High expression of Matrix Gla Protein in Schnyder corneal dystrophy patients points to an active role of vitamin K in corneal health

Purpose Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disorder characterized by corneal lipid accumulation and caused byUBIAD1pathogenic variants.UBIAD1encodes a vitamin K (VK) biosynthetic enzyme. To assess the corneal and vascular VK status in SCD patients, we focused on matrix Gla protein (MGP), a VK-dependent protein. Methods Conformation-specific immunostainings of different MGP maturation forms were performed on corneal sections and primary keratocytes from corneal buttons of two SCD patients with UBIAD1 p.Asp112Asn and p.Asn102Ser pathogenic variants and unrelated donors. Native or UBIAD1-transfected keratocytes were used for gene expression analysis. Plasma samples from SCD patients (n = 12) and control individuals (n = 117) were subjected for inactive desphospho-uncarboxylated MGP level measurements with an ELISA assay. Results Substantial amounts of MGP were identified in human cornea and most of it in its fully matured and active form. The level of mature MGP did not differ between SCD and control corneas. In primary keratocytes from SCD patients, a highly increased MGP expression and presence of immature MGP forms were detected. Significantly elevated plasma concentration of inactive MGP was found in SCD patients. Conclusion High amount of MGP and the predominance of mature MGP forms in human cornea indicate that VK metabolism is active in the visual system. Availability of MGP seems of vital importance for a healthy cornea and may be related to protection against corneal calcification. Systemic MGP findings reveal a poor vascular VK status in SCD patients and indicate that SCD may lead to cardiovascular consequences

The Papillomavirus E2 Protein Binds to and Synergizes with C/EBP Factors Involved in Keratinocyte Differentiation

The papillomavirus life cycle is closely linked to the differentiation program of the host keratinocyte. Thus, late gene expression and viral maturation are restricted to terminally differentiated keratinocytes. A variety of cellular transcription factors including those of the C/EBP family are involved in the regulation of keratinocyte differentiation. In this study we show that the papillomavirus transcription factor E2 cooperates with C/EBPα and -β in transcriptional activation. This synergism was independent of an E2 binding site. E2 and C/EBP factors synergistically transactivated a synthetic promoter construct containing classical C/EBPβ sites and the C/EBPα-responsive proximal promoter of the involucrin gene, which is naturally expressed in differentiating keratinocytes. C/EBPα or -β coprecipitated with E2 proteins derived from human papillomavirus type 8 (HPV8), HPV16, HPV18, and bovine papillomavirus type 1 in vitro and in vivo, indicating complex formation by the cellular and viral factors. The interaction domains could be mapped to the C terminus of E2 and amino acids 261 to 302 located within the bZIP motif of C/EBPβ. Our data suggest that E2, via its interaction with C/EBP factors, may contribute to enhancing keratinocyte differentiation, which is suppressed by the viral oncoproteins E6 and E7 in HPV-induced lesions

The Human Papillomavirus Type 8 E2 Protein Suppresses β4-Integrin Expression in Primary Human Keratinocytes

Human papillomaviruses (HPVs) infect keratinocytes of skin and mucosa. Homeostasis of these constantly renewing, stratified epithelia is maintained by balanced keratinocyte proliferation and terminal differentiation. Instructions from the extracellular matrix engaging integrins strongly regulate these keratinocyte functions. The papillomavirus life cycle parallels the differentiation program of stratified epithelia, and viral progeny is produced only in terminally differentiating keratinocytes. Whereas papillomavirus oncoproteins can inhibit keratinocyte differentiation, the viral transcription factor E2 seems to counterbalance the impact of oncoproteins. In this study we show that high expression of HPV type 8 (HPV8) E2 in cultured primary keratinocytes leads to strong down-regulation of β4-integrin expression levels, partial reduction of β1-integrin, and detachment of transfected keratinocytes from underlying structures. Unlike HPV18 E2-expressing keratinocytes, HPV8 E2 transfectants did not primarily undergo apoptosis. HPV8 E2 partially suppressed β4-integrin promoter activity by binding to a specific E2 binding site leading to displacement of at least one cellular DNA binding factor. To our knowledge, we show for the first time that specific E2 binding contributes to regulation of a cellular promoter. In vivo, decreased β4-integrin expression is associated with detachment of keratinocytes from the underlying basement membrane and their egress from the basal to suprabasal layers. In papillomavirus disease, β4-integrin down-regulation in keratinocytes with higher E2 expression may push virally infected cells into the transit-amplifying compartment and ensure their commitment to the differentiation process required for virus replication

