Monika Ołdak

M34T and V37I mutations in GJB2 associated hearing impairment: Evidence for pathogenicity and reduced penetrance

Despite research the role of the M34T and V37I variants of GJB2 in causing hearing impairment (HI) remains controversial. Our purpose was to test a hypothesis that M34T and V37I are pathogenic but have distinct features resulting in a reduced penetrance. We screened for known GJB2/GJB6 mutations 233 Polish consecutive unrelated subjects with non‐syndromic, sensorineural HI who were previously found to carry 35delG mutation on one chromosome. The most frequent mutations were also analyzed in ∼1,000 controls. We found that M34T and V37I were significantly (P ≪ 10−6) overrepresented among patients, but their penetrance was estimated as 1/10 relative to mutations of undisputed pathogenicity. This finding apparently could not be explained by low degree of HI associated with M34T and V37I since another mutation causing comparably mild HI (L90P) did not have reduced penetrance. Subsequent analyses showed that the patients with M34T/35delG and V37I/35delG had significantly later onset of HI than patients with other genotypes (P < 10−6) including the L90P/35delG (P = 0.006). Also, among these patients (but not others) a strong correlation between the degree of HI and its duration was found (r = 0.79, P < 10−5). We tentatively suggest that M34T and V37I might cause mild HI characterized by relatively late onset and progression.

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

ABSTRACT 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

ABSTRACT 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.

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss.

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.

Upregulation of the WNT pathway in tuberous sclerosis-associated subependymal giant cell astrocytomas

Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10-15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that beta-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by beta-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs.

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

ABSTRACT 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.

Novel and De Novo Mutations Extend Association of POU3F4 with Distinct Clinical and Radiological Phenotype of Hearing Loss.

POU3F4 mutations (DFNX2) are the most prevalent among non-syndromic X-linked hearing loss (HL) identified to date. Clinical manifestations of DFNX2 usually comprise congenital HL either sensorineural or mixed, a tendency towards perilymphatic gusher during otologic surgery and temporal bone malformations. The aim of the present study was to screen for POU3F4 mutations in a group of 30 subjects with a suggestive clinical phenotype as well as a group (N = 1671–2018) of unselected hearing loss patients. We also planned to analyze audiological and radiological features in patients with HL caused by POU3F4 defects. The molecular techniques used to detect POU3F4 mutations included whole exome sequencing (WES), Sanger sequencing and real-time polymerase chain reaction. Hearing status was assessed with pure-tone audiometry and auditory brainstem response. Computer tomography scans were evaluated to define the pattern of structural changes in the temporal bones. Six novel (p.Gln27*, p.Glu187*, p.Leu217*, p.Gln275*, p.Gln306*, p.Val324Asp) and two known (p.Ala116fs141*, p.Leu208*) POU3F4 mutations were detected in the studied cohort. All probands with POU3F4 defects suffered from bilateral, prelingual, severe to profound HL. Morphological changes of the temporal bone in these patients presented a similar pattern, including malformations of the internal auditory canal, vestibular aqueduct, modiolus and vestibule. Despite different localization in the POU3F4 gene all mutations severely impair the protein structure affecting at least one functional POU3F4 domain, and results in similar and severe clinical manifestations. Sequencing of the entire POU3F4 gene is recommended in patients with characteristic temporal bone malformations. Results of POU3F4 mutation testing are important not only for a proper genetic counseling, but also for adequate preparation and conduction of a surgical procedure.

Evidence Against RAB40AL Being the Locus for Martin–Probst X‐Linked Deafness–Intellectual Disability Syndrome

RAB40AL has been reported as the locus for Martin–Probst syndrome (MPS), an X‐linked deafness–intellectual disability syndrome. The report was based on segregation of a missense change p.D59G with the disease in a single family and in vitro localization studies. We found the p.D59G variant by whole‐exome sequencing in two patients; however, the diagnosis of MPS was excluded in both cases. Furthermore, screening of control DNA samples (n = 810) from a general Polish population, using allele‐specific PCR and direct DNA sequencing for verification, identified p.D59G in 8/405 males and 12/405 females. High prevalence of the p.D59G variant (2.47%) is typical for a common genetic variation observed in asymptomatic individuals. Our data question the role of RAB40AL mutation as a disease‐causing change and the involvement of RAB40AL in MPS. Considering an increasing use of next‐generation sequencing in the clinical setting, our finding is of practical diagnostic importance.

Phacoemulsification with corneal astigmatism correction with the use of a toric intraocular lens in a case of megalocornea

&NA; We report the case of a 49‐year‐old patient with megalocornea and coexisting corneal astigmatism. The corneal diameter in the right eye was 15.0 mm and in the left eye, 14.9 mm. In both eyes, a nuclear sclerotic cataract developed, with the tendency toward cortical mass swelling in the right eye. The aim of surgical treatment was to remove the cataract with simultaneous correction of corneal astigmatism by implanting an Acrysof toric intraocular lens (IOL). Intraocular lens stabilization was obtained by suturing it to an capsular tension ring (CTR) in the anterior chamber. The IOL–CTR complex was rotated into the lens capsule and aligned with the steep meridian of corneal astigmatism. The surgical technique provides a stable refractive and functional effect in patients with megalocornea and coexisting cataract and corneal astigmatism. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Fuchs Endothelial Corneal Dystrophy: Strong Association with rs613872 Not Paralleled by Changes in Corneal Endothelial TCF4 mRNA Level

Fuchs endothelial corneal dystrophy (FECD) is a common corneal endotheliopathy with a complex and heterogeneous genetic background. Different variants in the TCF4 gene have been strongly associated with the development of FECD. TCF4 encodes the E2-2 transcription factor but the link between the strong susceptibility locus and disease mechanism remains elusive. Here, we confirm a strong positive association between TCF4 single nucleotide polymorphism rs613872 and FECD in Polish patients (OR = 12.95, 95% CI: 8.63–19.42, χ 2 = 189.5, p < 0.0001). We show that TCF4 expression at the mRNA level in corneal endothelium (n = 63) does not differ significantly between individuals with a particular TCF4 genotype. It is also not altered in FECD patients as compared to control samples. The data suggest that changes in the transcript level containing constitutive TCF4 exon encoding the amino-terminal part of the protein seem not to contribute to disease pathogenesis. However, considering the strong association of TCF4 allelic variants with FECD, genotyping of TCF4 risk alleles may be important in the clinical practice.