Defects In Eye Development Research Articles

Chlorpyrifos and cypermethrin combination disrupts sonic hedgehog signaling and associated regulatory molecules, leading to congenital eye defects in chick embryos

Pesticides are reported to be teratogenic for non-target species. Our studies have unraveled the teratogenicity of 50 % Chlorpyrifos & 5 % Cypermethrin combination (Ci) in developing chick embryos. A sub-lethal dose of this combination when administered to chick embryos, caused several developmental anomalies, with defects in eye development being frequent. Eye development begins at an early embryonic stage, with Sonic hedgehog (Shh) serving as a crucial signaling molecule. Shh plays a pivotal role in the early development of multiple organs, including the eye, by interacting with Pax6 and other regulatory molecules to guide the proper patterning of the eye. Thus, we hypothesized that Ci administration may lead to alteration in Shh expression which subsequently hampers downstream signaling molecules potentially contributing to congenital eye defects. Morphological, anatomical, histological, transcriptional and protein level analyses at various stages (Days 1,2,4 and 10) were carried out to evaluate the hypothesis. The results revealed a remarkable alteration of key regulators in treated embryos compared to control, providing insights into plausible causative mechanisms underlying Ci-induced congenital eye defects.

Genetic Mapping of prod E.3.3 , a New Lethal Allele of prod.

The E.3.3 mutation was generated in a Flp/FRT EMS screen for conditional mutations that cause growth and developmental defects in a genetic background that blocks apoptosis. The mutations were conditional, based on the Dark 82 allele being present on the starting chromosome, and blocking canonical apoptosis in a homozygous state. The E.3.3 mosaic eyes exhibit defects in eye development including patches of rough eye and irregular surface structure. Whole Genome Sequencing and complementation mapping revealed E.3.3 as an allele of prod . Prod is a DNA-binding protein that binds satellite repeats and is involved in chromocenter formation during mitosis. Here we present a novel allele of prod , prod E.3.3 , that disrupts the functional region of the Prod protein resulting in disruption of typical eye structure, likely due to disruption of chromatid separation during development.

Medaka (Oryzias latipes) Olpax6.2 acquires maternal inheritance and germ cells expression, but functionally degenerate in the eye

Gene duplication provides raw material for the evolution of genetic and phenotypic complexity. It has remained a long-standing mystery how duplicated genes evolve into new genes by neofunctionalization via the acquisition of new expression and/or activity and simultaneous loss of the old expression and activity. Fishes have many gene duplicates from whole genome duplication, making them excellent for studying the evolution of gene duplicates. In the fish medaka (Oryzias latipes), an ancestral pax6 gene has given rise to Olpax6.1 and Olpax6.2. Here we report that medaka Olpax6.2 is evolving towards neofunctionalization. A chromosomal syntenic analysis indicated that Olpax6.1 and Olpax6.2 are structurally co-homologous to the single pax6 in other organisms. Interestingly, Olpax6.2 maintains all conserved coding exons but loses the non-coding exons of Olpax6.1, and has 4 promoters versus 8 in Olpax6.1. RT-PCR revealed that Olpax6.2 maintains expression in the brain eye, pancreas as Olpax6.1. Surprisingly, Olpax6.2 also exhibits maternal inheritance and gonadal expression by RT-PCR, in situ hybridization and RNA transcriptome analysis. The expression and distribution of Olpax6.2 is not different from Olpax6.1 in the adult brain, eye and pancreas, but exhibited overlapping and distinct expression in early embryogenesis. We show that ovarian Olpax6.2 expression occurs in female germ cells. Olpax6.2 knockout shows no obvious defect in eye development, while Olpax6.1 F0 mutant have severe defects in eye development. Thus, Olpax6.2 acquires maternal inheritance and germ cell expression, but functionally degenerates in the eye, making this gene as an excellent model to study the neofunctionalization of duplicated genes.

