Monitoring TIGIT/DNAM-1 and PVR/PVRL2 Immune Checkpoint Expression Levels in Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia.

After allogeneic stem cell transplantation (alloSCT), several immune checkpoints play an important role in the antileukemic immune response in the bone marrow (BM) microenvironment. However, immune checkpoint expression levels in the BM have not been reported after alloSCT in patients with acute myeloid leukemia (AML). We investigated the clinical impact of immune checkpoint expression in BM samples after alloSCT for AML. Higher expression of T cell immunoreceptor with Ig and ITIM domains (TIGIT) was associated with a decreased incidence of acute graft-versus-host disease (P = .048) and poor overall (P = .046) and progression-free survival (P = 0.024). In addition, higher expression of TIGIT at engraftment after alloSCT was correlated with a decreased number of natural killer cells in BM (P = .019). Monitoring TIGIT expression in the BM could be useful for predicting outcome after alloSCT for AML. Our findings raise the possibility that blockade of TIGIT would improve survival.

Identification of OAF and PVRL1 as candidate genes for an ocular anomaly characterized by Peters anomaly type 2 and ectopia lentis.

Keratolenticular dysgenesis (KLD) and ectopia lentis are congenital eye defects. The aim of this study is the identification of molecular genetic alterations responsible for those ocular anomalies with neurologic impairment in an individual with a de novo balanced chromosome translocation t(11;18)(q23.3;q11.2)dn. Disruption of OAF, the human orthologue of the Drosophila oaf, by the 11q23.3 breakpoint results in reduced expression of this transcriptional regulator. Furthermore, four most likely nonfunctional chimeric transcripts comprising up to OAF exon 3, derived from the der(11) allele, have also been identified. This locus has been implicated by publicly available genome-wide association data in corneal disease and corneal topography. The expression of the poliovirus receptor-related 1(PVRL1) or nectin cell adhesion molecule 1 (NECTIN1), a paralogue of nectin cell adhesion molecule 3 (PVRL3) associated with congenital ocular defects, situated 500 kb upstream from 11q23.3 breakpoint, is increased. The 18q11.2 breakpoint is localized between cutaneous T-cell lymphoma-associated antigen 1(CTAGE1) and retinoblastoma binding protein 8 (RBBP8) genes. Genomic imbalance that could contribute to the observed phenotype was excluded. Analysis of gene expression datasets throughout normal murine ocular lens embryogenesis suggests that OAF expression is significantly enriched in the lens from early stages of development through adulthood, whereas PVRL1 is lens-enriched until E12.5 and then down-regulated. This contrasts with the observation that the proposita's lymphoblastoid cell lines exhibit low OAF and high PVRL1 expression as compared to control, which offers further support that the alterations described above are most likely responsible for the clinical phenotype. Finally, gene interaction topology data for PVRL1 also agree with our proposal that disruption of OAF by the translocation breakpoint and misregulation of PVRL1 due to a position effect contribute to the observed ocular and neurological phenotype.

Distribution of cellular HSV-1 receptor expression in human brain.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

p63-dependent and independent mechanisms of nectin-1 and nectin-4 regulation in the epidermis.

Nectins are immunoglobulin-like cell adhesion molecules mainly localized in adherens junctions. The transcription factor p63 is a master regulator of gene expression in stratified epithelia and controls several molecular processes. As mutations in the Pvrl1 and Pvrl4 genes encoding for nectins cause genetic disorders with phenotypes similar to p63-related syndromes, we investigated whether these proteins might be under p63 transcriptional control. Here, we show that in p63-null skin, Pvrl1 gene expression is strongly reduced, whereas Pvrl4 expression is unaffected. In human and mouse primary keratinocytes p63 depletion leads to a specific downregulation of the Pvrl1 gene. Consistent with a direct regulation, chromatin immunoprecipitation experiments (ChIP) indicate that p63 binds to two conserved intronic Pvrl1 enhancer regions. Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder, caused by mutations in p63 gene, mainly characterized by skin fragility. To test whether nectins may be affected in AEC syndrome, their expression was measured in keratinocytes obtained from patients with AEC or from a conditional mouse model for AEC syndrome. Pvrl1 expression was reduced in AEC keratinocytes, consistent with impaired p63 function. Surprisingly, Pvrl4 expression was similarly affected, in parallel with decreased expression of the transcription factor Irf6. Consistent with the well-characterized role of Irf6 in keratinocyte differentiation and its strong downregulation in AEC syndrome, Irf6 depletion caused reduced expression of Pvrl4 in wild-type keratinocytes. Taken together, our results indicate that Pvrl1 is a bona fide target gene of the transcription factor p63, whereas Pvrl4 regulation is linked to epidermal differentiation and is under Irf6 control.

