Minocycline and Diacetyl Minocycline Eye Drops Reduce Ocular Neovascularization in Mice.

Purpose: To evaluate the efficacy of minocycline and a novel, modified minocycline analogue that lacks antimicrobial action, diacetyl minocycline (DAM), on choroidal neovascularization (CNV) in mice of both sexes. Methods: CNV was induced via laser injury in female and male C57BL/6J mice. Minocycline, DAM, or saline was administered via topical eye drops twice a day for 2 weeks starting the day after laser injury. CNV volume was measured using immunohistochemistry labeling and confocal microscopy. Results: Minocycline reduced lesion volume by 79% (P ≤ 0.0004) in female and male mice. DAM reduced lesion volume by 73% (P ≤ 0.001) in female and male mice. There was no significant difference in lesion volume between minocycline and DAM treatment groups or between female and male mice. Conclusions: Both minocycline and DAM eye drops significantly reduced laser-induced CNV lesion volume in female and male mice. While oral tetracyclines have been shown to mitigate pathologic neovascularization in both preclinical studies and clinical trials, the present data are the first to suggest that tetracycline derivatives may be effective to reduce pathologic CNV when administered via topical eye drops. However, the action is unrelated to antimicrobial action. Targeted delivery of these medications via eye drops may reduce the potential for systemic side effects. Translational Relevance: Topical administration of minocycline and/or DAM via eye drops may represent a novel therapeutic strategy for disorders involving pathologic CNV.

Investigation of oral, gastric, and duodenal microbiota in patients with upper gastrointestinal symptoms.

Disease-associated alterations of the intestinal microbiota composition, known as dysbiosis, have been well described in several functional gastrointestinal (GI) disorders. Several studies have described alterations in the gastric microbiota in functional dyspepsia, but very few have looked at the duodenum.Here, we explored the upper GI tract microbiota of inpatients with upper GI dyspeptic symptoms, and compared them to achalasia controls, as there is no indication for an esophagogastroduodenoscopy in healthy individuals.We found differences in the microbiota composition at the three sites evaluated (ie, saliva, stomach and duodenum). Changes observed in patients with dyspepsia included an increase in Veillonella in saliva, an oral shift in the composition of the gastric microbiota, and to some degree in the duodenum as well, where an important abundance of anaerobes was observed. Metabolic function prediction identified greater anaerobic metabolism in the stomach microbial community of patients with dyspepsia. Proton pump inhibitor use was not associated with any particular genus. Co-abundance analysis revealed Rothia as the main hub in the duodenum, a genus that significantly correlated with the relative abundance of Clostridium, Haemophilus, and ActinobacillusWe conclude that patients with upper GI symptoms consistent with dyspepsia have alterations in the microbiota of saliva, the stomach, and duodenum, which could contribute to symptoms of functional GI disorders.

The human jejunum has an endogenous microbiota that differs from those in the oral cavity and colon.

BACKGROUND: The upper half of the human small intestine, known as the jejunum, is the primary site for absorption of nutrient-derived carbohydrates, amino acids, small peptides, and vitamins. In contrast to the colon, which contains 1011-1012 colony forming units of bacteria per ml (CFU/ml), the normal jejunum generally ranges from 103 to 105 CFU per ml. Because invasive procedures are required to access the jejunum, much less is known about its bacterial microbiota. Bacteria inhabiting the jejunal lumen have been investigated by classical culture techniques, but not by culture-independent metagenomics. RESULTS: The lumen of the upper jejunum was sampled during enteroscopy of 20 research subjects. Culture on aerobic and anaerobic media gave live bacterial counts ranging from 5.8 × 103 CFU/ml to 8.0 × 106 CFU/ml. DNA from the same samples was analyzed by 16S rRNA gene-specific quantitative PCR, yielding values from 1.5 × 105 to 3.1 × 107 bacterial genomes per ml. When calculated for each sample, estimated bacterial viability ranged from effectively 100% to a low of 0.3%. 16S rRNA metagenomic analysis of uncultured bacteria by Illumina MiSeq sequencing gave detailed microbial composition by phylum, genus and species. The genera Streptococcus, Prevotella, Veillonella and Fusobacterium, were especially abundant, as well as non-oral genera including Escherichia, Klebsiella, and Citrobacter. The jejunum was devoid of the genera Alistipes, Ruminococcus, Faecalibacterium, and other extreme anaerobes abundant in the colon. In patients with higher bacterial loads, there was no significant change in microbial species composition. CONCLUSIONS: The jejunal lumen contains a distinctive bacterial population consisting primarily of facultative anaerobes and oxygen-tolerant obligate anaerobes similar to those found in the oral cavity. However, the frequent abundance of Enterobacteriaceae represents a major difference from oral microbiota. Although a few genera are shared with the colon, we found no evidence for retrograde movement of the most abundant colonic microbes to the jejunum. Some individuals had much higher bacterial loads, but this was not correlated with decreases in bacterial species diversity or other evidence of dysbiosis.