Identification and characterization of two novel noncoding tyrosinase (TYR) gene variants leading to oculocutaneous albinism type 1

Oculocutaneous albinism type 1 (OCA1), resulting from pathogenic variants in the tyrosinase (TYR) gene, refers to a group of phenotypically heterogeneous autosomal recessive disorders characterized by a partial or a complete absence of pigment in the skin/hair and is also associated with common developmental eye defects. In this study, we identified two novel compound heterozygous TYR variants from a Chinese hypopigmentary patient by whole-exome sequencing. Specifically, the two variants were c.-89T>G, located at the core of the initiator E-box (Inr E-box) of the TYR promoter, and p.S16Y (c.47C>A), located within the signal sequence. We performed both in silico analysis and experimental validation and verified these mutations as OCA1 variants that caused either impaired or complete loss of function of TYR. Mechanistically, the Inr E-box variant dampened TYR binding to microphthalmia-associated transcription factor, a master transcriptional regulator of the melanocyte development, whereas the S16Y variant contributed to endoplasmic reticulum retention, a common and principal cause of impaired TYR activity. Interestingly, we found that the Inr E-box variant creates novel protospacer adjacent motif sites, recognized by nucleases SpCas9 and SaCas9-KKH, respectively, without compromising the functional TYR coding sequence. We further used allele-specific genomic editing by CRISPR activation to specifically target the variant promoter and successfully activated its downstream gene expression, which could lead to potential therapeutic benefits. In conclusion, this study expands the spectrum of TYR variants, especially those within the promoter and noncoding regions, which can facilitate genetic counseling and clinical diagnosis of OCA1.

Towards a Better Vision of Retinoic Acid Signaling during Eye Development.

Retinoic acid (RA) functions as an essential signal for development of the vertebrate eye by controlling the transcriptional regulatory activity of RA receptors (RARs). During eye development, the optic vesicles and later the retina generate RA as a metabolite of vitamin A (retinol). Retinol is first converted to retinaldehyde by retinol dehydrogenase 10 (RDH10) and then to RA by all three retinaldehyde dehydrogenases (ALDH1A1, ALDH1A2, and ALDH1A3). In early mouse embryos, RA diffuses to tissues throughout the optic placode, optic vesicle, and adjacent mesenchyme to stimulate folding of the optic vesicle to form the optic cup. RA later generated by the retina is needed for further morphogenesis of the optic cup and surrounding perioptic mesenchyme; loss of RA at this stage leads to microphthalmia and cornea plus eyelid defects. RA functions by binding to nuclear RARs at RA response elements (RAREs) that either activate or repress transcription of key genes. Binding of RA to RARs regulates recruitment of transcriptional coregulators such as nuclear receptor coactivator (NCOA) or nuclear receptor corepressor (NCOR), which in turn control binding of the generic coactivator p300 or the generic corepressor PRC2. No genes have been identified as direct targets of RA signaling during eye development, so future studies need to focus on identifying such genes and their RAREs. Studies designed to learn how RA normally controls eye development in vivo will provide basic knowledge valuable for determining how developmental eye defects occur and for improving strategies to treat eye defects.

Di-butyl phthalate (DBP) induces defects during embryonic eye development in zebrafish.

Di-butyl phthalate (DBP) is a phthalate ester (PAEs) added during the manufacturing of plastics to make them stronger, yet more pliable. DBP is noncovalently bound to plastics resulting in leaching into the environment. Concerning concentrations of DBP have been noted in surface and groundwater, aquatic ecosystems, soil and atmospheric environments globally. Global production of phthalates and thus concomitant exposure has increased over the years making studies on the ecological and environmental safety needed. Most of the literature on DBP focuses on the endocrine disrupting properties of phthalate esters, but the developmental toxicity of DBP is an understudied area. Here, we treat gastrula staged zebrafish embryos with environmentally relevant concentrations of DBP (2.5 µM). We find defects in eye development at 96 h post fertilization including a decrease in the size of the lens and retina in DBP-treated embryos. Defects in eye vascularization as well as loss of the optic nerve and optic tectum were also noted. Here we conclude that exposure to environmentally relevant doses of DBP during early embryonic development is toxic to eye development.

Biallelic variants in the small optic lobe calpain CAPN15 are associated with congenital eye anomalies, deafness and other neurodevelopmental deficits.