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes ▿

The papillomavirus life cycle parallels keratinocyte differentiation in stratifying epithelia. We have previously shown that the human papillomavirus type 8 (HPV8) E2 protein downregulates β4-integrin expression in normal human keratinocytes, which may trigger subsequent differentiation steps. Here, we demonstrate that the DNA binding domain of HPV8 E2 is sufficient to displace a cellular factor from the β4-integrin promoter. We identified the E2-displaceable factor as activator protein 1 (AP-1), a heteromeric transcription factor with differentiation-specific expression in the epithelium. β4-Integrin-positive epithelial cells displayed strong AP-1 binding activity. Both AP-1 binding activity and β4-integrin expression were coregulated during keratinocyte differentiation suggesting the involvement of AP-1 in β4-integrin expression. In normal human keratinocytes the AP-1 complex was composed of JunB and Fra-1 subunits. Chromatin immunoprecipitation assays confirmed that JunB/Fra-1 proteins interact in vivo with the β4-integrin promoter and that JunB/Fra-1 promoter occupancy is reduced during keratinocyte differentiation as well as in HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the β4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 effect. In summary, we identified a novel mechanism of human β4-integrin regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle

First confirmatory study on PTPRQ as an autosomal dominant non-syndromic hearing loss gene

Background Biallelic PTPRQ pathogenic variants have been previously reported as causative for autosomal recessive non-syndromic hearing loss. In 2018 the first heterozygous PTPRQ variant has been implicated in the development of autosomal dominant non-syndromic hearing loss (ADNSHL) in a German family. The study presented the only, so far known, PTPRQ pathogenic variant (c.6881G>A) in ADNSHL. It is located in the last PTPRQ coding exon and introduces a premature stop codon (p.Trp2294*). Methods A five-generation Polish family with ADNSHL was recruited for the study (n = 14). Thorough audiological, neurotological and imaging studies were carried out to precisely define the phenotype. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted for the proband. Family segregation analysis of the identified variants was performed using Sanger sequencing. Single nucleotide polymorphism array on DNA samples from the Polish and the original German family was used for genome-wide linkage analysis. Results Combining clinical exome sequencing and family segregation analysis, we have identified the same (NM_001145026.2:c.6881G>A, NP_001138498.1:p.Trp2294*) PTPRQ alteration in the Polish ADNSHL family. Using genome-wide linkage analysis, we found that the studied family and the original German family derive from a common ancestor. Deep phenotyping of the affected individuals showed that in contrast to the recessive form, the PTPRQ-related ADNSHL is not associated with vestibular dysfunction. In both families ADNSHL was progressive, affected mainly high frequencies and had a variable age of onset. Conclusion Our data provide the first confirmation of PTPRQ involvement in ADNSHL. The finding strongly reinforces the inclusion of PTPRQ to the small set of genes leading to both autosomal recessive and dominant hearing loss

Association of Sex With Frequent and Mild ABCA4 Alleles in Stargardt Disease

IMPORTANCE The mechanisms behind the phenotypic variability and reduced penetrance in autosomal recessive Stargardt disease (STGD1), often a blinding disease, are poorly understood. Identification of the unknown disease modifiers can improve patient and family counseling and provide valuable information for disease management. OBJECTIVE To assess the association of incompletely penetrant ABCA4 alleles with sex in STGD1. DESIGN, SETTING, AND PARTICIPANTS Genetic data for this cross-sectional study were obtained from 2 multicenter genetic studies of 1162 patients with clinically suspected STGD1. Unrelated patients with genetically confirmed STGD1 were selected. The data were collected from June 2016 to June 2019, and post hoc analysis was performed between July 2019 and January 2020. MAIN OUTCOMES AND MEASURES Penetrance of reported mild ABCA4 variants was calculated by comparing the allele frequencies in the general population (obtained from the Genome Aggregation Database) with the genotyping data in the patient population (obtained from the ABCA4 Leiden Open Variation Database). The sex ratio among patients with and patients without an ABCA4 allele with incomplete penetrance was assessed. RESULTS A total of 550 patients were included in the study, among which the mean (SD) age was 45.7 (18.0) years and most patients were women (311 [57%]). Five of the 5 mild ABCA4 alleles, including c.5603A>T and c.5882G>A, were calculated to have incomplete penetrance. The women to men ratio in the subgroup carrying c.5603A>T was 1.7 to 1; the proportion of women in this group was higher compared with the subgroup not carrying a mild allele (difference, 13%; 95% CI, 3%-23%; P=.02). The women to men ratio in the c.5882G>A subgroup was 2.1 to 1, and the women were overrepresented compared with the group carrying no mild allele (difference, 18%; 95% CI, 6%-30%; P=.005). CONCLUSIONS AND RELEVANCE This study found an imbalance in observed sex ratio among patients harboring a mild ABCA4 allele, which concerns approximately 25% of all patients with STGD1, suggesting that STGD1 should be considered a polygenic or multifactorial disease rather than a disease caused by ABCA4 gene mutations alone. The findings suggest that sex should be considered as a potential disease-modifying variable in both basic research and clinical trials on STGD1