A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs.

Efficacy of topical dorzolamide therapy for cystoid macular edema in a patient with MFRP-related nanophthalmos-retinitis pigmentosa-foveoschisis-optic disk drusen syndrome.

PURPOSE: Mutations in the MFRP (membrane-type frizzled-related protein) gene leads to an entity characterized by retinitis pigmentosa, nanophthalmos, optic disk drusen, and macular changes, originally described as foveoschisis. Despite the association of MFRP gene mutation and increase in macular thickness, no treatment modality has been described for cystoid macular edema related to this particular entity so far. METHODS: In this case report, a 52-year-old woman presented with nanophthalmos, optic disk drusen, retinitis pigmentosa, and increase in macular thickness. Genetic analysis revealed an MFRP gene mutation. The patient was treated with topical carbonic anhydrase inhibitors. RESULTS: A progressive decrease in macular thickness and cystic changes was observed during the 2-month course of topical carbonic anhydrase inhibitor treatment, and best-corrected visual acuity improved from 20/100 to 20/50. Macular thickness remained stable after 6 months of follow-up. CONCLUSION: Cystoid macular edema is part of the macular changes noted in the MFRP mutation-related nanophthalmos-retinitis pigmentosa-foveoschisis-optic disk drusen, syndrome. Taking into account that resolution of cystoid macular edema in patients with retinitis pigmentosa may delay an irreversible decrease in visual acuity, treatment should be considered when cystic changes are suspected. Topical carbonic anhydrase inhibitor was effective in decreasing macular thickness and cystic changes in the patient reported.

Novel membrane frizzled-related protein gene mutation as cause of posterior microphthalmia resulting in high hyperopia with macular folds.

PURPOSE: We present a genetic and clinical analysis of two sisters, 3 and 4 years of age, with nanophthalmos and macular folds. METHODS: Ophthalmological examination, general paediatric examination and molecular genetic analysis of the MFRP gene were performed in both affected siblings. RESULTS: Clinical analysis showed high hyperopia (+11 D and +12 D), short axial lengths (15 mm) and the presence of macular folds and optic nerve head drusen. Autofluorescence of the retina was generally normal with subtle macular abnormalities. Sequence analysis showed compound heterozygosity for severe MFRP mutations in both sisters: a previously reported p.Asn167fs (c.498dupC) and a novel stop codon mutation p.Gln91X (c.271C>T). CONCLUSION: These are the youngest nanophthalmos patients in the literature identified with severe loss of MFRP function, showing already the known structural abnormalities for this disease. Adult patients affected by homozygous or compound heterozygous MFRP mutations generally show signs of retinal dystrophy, with ERG disturbances and RPE abnormalities on autofluorescence imaging. ERG examination could not be performed in these children, but extensive RPE abnormalities were not seen at this young age.

Developmental basis of nanophthalmos: MFRP Is required for both prenatal ocular growth and postnatal emmetropization.