Microphthalmia, coloboma and cataract are part of a spectrum of developmental eye disorders in humans affecting ~12 per 100 000 live births. Currently, variants in over 100 genes are known to underlie these conditions. However, at least 40% of affected individuals remain without a clinical genetic diagnosis, suggesting variants in additional genes may be responsible. Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, an important class of developmental proteins, as yet uncharacterized in vertebrates. We identified five individuals with microphthalmia and/or coloboma from four independent families carrying homozygous or compound heterozygous predicted damaging variants in CAPN15. Several individuals had additional phenotypes including growth deficits, developmental delay and hearing loss. We generated Capn15 knockout mice that exhibited similar severe developmental eye defects, including anophthalmia, microphthalmia and cataract, and diminished growth. We demonstrate widespread Capn15 expression throughout the brain and central nervous system, strongest during early development, and decreasing postnatally. Together, these findings demonstrate a critical role of CAPN15 in vertebrate developmental eye disorders, and may signify a new developmental pathway.

The Classic Lobe Eye Phenotype of Drosophila Is Caused by Transposon Insertion-Induced Misexpression of a Zinc-Finger Transcription Factor.

Drosophila Lobe (L) alleles were first discovered ∼100years ago as spontaneous dominant mutants with characteristic developmental eye defects. However, the molecular basis for L dominant eye phenotypes has not been clearly understood. A previous work reported identification of CG10109/PRAS40 as the L gene, but subsequent analyses suggested that PRAS40 may not be related to L Here, we revisited the L gene to clarify this discrepancy and understand the basis for the dominance of L mutations. Genetic analysis localized the L gene to Oaz, which encodes a homolog of the vertebrate zinc finger protein 423 (Zfp423) family transcriptional regulators. We demonstrate that RNAi knockdown of Oaz almost completely restores all L dominant alleles tested. Lrev6-3 , a revertant allele of the L2 dominant eye phenotype, has an inframe deletion in the Oaz coding sequence. Molecular analysis of L dominant mutants identified allele-specific insertions of natural transposons (roo[ ]L1 , hopper[ ]L5 , and roo[ ]Lr ) or alterations of a preexisting transposon (L2 -specific mutations in roo[ ]Mohr) in the Oaz region. In addition, we generated additional L2 -reversion alleles by CRISPR targeting at Oaz These new loss-of-function Oaz mutations suppress the dominant L eye phenotype. Oaz protein is not expressed in wild-type eye disc but is expressed ectopically in L2/+ mutant eye disc. We induced male recombination between Oaz-GAL4 insertions and the L2 mutation through homologous recombination. By using the L2 -recombined GAL4 reporters, we show that Oaz-GAL4 is expressed ectopically in L2 eye imaginal disc. Taken together, our data suggest that neomorphic L eye phenotypes are likely due to misregulation of Oaz by spontaneous transposon insertions.

The transcription factor Maz is essential for normal eye development.

ABSTRACTWnt/β-catenin signaling has an essential role in eye development. Faulty regulation of this pathway results in ocular malformations, owing to defects in cell-fate determination and differentiation. Herein, we show that disruption of Maz, the gene encoding Myc-associated zinc-finger transcription factor, produces developmental eye defects in mice and humans. Expression of key genes involved in the Wnt cascade, Sfrp2, Wnt2b and Fzd4, was significantly increased in mice with targeted inactivation of Maz, resulting in abnormal peripheral eye formation with reduced proliferation of the progenitor cells in the region. Paradoxically, the Wnt reporter TCF-Lef1 displayed a significant downregulation in Maz-deficient eyes. Molecular analysis indicates that Maz is necessary for the activation of the Wnt/β-catenin pathway and participates in the network controlling ciliary margin patterning. Copy-number variations and single-nucleotide variants of MAZ were identified in humans that result in abnormal ocular development. The data support MAZ as a key contributor to the eye comorbidities associated with chromosome 16p11.2 copy-number variants and as a transcriptional regulator of ocular development.

Biallelic Deletion of Pxdn in Mice Leads to Anophthalmia and Severe Eye Malformation.

Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5′ upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the β3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose β1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

The Oculome Panel Test: Next-Generation Sequencing to Diagnose a Diverse Range of Genetic Developmental Eye Disorders

To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. Evaluation of diagnostic test. Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. Collated clinical details and oculome molecular genetic results. The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8of 9 cases solved). The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.

