Konstantin Petrukhin

Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies.

Abstract Vitelliform macular dystrophy (VMD2, Best disease, MIM153700) is an early onset, autosomal, dominant macular degeneration characterized by the deposition of lipofuscin-like material within and below the retinal pigment epithelium (RPE); it is associated with degeneration of the RPE and overlying photoreceptors. Recently, we cloned the gene bestrophin, which is responsible for the disease, and identified a number of causative mutations in families with VMD2. Here, we report that the analysis of bestrophin in a collection of 259 age-related macular degeneration (AMD) patients provides evidence that mutations in the Best disease gene do not play a significant role in the predisposition of individuals to AMD. However, our results suggest that, in addition to Best disease, mutations within the bestrophin gene could be responsible for other forms of maculopathy with phenotypic characteristics similar to Best disease and for other diseases not included in the VMD category.

Assessment of mutations in the Best macular dystrophy (VMD2) gene in patients with adult-onset foveomacular vitelliform dystrophy, age-related maculopathy, and bull's-eye maculopathy.

PURPOSEnTo study the presence of Best macular dystrophy (VMD2) gene mutations in patients diagnosed with maculopathies other than classic Best disease and to describe the clinical characteristics of these subjects.nnnDESIGNnCase-comparison study of phenotype-genotype correlations.nnnMETHODSnPatients with either age-related maculopathy (ARM; n = 259) or maculopathies other than classic Best disease (n = 28) were screened for mutations in the Best gene (VMD2; OMIM 153700). These cases were compared with ethnically similar subjects in the same age range without maculopathy (n = 196). All patients underwent a complete dilated ocular examination, and all affected individuals underwent fundus photography. Phenotype-genotype comparisons were made.nnnMAIN OUTCOME MEASURESnPresence of mutations in the Best gene (VMD2; OMIM 153700) and the clinical phenotype.nnnRESULTSnThree of 259 patients (1%) with ARM and 2 of 28 patients (7%) with other maculopathies including 1 of 3 patients with adult-onset foveomacular vitelliform dystrophy and 1 of 5 patients with a bulls eye maculopathy, but none of the controls, were found to possess amino acid-changing variants in the VMD2 gene. These included a man with confluent drusen and retinal pigment epithelial detachments (variant in exon 6; T216I), a man with geographic atrophy and numerous soft drusen (variant in exon 10; L567F), a woman with drusen and retinal pigment epithelial alterations (variant in exon 10; L567F), a woman with drusen and retinal pigment epithelial alterations resembling bulls-eye maculopathy (variant in exon 4; E119Q), and a woman diagnosed with adult-onset foveomacular vitelliform dystrophy (variant in exon 4; A146K).nnnCONCLUSIONSnNovel mutations in the VMD2 gene were found in patients diagnosed with maculopathies other than classic Best disease. Some cases diagnosed as adult-onset vitelliform foveomacular dystrophy may represent a variant of Best disease with delayed onset. The VMD2 gene does not play a major role in the development of ARM.

The mutation spectrum of the bestrophin protein--functional implications.

Abstract Best’s macular dystrophy (BMD), also known as vitelliform macular degeneration typeu20022 (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration with mainly juvenile onset. BMD is characterized by the accumulation of lipofuscin within and beneath the retinal pigment epithelium. The gene causing the disease has been localized to 11q13 by recombination breakpoint mapping. Recently, we have identified the causative gene encoding a protein named bestrophin, and mutations have been found mainly to affect residues that are conserved from a family of genes in Caenorhabditis elegans. The function of bestrophin is so far unknown, and no reliable predictions can be made from sequence comparisons. We have investigated the bestrophin gene in 14 unrelated Swedish, Dutch, Danish, and Moroccan families affected with BMD and found eight new mutations. Including the previously published mutations, 15 different missense mutations have now been detected in 19 of the 22 families with BMD investigated by our laboratory. Interestingly, the mutations cluster in certain regions, and no nonsense mutations or mutations causing frame-shifts have been identified. Computer simulations of the structural elements in the bestrophin protein show that this protein is probably membrane bound, with four putative transmembrane regions.