BACKGROUND: Nanophthalmos is a genetic disorder characterized by very small, hyperopic eyes that are without gross structural defects. Recessive nanophthalmos is caused by severe mutations in the MFRP gene, which encodes a Frizzled-related transmembrane protein that is selectively expressed in the retinal pigment epithelium (RPE) and ciliary body. RESULTS: For two MFRP -/- adults, we have obtained records of refraction that begin in early childhood. At the age of 6 months, one patient's eyes already had a refractive error of +12.25 D, and over the next 20 years this slowly increased to +17.50 D. Adults homozygous for null mutations in MFRP have eyes with axial lengths shorter than those of normal newborns. Furthermore, the unusually high curvature of their corneas is consistent with eyes that had been smaller than normal during late fetal development. MFRP protein was first detected at 14 weeks of gestation, when it was restricted to the posterior pole RPE. By 20 weeks gestation, MFRP expression had spread laterally, and was found throughout the RPE. MFRP protein was detected in both posterior and lateral RPE of the adult eye. CONCLUSIONS: Embryonic function of the MFRP gene appears necessary for the eye to reach its full size at birth. Its onset of expression in the RPE during mid-gestation suggests that MFRP does not participate in early formation of the optic cup, and is consistent with a role in later growth and development of the eye. Patients without MFRP gene function exhibit no correction of refractive error during childhood, which suggests that this gene is essential for emmetropization, a complex process by which vision regulates axial growth of the eye.

betaA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development.

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague-Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the betaA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate betaA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

A common locus for late-onset Fuchs corneal dystrophy maps to 18q21.2-q21.32.

PURPOSE: To identify the genetic basis of late-onset Fuchs corneal dystrophy (FCD). METHODS: Phenotypes and genotypes at 1107 short tandem repeat polymorphism markers were obtained for 43 affected and 33 unaffected individuals in three large families. Two-point genetic linkage analysis was performed with MLINK and multipoint linkage with SimWalk 2.89. RESULTS: In each family, the most significant cluster of two-point lod scores mapped to chromosome 18, at 18q21.2-q21.3. The highest two-point lod score for each family was at D18S1129, with scores of 3.41, 2.89, and 2.45, with a combined two-point lod score of 7.70. Multipoint analysis yielded a maximum score of 5.94 at D18S1129 for a dominant Mendelian trait exhibiting 85% penetrance and 15% phenocopy rate. Disease interval haplotypes for each family are different. CONCLUSIONS: FCD2, at 18q21, is the second genetic locus identified for late-onset FCD. Presence of this same locus in all three families may indicate its widespread involvement in late-onset FCD. Allelic differences between disease-associated haplotypes in the families leave open the possibility of independent mutations in the same gene. The incomplete penetrance and high phenocopy rate observed at FCD2 suggest that the origin of FCD in these three families is complex and also depends on other genetic loci or environmental factors.

Analysis and documentation of progression of Fuchs corneal dystrophy with retroillumination photography.

PURPOSE: Fuchs corneal dystrophy (FCD) is a degenerative disorder of the cornea that is characterized by the progressive accumulation of guttae, which are small excrescences of Descemet's membrane. We describe a method for documenting the location and number of guttae, and ask whether disease progression can be observed during relatively short periods. METHODS: Patients with FCD were imaged by standard retroillumination photography with a slit lamp. Scanned photographs were analyzed by using NIH ImageJ software to determine the number of individual guttae and areas of confluence. RESULTS: In 4 FCD patients, photographs taken 23 to 30 months apart revealed that, once formed, individual guttae and their relative positions persisted during this period. Very few guttae disappeared, and the emergence of many new guttae was observed. Determination of the area with confluent guttae was used to quantify disease stage. CONCLUSIONS: Computer-assisted analysis of retroillumination photographs is proposed as an effective way to document the number and distribution of individual guttae. Although the disease typically progresses slowly during decades, we have been able to detect the formation of new guttae within only 2 years. This rapid assessment of disease progression could be used to measure phenotypic differences between genetic subtypes of FCD. It also could provide important baseline information and methodology for clinical trials of therapeutic options, should these become available.

Linkage of late-onset Fuchs corneal dystrophy to a novel locus at 13pTel-13q12.13.