Mutations in VSX2, SOX2, and FOXE3 Identified in Patients with Micro-/Anophthalmia.

Anophthalmia and microphthalmia (A/M) are rare distinct phenotypes that represent a continuum of structural developmental eye defects. Here, we describe three probands from an Egyptian population with various forms of A/M: two patients with bilateral anophthalmia and one with bilateral microphthalmia that were investigated using whole exome sequencing (WES). We identified three causative mutations in three different genes. A new homozygous frameshift mutation c.[422delA];[422delA], p.[N141Ifs∗19];[N141Ifs∗19] in VSX2 was identified in a patient showing bilateral anophthalmia. A previously reported SOX2 deletion c.[70_89del20] p.[N24Rfs∗65];[=] was found in one subject with bilateral anophthalmia. A novel homozygous in-frame mutation c.[431_433delACT];[431_433delACT], p.[Y144del]; [Y144del]) in FOXE3 was identified in a patient with severe bilateral microphthalmia and anterior segment dysgenesis. This study shows that whole exome sequencing (WES) is a reliable and effective strategy for the molecular diagnosis of A/M. Our results expand its allelic heterogeneity and highlight the need for the testing of patient with this developmental anomaly.

Novel mutations in ALDH1A3 associated with autosomal recessive anophthalmia/microphthalmia, and review of the literature

BackgroundAutosomal recessive anophthalmia and microphthalmia are rare developmental eye defects occurring during early fetal development. Syndromic and non-syndromic forms of anophthalmia and microphthalmia demonstrate extensive genetic and allelic heterogeneity. To date, disease mutations have been identified in 29 causative genes associated with anophthalmia and microphthalmia, with autosomal dominant, autosomal recessive and X-linked inheritance patterns described. Biallelic ALDH1A3 gene variants are the leading genetic causes of autosomal recessive anophthalmia and microphthalmia in countries with frequent parental consanguinity.MethodsThis study describes genetic investigations in two consanguineous Pakistani families with a total of seven affected individuals with bilateral non-syndromic clinical anophthalmia.ResultsUsing whole exome and Sanger sequencing, we identified two novel homozygous ALDH1A3 sequence variants as likely responsible for the condition in each family; missense mutation [NM_000693.3:c.1240G > C, p.Gly414Arg; Chr15:101447332G > C (GRCh37)] in exon 11 (family 1), and, a frameshift mutation [NM_000693.3:c.172dup, p.Glu58Glyfs*5; Chr15:101425544dup (GRCh37)] in exon 2 predicted to result in protein truncation (family 2).ConclusionsThis study expands the molecular spectrum of pathogenic ALDH1A3 variants associated with anophthalmia and microphthalmia, and provides further insight of the key role of the ALDH1A3 in human eye development.

Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3.

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

Identification of Novel G Protein-Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism.

PurposeGPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein–coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators.MethodsGPR143 interacts with β-arrestin; we therefore established a β-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation.ResultsGPR143, which showed high constitutive activity in the β-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity.ConclusionsX-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents.

A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy

BackgroundThe identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene led us to study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of the well-studied model organism Drosophila melanogaster. The multitude of biological functions attributable to the vitamers catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk and we used RNA interference to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. We further investigated any association between this enhanced disease risk gene with the established PD model induced by expression of α-synuclein in the same neurons. We relied on the pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, to rescue the Pdxk-induced phenotypes.ResultsTo drive the expression of Pdxk RNA interference in DA neurons of Drosophila, we used Ddc-Gal4 which drives expression in both dopaminergic and serotonergic neurons, to result in decreased longevity and compromised climbing ability, phenotypes that are strongly associated with Drosophila models of PD. The inhibition of Pdxk in the α-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when we inhibited Pdxk in the developing eye using GMR-Gal4, we found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the α-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array.ConclusionsThough Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD.

