Epidemiology of PAX6 Gene Pathogenic Variants and Expected Prevalence of PAX6-Associated Congenital Aniridia across the Russian Federation: A Nationwide Study.

This study investigates the distribution of PAX6-associated congenital aniridia (AN) and WAGR syndrome across Russian Federation (RF) districts while characterizing PAX6 gene variants. We contribute novel PAX6 pathogenic variants and 11p13 chromosome region rearrangements to international databases based on a cohort of 379 AN patients (295 families, 295 probands) in Russia. We detail 100 newly characterized families (129 patients) recruited from clinical practice and specialized screening studies. Our methodology involves multiplex ligase-dependent probe amplification (MLPA) analysis of the 11p13 chromosome, PAX6 gene Sanger sequencing, and karyotype analysis. We report novel findings on PAX6 gene variations, including 67 intragenic PAX6 variants and 33 chromosome deletions in the 100 newly characterized families. Our expanded sample of 295 AN families with 379 patients reveals a consistent global PAX6 variant spectrum, including CNVs (copy number variants) of the 11p13 chromosome (31%), complex rearrangements (1.4%), nonsense (25%), frameshift (18%), and splicing variants (15%). No genetic cause of AN is defined in 10 patients. The distribution of patients across the Russian Federation varies, likely due to sample completeness. This study offers the first AN epidemiological data for the RF, providing a comprehensive PAX6 variants spectrum. Based on earlier assessment of AN prevalence in the RF (1:98,943) we have revealed unexamined patients ranging from 55% to 87%, that emphases the need for increased awareness and comprehensive diagnostics in AN patient care in Russia.

Upstream ORF frameshift variants in the PAX6 5'UTR cause congenital aniridia.

Congenital aniridia (AN) is a severe autosomal dominant panocular disorder associated with pathogenic variants in the PAX6 gene. Previously, we performed a molecular genetic study of a large cohort of Russian patients with AN and revealed four noncoding nucleotide variants in the PAX6 5'UTR. 14 additional PAX6-5'UTR variants were also reported in the literature, but the mechanism of their pathogenicity remained unclear. In the present study, we experimentally analyze five patient-derived PAX6 5'UTR-variants: four variants that we identified in Russian patients (c.-128-2delA, c.-125dupG, c.-122dupG, c.-118_-117del) and one previously reported (c.-52+5G>C). We show that the variants lead to a decrease in the protein translation efficiency, while mRNA expression level is not significantly reduced. Two of these variants also affect splicing. Furthermore, we predict and experimentally validate the presence of an evolutionarily conserved small uORF in the PAX6 5'UTR. All studied variants lead to the frameshift of the uORF, resulting in its extension. This extended out-of-frame uORF overlaps with the downstream CDS and thereby reduces its translation efficiency. We conclude that the uORF frameshift may be the main mechanism of pathogenicity for at least 15 out of 18 known PAX6 5'UTR variants. Moreover, we predict additional uORFs in the PAX6 5'UTR.

Analysis of genotype-phenotype correlations in PAX6-associated aniridia.

BackgroundAniridia is a severe autosomal dominant panocular disorder associated with pathogenic sequence variants of the PAX6 gene or 11p13 chromosomal aberrations encompassing the coding and/or regulatory regions of the PAX6 gene in a heterozygous state. Patients with aniridia display several ocular anomalies including foveal hypoplasia, cataract, keratopathy, and glaucoma, which can vary in severity and combination.MethodsA cohort of 155 patients from 125 unrelated families with identified point PAX6 pathogenic variants (118 patients) or large chromosomal 11p13 deletions (37 patients) was analyzed. Genetic causes were divided into 6 types. The occurrence of 6 aniridic eye anomalies was analyzed. Fisher's exact test was applied for 2×2 contingency tables assigning numbers of patients with/without each sign and each type of the PAX6 variants or 11p13 deletions with Benjamini-Hochberg correction. The age of patients with different types of mutation did not differ.ResultsPatients with 3'-cis-regulatory region deletions had a milder aniridia phenotype without keratopathy, nystagmus, or foveal hypoplasia. The phenotypes of the patients with other rearrangements involving 11p13 do not significantly differ from those associated with point pathogenic variants in the PAX6 gene. Missense mutations and genetic variants disrupting splicing are associated with a severe aniridia phenotype and resemble loss-of-function mutations. It is particularly important that in all examined patients, PAX6 mutations were found to be associated with multiple eye malformations. The age of patients with keratopathy, cataract, and glaucoma was significantly higher than the age of patients without these signs.ConclusionWe got clear statistically significant genotype-phenotype correlations in congenital aniridia and evident that aniridia severity indeed had worsened with age.

Morphometric analysis of the lens in human aniridia and mouse Small eye.

Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia. Here, the ocular morphometry of aniridia patients is compared with the lens phenotype of Pax6+/tm1/Pgr mice to reveal whether there are species differences in Pax6 regulation of lens development and homeostasis. Ultrasound biometry (UBM) revealed that eleven percent of aniridia patients exhibited mild microphthalmia while the anterior chamber depth of aniridic eyes was significantly reduced from 6 months of age onward. Although aniridic lens thickness was normal from birth, it was significantly decreased in aniridic lenses older than 30. Notably, 86% of aniridic lenses exhibited cataractous changes in this cohort. In addition, a significant proportion of aniridia patients develop lens subluxation as they age associated with reduced lens diameter as measured by anterior segment optical coherence tomography (AS-OCT). Analysis of young adult Pax6+/tm1/Pgr mouse lenses by micro-computed tomography (microCT), bright field and dark field imaging revealed that they are reduced in size but did not exhibit overt cataracts at this age. Overall, this study reveals that congenital microphthalmia as assessed by axial length, or microphakia, as assessed by lens thickness, are not typical in human aniridia, although these are primary manifestations of Pax6 mutations in mice, suggesting that PAX6 regulates some aspects of lens development differently between these species.

Expanding the phenotype of CRYAA nucleotide variants to a complex presentation of anterior segment dysgenesis.

BACKGROUND: Mutations in CRYAA, which encodes the α-crystallin protein, are associated with a spectrum of congenital cataract-microcornea syndromes. RESULTS: In this study, we performed clinical examination and subsequent genetic analysis in two unrelated sporadic cases of different geographical origins presenting with a complex phenotype of ocular malformation. Both cases manifested bilateral microphthalmia and severe anterior segment dysgenesis, primarily characterized by congenital aphakia, microcornea, and iris hypoplasia/aniridia. NGS-based analysis revealed two novel single nucleotide variants occurring de novo and affecting the translation termination codon of the CRYAA gene, c.520T > C and c.521A > C. Both variants are predicted to elongate the C-terminal protein domain by one-third of the original length. CONCLUSIONS: Our report not only expands the mutational spectrum of CRYAA but also identifies the genetic cause of the unusual ocular phenotype described in this report.

LMO2 gene deletions significantly worsen the prognosis of Wilms' tumor development in patients with WAGR syndrome.

WAGR syndrome (OMIM #194072) is a rare genetic disorder that consists of development of Wilms' tumor (nephroblastoma), aniridia, genitourinary anomalies and intellectual disability (mental retardation). It is associated with WAGR-region deletions in the 11p13 chromosome region. Our previous study of congenital aniridia patients revealed a noticeable number of aniridia patients with WAGR-region deletions but without Wilms' tumor in their medical history. We assessed the involvement of other neighboring genes from affected chromosome regions in the patients with and without Wilms' tumor. Reliable confidence was obtained for the LMO2 gene, which is significantly more often deleted in patients with nephroblastoma. Thus, our study presents genetic evidence that the development of Wilms tumors in WAGR syndrome patients should be attributed to the deletion of WT1 and LMO2 rather than WT1 only.

Functional reassessment of PAX6 single nucleotide variants by in vitro splicing assay.

Nucleotide variants that disrupt normal splicing might be the cause of a large number of diseases. Nevertheless, because of the complexity of splicing regulation, it is not always possible to accurately predict the effect of nucleotide sequence changes on splicing events and mRNA structure. Thereby, a number of newly identified nucleotide variants are falsely classified as VUS (a variant of uncertain significance). In the present study we used the minigene assay to analyze the functional consequences of six intronic (c.142-5T>G, c.142-14C>G, c.142-64A>C, c.141+4A>G, c.1032+ 6T>G, c.682+4delA), one missense (c.140A>G) and one synonymous (c.174C>T) variants in the PAX6 gene found in patients with congenital aniridia. We revealed that all except one (c.142-64A>C) variants lead to the disruption of normal splicing patterns resulting in premature termination codon formation followed by mRNA degradation through the nonsense mediated decay pathway. This produces a null allele of the PAX6 gene. That allowed us to reclassify the analyzed variants as loss-of-function and to establish their functional role.

Clinical and morphological manifestations of aniridia-associated keratopathy on anterior segment optical coherence tomography and in vivo confocal microscopy.

PURPOSE: The study aimed to evaluate clinical and morphological changes in the limbal palisades of Vogt (POV) at different stages of aniridia-associated keratopathy (AAK) and to assess possible utility of anterior segment optical coherence tomography (AS-OCT) for the visualization of limbal progenitor structures as it correlates to laser scanning confocal microscopy (LSCM) data. METHODS: The study involved 32 patients (59 eyes) with congenital aniridia. AAK stage was defined based on biomicroscopy. Assessment of limbal zone and detection of POVs in identical areas was performed by LSCM (HRT3) and AS-OCT (RTVue XR Avanti) using 3D Cornea (En Face mode) and Cornea Cross Line protocols. RESULTS: Intact and changed POVs were found in 8/8 stage 0 eyes, in 1/21 stage I and 2/13 stage II eyes. Spearman's correlation coefficient in assessing the consistency of the POV diagnostic results by LSCM and AS-OCT for the inferior limbus was rS = 0.85 (P < 0.05), for the superior limbus - rS = 0.53 (P < 0.05). AS-OCT was less sensitive for detection of partially present POVs in superior limbus. The negative correlation between AAK stage and POV preservation was determined (rS = -0.5, P < 0.05). There was no correlation between AAK stage and patient age (rS = 0.235, P = 0.209). Three patients with PAX6 3' deletion showed stage 0 AAK with intact or slightly disturbed POVs morphology and transparent cornea. CONCLUSION: AS-OCT may be an additional diagnostic tool for POV visualization in vivo in aniridic patients. Its diagnostic accuracy is subject to selection of anatomic region, nystagmus and the degree of POV degradation.