PURPOSE: To identify the gene locus underlying the inheritance of late-onset Fuchs corneal dystrophy (FCD) in a large white kindred. METHODS: Genotypes of small tandem repeat polymorphisms were obtained from 17 affected and 3 unaffected family members, followed by genetic linkage analysis. RESULTS: In this family, classic late-onset FCD appeared to be inherited as a single, dominant Mendelian trait. In two exceptional sibships, however, children aged 10 and 13 years had FCD. In each sibship, both parents were found to be affected, opening the possibility that this unusually early age of onset was the result of homozygosity for an FCD mutation. Genotype results, however, were not consistent with consanguinity of the parents, who appear to have independent cases of FCD. A whole-genome linkage scan mapped FCD to a single locus at 13pTel-13q12.13, with significant two-point LOD scores of 3.91 at D13S1236 and 3.80 at D13S1304. The 26.4-Mb disease interval contains the chromosome 13 nucleolus organizer (RNR1), the centromere, and 44 annotated protein-encoding genes. So far, exons of 10 of these genes have been screened, but no mutations have been found. CONCLUSIONS: FCD1 is the first genetic locus to be identified for late-onset FCD, a common disease of the aging cornea. The exceptional early onset of the disease observed in two children is unusual and might be the result of digenic interaction between FCD1 and an independent late-onset FCD mutation.

Immunohistochemistry and electron microscopy of early-onset fuchs corneal dystrophy in three cases with the same L450W COL8A2 mutation.

PURPOSE: A rare, familial early-onset form of Fuchs corneal dystrophy (FCD) is caused by mutation in the COL8A2 gene. This study describes the aberrant pattern of distribution of collagen type VIII and basement membrane components in Descemet's membrane (DM) and endothelium of three individuals with the same L450W mutation that represent different stages of early-onset FCD. METHODS: Immunohistochemical studies with bright field, fluorescence, and confocal microscopy characterized the pathology of sectioned corneal buttons with antibodies against COL8A1, COL8A2, COL4, laminin, and fibronectin. A portion of each was processed for electron microscopy. RESULTS: Histologic examination of pathologic changes in case 1 demonstrated relative preservation of the endothelium, whereas in case 2 much of this layer was atrophic and in case 3 there was complete loss of the endothelium. DM also increased in thickness to 25 mum for case 1, to 31 mum for case 2, and to 38 mum for case 3. Case 1 was the only specimen to reveal shallow warts along the posterior surface of DM, whereas the most advanced specimen, case 3, showed evidence of earlier nodularity that had been buried by the accretion of further extracellular matrix material. The posterior aspect of DM in this specimen had the unusual property of lighter staining relative to the anterior region of DM, laid down earlier in life. Immunocytochemistry revealed increased expression and complex, sharply defined patterns of deposition of collagen VIII, collagen IV, laminin, and fibronectin. Ultrastructurally, the posterior nonbanded layer of DM was intermixed with banded collagen, and the posterior region of DM showed a high density of foci of spindle-shaped structures with intense-staining bands, spaced at approximately 120 nm. Finally, ultrastructural studies of the endothelium in case 1 revealed unusual accumulation of swelling mitochondria. The endothelial cells also had large amounts of abnormal prominent rough endoplasmic reticulum. Type VIII collagen alpha 2 immunogold signal was associated with the highly granular ribosomes of the rough endoplasmic reticulum of these patients. CONCLUSIONS: Microscopic and electron microscopic examination revealed pathological changes in DM of L450W COL8A2 mutants that were consistent with several-fold increased growth of the extracellular matrix and progressive deposition and synthesis of extracellular material by endothelial cells. As with late-onset FCD, this is accompanied by attenuation and eventual loss of the endothelium itself. Whether the abnormal deposition of collagen, laminin, and fibronectin contributes to the dysfunction and death of the endothelium remains to be determined.

Posterior polar cataract: genetic analysis of a large family.