Association of Nucleotide Variants of GRHL3, IRF6, NAT2, SDC2, BCL3, and PVRL1 Genes with Nonsyndromic Cleft Lip With/Without Cleft Palate in Multigenerational Families: A Retrospective Study.

BACKGROUND: Several genes are associated with the etiology of cleft lip and palate (CLP) in different populations. Many nucleotide variants on genes such as GRHL3, IRF6, NAT2, SDC2, BCL3, and PVRL1 were reported in different populations, but not studied in multigenerational cases in the Indian population. AIM AND OBJECTIVE: The aim of this study is to evaluate whether nucleotide variants rs41268753, rs861020, rs1041983, rs1042381, rs2965169, and rs10790332 are involved in the etiology of nonsyndromic CLP (NSCLP) in multigenerational Indian families. STUDY DESIGN: Retrospective genetic study. MATERIALS AND METHODS: Based on inclusion and exclusion criteria, 20 multigenerational families with nonsyndromic cleft lip with or without cleft palate (NSCL/P) were selected. Blood samples from both affected and unaffected participants were collected as a source of genomic DNA. Six nucleotide variants on these genes were genotyped to test for the association with NSCL/P. Genotyping was performed with the MassArray method. Genotype distribution was used to calculate the Hardy-Weinberg equilibrium using PLINK, a whole-genome association analysis toolset. The allelic association was compared among cases and controls using Chi-square test as implemented in PLINK. P ≤ 0.05 indicates statistical differences between groups. RESULTS: No significant associations were found between individual single-nucleotide polymorphisms and NSCL/P. The odds ratio was 1.531, 1.198, 0.8082, 1.418, 1, and 0.5929 for polymorphisms rs41268753, rs861020, rs1041983, rs1042381, rs2965169, and rs10790332, respectively. CONCLUSION: Our findings suggest that among the multigenerational families in our population, the high-risk nucleotide variants GRHL3 rs41268753, IRF6 rs861020, NAT2 rs1041983, SDC2 rs1042381, BCL3 rs2965169, and PVRL1 rs10790332 are not associated with increased risk of NSCL/P.

Salivary microRNAs as new molecular markers in cleft lip and palate: a new frontier in molecular medicine.

MicroRNAs (miRNAs) are endogenous non-coding RNAs of about twenty-two nucleotides that regulate gene expression through post-transcriptional control. The purpose of the present study was to identify and describe the salivary miRNAs in cleft lip and palate (CLP) patients comparing them with a control healthy group. Twelve patients (mean age 11.9 ± 2.42 years; 6M/6F) formed the study group. The control group was created selecting twelve healthy subjects matched for age and sex with study group. We recorded differences in miRNA expression profile between the saliva of CLP patients and the control group. Specifically, miR-141, miR-223, and miR-324-3p were mostly deregulated between the study and control groups. Interestingly, these three miRNAs are the regulators of the following genes correlated to cleft palate and lip development: MTHFR, SATB2, PVRL1. The present study showed that collecting saliva samples is a non-invasive procedure and is well accepted by CLP patients. MiRNAs can be easily isolated and identified. The differences in regulation of miR-141, miR-223 and miR-324-3p between the two groups of salivary samples suggest that these molecules are valid prognostic biomarkers and therapy dynamic response indicators, also for the accuracy and non-invasive sampling and dosing system.

microRNA-4516 Contributes to Different Functions of Epithelial Permeability Barrier by Targeting Poliovirus Receptor Related Protein 1 in Enterovirus 71 and Coxsackievirus A16 Infections.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) remain the predominant etiological agents of hand, foot, and mouth disease (HFMD). The clinical manifestations caused by the two viruses are obviously different. CV-A16 usually triggers a repeated infection, and airway epithelial integrity is often the potential causative factor of respiratory repeated infections. Our previous studies have demonstrated that there were some differentially expressed miRNAs involved in the regulation of adhesion function of epithelial barrier in EV-A71 and CV-A16 infections. In this study, we compared the differences between EV-A71 and CV-A16 infections on the airway epithelial barrier function in human bronchial epithelial (16HBE) cells and further screened the key miRNA which leaded to the formation of these differences. Our results showed that more rapid proliferation, more serious destruction of 16HBE cells permeability, more apoptosis and disruption of intercellular adhesion-associated molecules were found in CV-A16 infection as compared to EV-A71 infection. Furthermore, we also identified that microRNA-4516 (miR-4516), which presented down-regulation in EV-A71 infection and up-regulation in CV-A16 infection was an important regulator of intercellular junctions by targeting Poliovirus receptor related protein 1(PVRL1). The expressions of PVRL1, claudin4, ZO-1 and E-cadherin in CV-A16-infected cells were significantly less than those in EV-A71-infected cells, while the expressions of these proteins were subverted when pre-treated with miR-4516-overexpression plasmid in EV-A71 infected and miR-4516-knockdown plasmid in CV-A16 infected 16HBE cells. Thus, these data suggested that the opposite expression of miR-4516 in EV-A71 and CV-A16 infections might be the initial steps leading to different epithelial impairments of 16HBE cells by destroying intercellular adhesion, which finally resulted in different outcomes of EV-A71 and CV-A16 infections.