Diagnostic exome sequencing in 266 Dutch patients with visual impairment

Inherited eye disorders have a large clinical and genetic heterogeneity, which makes genetic diagnosis cumbersome. An exome-sequencing approach was developed in which data analysis was divided into two steps: the vision gene panel and exome analysis. In the vision gene panel analysis, variants in genes known to cause inherited eye disorders were assessed for pathogenicity. If no causative variants were detected and when the patient consented, the entire exome data was analyzed. A total of 266 Dutch patients with different types of inherited eye disorders, including inherited retinal dystrophies, cataract, developmental eye disorders and optic atrophy, were investigated. In the vision gene panel analysis (likely), causative variants were detected in 49% and in the exome analysis in an additional 2% of the patients. The highest detection rate of (likely) causative variants was in patients with inherited retinal dystrophies, for instance a yield of 63% in patients with retinitis pigmentosa. In patients with developmental eye defects, cataract and optic atrophy, the detection rate was 50, 33 and 17%, respectively. An exome-sequencing approach enables a genetic diagnosis in patients with different types of inherited eye disorders using one test. The exome approach has the same detection rate as targeted panel sequencing tests, but offers a number of advantages. For instance, the vision gene panel can be frequently and easily updated with additional (novel) eye disorder genes. Determination of the genetic diagnosis improved the clinical diagnosis, regarding the assessment of the inheritance pattern as well as future disease perspective.

Inhibition of mitochondrial calcium uptake 1 in Drosophila neurons.

The mitochondrial calcium uptake 1 (MICU1) is a regulatory subunit of the mitochondrial calcium uniporter that plays an important role in calcium sensing. It contains two EF-hand domains that are well conserved across diverse species from protozoa to plants and metazoans. The loss of MICU1 function in mammals is attributed to several neurological disorders that involve movement dysfunction. The CG4495 gene in Drosophila melanogaster was identified as a putative homolog of MICU1 in the HomoloGene database of the National Centre for Biotechnology Information (NCBI). In agreement with previous studies that have shown the development of neurological disorders and movement defects in MICU1 loss-of-function organisms, we attempted to identify the function of CG4495/MICU1 in Drosophila neurons. We analyzed survival and locomotor ability of these flies and additionally performed biometric analysis of the Drosophila developing eye. The inducible RNA interference-mediated inhibition of CG4495/MICU1 in the Ddc-Gal4-expressing neurons of Drosophila presented with reduction in survival coupled with a precocious loss of locomotor ability. Since the pro-survival Bcl-2 family genes have been shown to be protective towards mitochondria, and CG4495/MICU1 has a mitochondrial targeting sequence, we attempted to rescue the phenotypes resulting from the inhibition of CG4495/MICU1 by overexpressing Buffy, the sole Bcl-2 homologue in Drosophila. The co-expression of CG4495/MICU1-RNAi along with Buffy resulted in the suppression of the phenotypes induced by the inhibition of CG4495/MICU1. Subsequently, the inhibition of CG4495/MICU1 in the Drosophila developing eye, a neuron-rich organ, resulted in reduced number of ommatidia and a highly fused ommatidial array. These developmental eye defects were rescued by the overexpression of Buffy. Our study suggests an important role for MICU1 in the normal function of neurons in Drosophila.

Overexpression of Buffy enhances the loss of parkin and suppresses the loss of Pink1 phenotypes in Drosophila

Mutations in parkin (PARK2) and Pink1 (PARK6) are responsible for autosomal recessive forms of early onset Parkinson's disease (PD). Attributed to the failure of neurons to clear dysfunctional mitochondria, loss of gene expression leads to loss of nigrostriatal neurons. The Pink1/parkin pathway plays a role in the quality control mechanism aimed at eliminating defective mitochondria, and the failure of this mechanism results in a reduced lifespan and impaired locomotor ability, among other phenotypes. Inhibition of parkin or Pink1 through the induction of stable RNAi transgene in the Ddc-Gal4-expressing neurons results in such phenotypes to model PD. To further evaluate the effects of the overexpression of the Bcl-2 homologue Buffy, we analysed lifespan and climbing ability in both parkin-RNAi- and Pink1-RNAi-expressing flies. In addition, the effect of Buffy overexpression upon parkin-induced developmental eye defects was examined through GMR-Gal4-dependent expression. Curiously, Buffy overexpression produced very different effects: the parkin-induced phenotypes were enhanced, whereas the Pink1-enhanced phenotypes were suppressed. Interestingly, the overexpression of Buffy along with the inhibition of parkin in the neuron-rich eye results in the suppression of the developmental eye defects.