Congenital cataracts are clinically and genetically heterogeneous. Loci for autosomal dominant posterior polar cataracts have been mapped to chromosomes 1p36, 11q22-q22.3, 16q22, and 20p12-q12. We investigated a large four-generation family with 20 individuals affected with congenital posterior polar cataracts. After exclusion of known loci for posterior polar cataracts, a genome-wide screen was conducted. In this family, we mapped dominant congenital posterior polar cataracts to chromosome 10q24. On haplotype analysis, we identified an 11-cM interval between loci D10S1680 and D10S467, which included the PITX3 gene. On sequencing the coding region of PITX3, we found a 17-base-pair duplication in exon 4. Although the same genotype was described in a family with ASMD and cataracts, the common phenotype of this mutation is probably posterior polar cataract; a modifier gene is presumed to cause anterior segment abnormalities in the previously described patients. The same mutation was recently identified in four families with congenital cataracts. This study provides further evidence of genetic heterogeneity of autosomal dominant posterior polar cataract.

Fuchs corneal dystrophy: aberrant collagen distribution in an L450W mutant of the COL8A2 gene.

PURPOSE: To characterize histologically Descemet's membrane in an early-onset Fuchs corneal dystrophy (FCD) COL8A2 mutant and compare these findings with corneas from late-onset FCD and normal corneas. METHODS: A corneal explant from a patient with the L450W COL8A2 mutation and others with late-onset disease were studied with antibodies to collagens IV, VIIIA1, VIIIA2, fibronectin, and laminin. Transmission electron microscopy was performed on a portion of the explant. Control explants included eye bank corneas without known disease and surgical explants from unrelated conditions. RESULTS: In normal corneas, a regular array of colocalized COL8A1 and COL8A2 was observed in the anterior half of Descemet's membrane. In the L450W mutant, Descemet's membrane was several times thicker than normal and traversed by refractile strands and blebs that stained intensely for COL8A2, a feature also observed in late-onset FCD. Both the alpha1 and alpha2 subtypes of collagen VIII were observed at high levels along the anterior edge of Descemet's, another abnormal feature also found in late-onset FCD. Ultrastructure of the L450W cornea revealed a well-formed anterior banded layer more than three times thicker than normal. An unusual, thin internal layer was rich in patches of wide-spaced collagen. The layer is a distinctive pathologic structure that is associated with FCD and is characterized by approximately 120 nm periodicity and the presence of collagen VIII. Depositions of collagen IV, fibronectin, and laminin were also greatly increased in the of posterior Descemet's membrane, yet another general feature shared between early- and late-onset disease. CONCLUSIONS: Early-onset COL8A2 L450W disease involves massive accumulation and abnormal assembly of collagen VIII within Descemet's membrane, a process that is presumed to begin during fetal development. Both early- and late-onset subtypes of FCD appear to be the result of abnormal basement membrane assembly rather than a primary defect in endothelial metabolism.

Extreme hyperopia is the result of null mutations in MFRP, which encodes a Frizzled-related protein.

Nanophthalmos is a rare disorder of eye development characterized by extreme hyperopia (farsightedness), with refractive error in the range of +8.00 to +25.00 diopters. Because the cornea and lens are normal in size and shape, hyperopia occurs because insufficient growth along the visual axis places these lensing components too close to the retina. Nanophthalmic eyes show considerable thickening of both the choroidal vascular bed and scleral coat, which provide nutritive and structural support for the retina. Thickening of these tissues is a general feature of axial hyperopia, whereas the opposite occurs in myopia. We have mapped recessive nanophthalmos to a unique locus at 11q23.3 and identified four independent mutations in MFRP, a gene that is selectively expressed in the eye and encodes a protein with homology to Tolloid proteases and the Wnt-binding domain of the Frizzled transmembrane receptors. This gene is not critical for retinal function, as patients entirely lacking MFRP can still have good refraction-corrected vision, produce clinically normal electro-retinograms, and show only modest anomalies in the dark adaptation of photoreceptors. MFRP appears primarily devoted to regulating axial length of the eye. It remains to be determined whether natural variation in its activity plays a role in common refractive errors.