Expression and Function of hsa-miR-6165 in Human Cell Lines and During the NT2 Cell Neural Differentiation Process.

MicroRNAs are small non-coding RNAs that posttranscriptionally regulate mRNA expression. hsa-miR-6165 which was previously discovered in our group is located in the forth intron of p75NTR gene and its function is still under investigation. As P75NTR has diverse cellular functions, some of the complexity of its function could be attributed to the internally located microRNA. Our analysis revealed that treatment of HCT116 cells with 5-azacytidine promoted differential expression of hsa-miR-6165 from its host gene which is consistent with the bioinformatic prediction of an independent promoter for hsa-miR-6165. In addition, hsa-miR-6165 promoter is capable of driving GFP reporter gene in HeLa cells. The putative target gene expression level which was detected using RT-qPCR is inversely proportional to the expression level of hsa-miR-6165 during NT2 cell neural differentiation. Furthermore, hsa-miR-6165 overexpression resulted in significant downregulation of ABLIM-1, PVRL1, and PDK1 target genes, while it attenuates NT2 neural differentiation. Hsa-miR-6165 overexpression in SW480 cells also resulted in significant downregulation of PKD1, DAGLA, and PLXNA2 putative target genes, while it increases the sub-G1 cell population of SW480 and HEK293T cells as detected by flow cytometry. Overall, in this study, we report an independent promoter for hsa-miR-6165 which is active in HeLa cells. Additionally, hsa-miR-6165 targets ABLIM-1, PVRL1, PKD1, PLXNA2, and PDK1 genes, and unlike in HEK293T and SW480 cells, hsa-miR-6165 overexpression does not affect HeLa cells while its downregulation reduces sub-G1 cell population. Our results validate that hsa-miR-6165 affects the cell cycle progression and could increase apoptosis in human cell lines.

Novel homozygous mutation, c.400C>T (p.Arg134*), in the PVRL1 gene underlies cleft lip/palate-ectodermal dysplasia syndrome in an Asian patient.

Cleft lip/palate-ectodermal dysplasia syndrome is a rare, autosomal recessive disorder caused by homozygous loss-of-function mutations of the poliovirus receptor-like 1 (PVRL1) gene encoding nectin-1. Nectin-1 is a cell-cell adhesion molecule that is important for the initial step in the formation of adherens junctions and tight junctions; it is expressed in keratinocytes, neurons, and the developing face and palate. Clinical manifestations comprise a unique facial appearance with cleft lip/palate, ectodermal dysplasia, cutaneous syndactyly of the fingers and/or toes, and in some cases, mental retardation. We present the first report, to our knowledge, of an Asian individual with cleft lip/palate-ectodermal dysplasia syndrome with a novel PVRL1 mutation. A 7-year-old Japanese boy, the first child of a consanguineous marriage, showed hypohidrotic ectodermal dysplasia with sparse, brittle, fine, dry hair and hypodontia, the unique facial appearance with cleft lip/palate, cutaneous syndactyly of the fingers and mild mental retardation. Scanning electron microscopic examination of the hair demonstrated pili torti and pili trianguli et canaliculi. Mutation analysis of exon 2 of PVRL1 revealed a novel homozygous nonsense mutation, c.400C>T (p.Arg134*). His parents were heterozygous for the mutant alleles. All four PVRL1 mutations identified in cleft lip/palate-ectodermal dysplasia syndrome to date, including this study, resulted in truncated proteins that lack the transmembrane domain and intracellular domain of nectin-1, which is necessary to initiate the cell-cell adhesion process.

Novel insertion mutation in the PVRL1 gene in Turkish patients with non-syndromic cleft lip with/without cleft palate.

OBJECTIVES: Non-syndromic cleft lip with/without cleft palate (nsCL/P) has a complex aetiology involving both genetic and environmental factors. The aim of this study was to investigate the association between PVRL1 gene mutations and Turkish patients with nsCL/P. DESIGN: In this study, 80 Turkish patients with nsCL/P and 125 unrealeted individuals were analyzed. Mutations were detected using polymerase chain reactions and DNA sequencing. RESULTS: We found a novel GGA insertion between nucleotide positions c.1311_1313delGGA in exon 6 of the PVRL1 gene. Fifteen of the 80 patients with nsCL/P had the GGA insertion, although no mutation was found in the 125 unrelated individuals. CONCLUSION: We identified new supportive evidence that the association between PVRL1 gene and nsCL/P.