Mass spectrometric signatures of the blood plasma metabolome for disease diagnostics.

In metabolomics, a large number of small molecules can be detected in a single run. However, metabolomic data do not include the absolute concentrations of each metabolite. Generally, mass spectrometry analyses provide metabolite concentrations that are derived from mass peak intensities, and the peak intensities are strictly dependent on the type of mass spectrometer used, as well as the technical characteristics, options and protocols applied. To convert mass peak intensities to actual concentrations, calibration curves have to be generated for each metabolite, and this represents a significant challenge depending on the number of metabolites that are detected and involved in metabolome-based diagnostics. To overcome this limitation, and to facilitate the development of diagnostic tests based on metabolomics, mass peak intensities may be expressed in quintiles. The present study demonstrates the advantage of this approach. The examples of diagnostic signatures, which were designed in accordance to this approach, are provided for lung and prostate cancer (leading causes of mortality due to cancer in developed countries) and impaired glucose tolerance (which precedes type 2 diabetes, the most common endocrinology disease worldwide).

Initial results of trifocal diffractive IOL implantation.

BACKGROUND: Bifocal intraocular lenses (IOL) are designed to reduce dependence on eyeglasses after surgery, but they cannot provide full range of vision, and have a drop of visual acuity in the intermediate distance. This problem may be solved with a trifocal lens, which produces three useful focal points. This study was conducted to evaluate clinical outcomes, functional vision and spectacle independence in patients with trifocal diffractive IOLs. METHODS: Thirty-six eyes after implantation of diffractive intraocular lens MIOL-Record were evaluated. This IOL has a diffractive lens profile binary in phase and produces three useful foci. Outcome measures recorded 6 months postoperatively were uncorrected and distance-corrected (far, near, intermediate) visual acuities, contrast sensitivity (under photopic and mesopic condition), root-mean-square (RMS) of higher order aberrations (HOA), patients' quality of life (visual function questionnaire VFQ-25, halos and glare presence, overall satisfaction), and spectacle independence. RESULTS: Postoperatively, monocularly, the uncorrected (UDVA) and the corrected distance visual acuity (CDVA) were 0.74 +/- 0.21 and 0.86 +/- 0.23 respectively (P < 0.05). No differences were found between uncorrected (UNVA)-0.85 +/- 0.13 and best distance-corrected near visual acuity (CNVA) 0.89 +/- 0.12 (P > 0.05). The uncorrected (UIVA) and best distance-corrected acuities of intermediate vision (CIVA) at 50 cm were 0.58 +/- 0.16 and 0.6 +/- 0.2 respectively. The photopic contrast sensitivity was within the standard normal range. But under dim light conditions it was inferior at all spatial frequencies by more than 0.2 logarithmic units. The mean aberration values were: total HOA RMS-0.816 +/- 0.362 mum, spherical aberration (SA)-0.207 +/- 0.136 mum, coma-0.335 +/- 0.206 mum. The percentage of patients achieving spectacle freedom was 94%. On the questionnaire, 25% of patient noted nighttime halos; glare difficulty was reported by 16.7% of patients, difficulties with night vision persisted in 22.3% of cases. CONCLUSION: Diffractive trifocal intraocular lens MIOL-Record provided good uncorrected distance, intermediate and near vision, gave high rates of spectacle freedom and overall patient satisfaction.

Preparation and characterization of amphiphilic poly-N-vinylpyrrolidone nanoparticles containing indomethacin.

Amphiphilic poly-N-vinylpyrrolidone derivatives (Amph-PVP) with different molecular weight of hydrophilic PVP fragment and one terminal hydrophobic n-alkyl fragment of different length were synthesized for preparation of nano-scaled particles in aqueous media. To estimate novel polymer efficiency and perspective as basis for drug delivery systems, the polymeric micelle-like particles were prepared by dialysis and solvent evaporation methods. Indomethacin was incorporated into hydrophobic inner core of these nanoparticles as a typical model drug. From the dynamic light-scattering measurements, the size of particles formed was less than 200 nm with narrow monodisperse size distribution and nanoparticles size slightly increased with the amount of indomethacin encapsulated into inner core of Amph-PVP particles. The critical aggregation concentration values for prepared polymer samples determined by fluorescence spectroscopy were in micromole range which is lower than it is for common low molecular weight surfactants. As the hydrophobic fragment of amphiphilic polymer increased, the critical aggregation concentration values decreased. An increase of polymer hydrophilic fragment molecular weight produced larger nanoaggregates. In vitro release experiments using indomethacin-loaded Amph-PVP nanoparticles exhibited the sustained release behavior without any burst effect for most polymer samples.