Inheritance of a novel COL8A2 mutation defines a distinct early-onset subtype of fuchs corneal dystrophy.

PURPOSE: To characterize the genetic basis and phenotype of inherited Fuchs corneal dystrophy (FCD). METHODS: DNA from blood was used for genome-wide linkage scans with tandem repeat polymorphisms. Mutation detection involved sequencing PCR-amplified exons. Families with FCD were clinically evaluated and graded on the Krachmer severity scale. Confocal specular microscopy visualized the morphology of endothelial guttae, small protrusions of Descemet's membrane that are characteristic of FCD. RESULTS: Linkage was obtained to 1p34.3-p32 for the autosomal dominant kindred originally reported by Magovern in 1979. All 21 cases with FCD and one with posterior polymorphous dystrophy were heterozygous for L450W, a novel point mutation in the COL8A2 gene. Of 62 independent cases of familial FCD, none had the previously reported mutations in COL8A2. Corneal guttae in COL8A2 patients were small, rounded, and associated with the endothelial cell center. This contrasts with common FCD, in which guttae were larger, sharply peaked, and initially positioned at edges of endothelial cells. The profile of age and disease severity for the L450W FCD kindred suggested that disease onset occurred in infancy, compared with an average age of onset of 50 years estimated for 201 familial FCD patients in 62 other families. CONCLUSIONS: A novel pathogenic L450W COL8A2 mutation was identified and its highly distinctive pathology characterized. This indicates that COL8A2 mutations give rise to a rare subtype of FCD. This study also provides the first direct evidence that COL8A2-FCD progresses from early to late stages in 25 years, a rate similar to that estimated for late-onset FCD.

A spontaneous mutation affects programmed cell death during development of the rat eye.

We have discovered a spontaneous mutation in the Sprague-Dawley rat with a novel eye phenotype that we have named Nuc1. The Nuc1 mutation behaves as a single semi-dominant locus with an intermediate phenotype in the heterozygotes. Heterozygotes exhibit nuclear cataracts. Homozygous Nuc1 rats are fully viable and have microphthalmia, retinal abnormalities and disruption of lens structure shortly before birth. The homozygous mutant shows no obvious pathology outside of the eye, indicating that the mutation is highly eye specific in its effects. An unusual feature of the mutation is that it prevents the normal programmed loss of nuclei from lens fiber cells, but does not affect the loss of other organelles. TUNEL, light, and electron microscopic studies show normal intact nuclei in lens fibers, in contrast to many other models with degenerate nuclei and unlike normal lenses where no such nuclei remain. The beaded filament protein, filensin, is down-regulated in fibers of Nuc1, while heat shock cognate 70 is up-regulated. Homozygous retinas are thicker than normal, and TUNEL labeling indicates roughly half the number of apoptotic cells compared to a wild-type retina. The transient layer of Chievitz persists in adult Nuc1 retina, indicative of delayed development. Hence, Nuc1 is a novel mutation that could be an eye-specific regulator of apoptosis.

A missense mutation in GUCY2D acts as a genetic modifier in RPE65-related Leber Congenital Amaurosis.

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous severe retinal dystrophy presenting in infancy. To explain the phenotypical variability observed in two affected siblings of a consanguineous pedigree diagnosed with LCA and establish a genotype-phenotype correlation, we screened GUCY2D, RPE65, CRX, AIPL1, and RPGRIP1 for mutations. The more severely affected sibling carried a heterozygous missense mutation in the GUCY2D gene (Ile539Val), which did not segregate with the disease phenotype. Subsequently, a homozygous nonsense mutation (Glu102STOP) in the RPE65 gene was identified in both affected siblings, thus identifying the causative gene. This data provides evidence for the presence of genetic modulation in LCA. It appears that the heterozygous GUCY2D mutation further disrupts the already compromised photoreceptor function resulting in more severe retinal dysfunction in the older sibling. We suggest that the unusual phenotypic variability in these two siblings with LCA is caused by the modifying effect of a heterozygous GUCY2D mutation observed against the disease background of a homozygous RPE65 mutation.