Evaluation of the ELOVL4 gene in patients with age-related macular degeneration

Stargardt-like macular degeneration (STGD3) and autosomal dominant macular degeneration (adMD) share phenotypic characters with atrophic age-related macular degeneration (AMD). Mutations in a photoreceptor cell-specific factor involved in the elongation of very long chain fatty acids ( ELOVL4 ) were shown to be associated with STGD3, adMD, and pattern dystrophy. We screened 778 patients with AMD and 551 age-matched controls to define the role of sequence variants in the ELOVL4 gene in age-related macular degeneration. We detected three sequence variants in the non-coding region and eight variants in the coding region. No statistically significant association was observed between sequence variants in the ELOVL4 gene and susceptibility to AMD. However, for the detection of modest effects of multiple alleles in a complex disease, the analysis of larger cohorts of patients may be required.

NEW THERAPEUTIC TARGETS IN ATROPHIC AGE-RELATED MACULAR DEGENERATION

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. There is no effective treatment for the most prevalent atrophic (dry) form of AMD. Atrophic AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and normally provides critical metabolic support to these light-sensing cells. Secondary to RPE dysfunction, macular rods and cones degenerate leading to the irreversible loss of vision. Oxidative stress, formation of drusen, accumulation of lipofuscin, local inflammation and reactive gliosis represent the pathologic processes implicated in pathogenesis of atrophic AMD. This review discusses potential target areas for small-molecule and biologic intervention, which may lead to development of new therapeutic treatments for atrophic AMD.

In Pursuit of Synthetic Modulators for the Orphan Retina-Specific Nuclear Receptor NR2E3

PURPOSEnNR2E3 is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. NR2E3-specific modulators may prolong photoreceptor survival in patients with dry age-related macular degeneration and other forms of retinal degeneration. To definitively establish NR2E3 as a photoreceptor protection target, identification of small-molecule NR2E3 modulators and their testing in animal models of retinal degeneration are required. Development of the high-throughput screen (HTS)-compatible screen for small-molecule NR2E3 modulators is the first step toward this goal.nnnMETHODSnPurification protocol for isolation of the functionally competent soluble NR2E3 protein after its expression in the insect Sf9 cells was developed. The time-resolved fluorescence energy-transfer (TR-FRET) assay assessing agonist-sensitive interaction between apo-NR2E3 and transcriptional corepressor RetCOR was used for characterization of the previously reported putative NR2E3 agonist, Compound 11a, and to conduct the HTS for novel small-molecule NR2E3 modulators (direct and inverse agonists). A counterscreen TR-FRET assay that measures the affect of test compounds on PPARγ interaction with corepressor NCOR was used for assessing the specificity of compounds identified in the HTS.nnnRESULTSnWe developed the cell-free TR-FRET assay for small-molecule NR2E3 modulators, which is based on agonist-induced disruption of the interaction between GST-tagged apo-NR2E3 and MBP-tagged fragment of transcriptional corepressor RetCOR. Compound 11a, a putative NR2E3 agonist, did not affect the NR2E3-RetCOR interaction, as was established by its titration in the developed assay. The assay was miniaturized for an ultralow-volume 1,536-well format and automated into 3 simple pipetting steps. Consistent with excellent assay performance, the test runs established a Z-score within the 0.6-0.8 range. Analysis of the mid-size National Institutes of Health collection of 315,001 structurally diverse drug-like compounds confirmed excellent assay performance, but did not reveal NR2E3-specific agonists or inverse agonists.nnnCONCLUSIONSnA robust and reliable TR-FRET assay for small-molecule NR2E3-specific modulators suitable for the analysis of million compound-strong HTS libraries was developed. A previously described putative NR2E3 agonist, Compound 11a, is unlikely to represent a direct NR2E3 agonist. Application of the developed assay for screening of a more abundant and diverse compound collection be required for identification of synthetic NR2E3 ligands.

Heterologous expression of the metal-binding domains of human copper-transporting ATPases (P1-ATPases).

