm. Micro(mi)RNA-interfered cancer progression is crucial, yet its migration machinery of OSCC is still unknown. To access whether the possible miRNA prognostic markers and underlying mechanisms, we developed a highly migratory TW2.6 MS-10 cells from TW2.6 cells to investigate the issue. MATERIALS AND METHODS: miRNA profiling was performed on TW2.6 and TW2.6 MS-10. Target miRNA was correlated to pathological status in OSCC patients by real-time RT-PCR. A downstream effector was identified using a bioinformatics analysis, and a 3'-untranslated region (UTR) reporter assay was used. RESULTS: An miRNA cluster, miR-17-92, including miR-17, miR-19b, miR-20a, and miR-92a, was found to be significantly down-regulated in TW2.6 MS-10 compared to TW2.6 cells. Overexpression of this cluster decreased the migratory ability of OSCC cell lines. We further demonstrated that miR-17 and miR-20a are the main miRNAs of miR-17-92 cluster which modulate OSCC migration. Clinically, miR-17/20a showed negative correlation with TNM stage and lymphatic metastasis. Through a bioinformatics screening analysis and 3'UTR reporter assay, we confirmed the integrin (ITG) ß8 as a direct target of miR-17/20a, and knockdown of ITGß8 reduced cell migratory capability of OSCC. CONCLUSIONS: miR-17/20a acts as a prognostic predictor of OSCC patients' outcome and a tumor migration suppressor miRNA.