Human copper-transporting ATPases (Wilson and Menkes disease proteins) are members of the PI-subfamily of P-type ATPases. ATPases in this group have unique structural features14 such as (1) a characteristic hydropathy profile with eight putative transmembrane segments, three pairs before the DKTG motif and only one transmembrane hairpin after the ATP-binding domain; (2) heavy-metal binding motif at the NH2-terminus, which is repeated one to six times depending on the species; (3) CPC/H sequence in the transmembrane segment; and (4) SEHPL motif, which seems to be characteristic of Cu-ATPases5 and is located after phosphorylation site DKTG. Based on the symptoms of the associated metabolic diseases, both Wilson (WND) and Menkes (MNK) disease gene products are presumed to be copper-specific proteins. However, the exact cation specificity of these two proteins remains to be determined. The presence of the GMTCxxC motif repeated six times at the NH2terminus suggests that the NHz-terminal domain plays an important role in the selective binding andor transport of heavy metals by these proteins. Alignment of all six repeats of the MNK and WND proteins5 revealed that the overall level of identity between repeats varies between 15 and 42%, suggesting that affinity towards heavy metals as well as specificity may vary for the MNK and WND proteins. Here we report expression of the NH2-terminal domains of the WND and MNK proteins as fusions with maltose binding protein (MBP) and characterization of their metal-binding properties. Regions corresponding to the NH2-tenninal domain, including all six repeats, were incorporated into pMAL-c2 and pMAL-p2 vectors (New England Biolabs) at the 3-end of coding sequence for MBP, after the Factor Xa cleavage site. To do so, segments corresponding to 64-1 868 bp of WND cDNA and 1-1836 bp of MNK cDNA were amplified by the polymerase chain reaction. Stop codons were engineered at the 3-end and unique restriction sites (Hind1 at 5-end and Hind11 at 3-end) were added to create a 5 blunt end, 3 sticky end-cloning fragment.

In vivo effect of mutant ELOVL4 on the expression and function of wild-type ELOVL4.

PURPOSEnMutations in the elongation of very long chain fatty acids 4 (ELOVL4) gene cause human Stargardts macular dystrophy 3 (STGD3), a juvenile onset dominant form of macular degeneration. To understand the role of the ELOVL4 protein in retinal function, several mouse models have been developed by using transgenic (TG), knock-in (Elovl4(+/mut)), and knockout (Elovl4(+/-)) approaches. Here we analyzed quantitatively the ELOVL4 protein and its enzymatic products (very long chain saturated fatty acid [VLC-FA] and VLC-polyunsaturated fatty acid [VLC-PUFA]) in the retinas of 8 to 10-week-old TG1(+), TG2(+), and Elovl4(+/mut) mice that harbor the mutant ELOVL4 and compared them to their wild-type littermates and Elovl4(+/-) that do not express the mutant protein. We also analyzed skin from these mice to gain insight into the pathogenesis resulting from the ELOVL4 mutation.nnnMETHODSnELOVL4 protein localization in the retina was determined by immunohistochemistry. Levels of wild-type ELOVL4 protein in skin and retinas were determined by Western blotting. Total lipids from skin and retinas were measured by gas chromatography-mass spectrometry (GC-MS). Retinal glycerophosphatidylcholines (PC) were analyzed by tandem mass spectrometry.nnnRESULTSnImmunohistochemical and Western analysis indicated that wild-type ELOVL4 protein was reduced in heterozygous Elovl4(+/mut) and Elovl4(+/-) retinas, but not in TG2(+) retinas. We found that VLC-FA was reduced by 50% in the skin of Elovl4(+/-) and by 60% to 65% in Elovl4(+/mut). We found VLC-PUFA levels at ∼ 50% in both the retinas, and wild-type levels of VLC-PUFA in TG2(+) retinas.nnnCONCLUSIONSnWe conclude that the presence of the mutant ELOVL4 does not affect the function of wild-type ELOVL4 in the fully developed 8- to 10-week-old retinas.

Preparation and characterization of calibration beads for sorting cells expressing a β-lactamase gene reporter

Modern drug discovery has been based on high‐throughput screening using whole‐cell assays. A prominent role has been assigned to the reporter gene technology based on a β‐lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence‐activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the β‐lactamase gene using the CCF2 substrate.

A novel RPE65 inhibitor CU239 suppresses visual cycle and prevents retinal degeneration

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6u202fμM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.