Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD.

Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4-/-) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.

Multimodal Regulation Orchestrates Normal and Complex Disease States in the Retina.

Regulation of biological processes occurs through complex, synergistic mechanisms. In this study, we discovered the synergistic orchestration of multiple mechanisms regulating the normal and diseased state (age related macular degeneration, AMD) in the retina. We uncovered gene networks with overlapping feedback loops that are modulated by nuclear hormone receptors (NHR), miRNAs, and epigenetic factors. We utilized a comprehensive filtering and pathway analysis strategy comparing miRNA and microarray data between three mouse models and human donor eyes (normal and AMD). The mouse models lack key NHRS (Nr2e3, RORA) or epigenetic (Ezh2) factors. Fifty-four total miRNAs were differentially expressed, potentially targeting over 150 genes in 18 major representative networks including angiogenesis, metabolism, and immunity. We identified sixty-eight genes and 5 miRNAS directly regulated by NR2E3 and/or RORA. After a comprehensive analysis, we discovered multimodal regulation by miRNA, NHRs, and epigenetic factors of three miRNAs (miR-466, miR1187, and miR-710) and two genes (Ell2 and Entpd1) that are also associated with AMD. These studies provide insight into the complex, dynamic modulation of gene networks as well as their impact on human disease, and provide novel data for the development of innovative and more effective therapeutics.

Excess cone cell proliferation due to lack of a functional NR2E3 causes retinal dysplasia and degeneration in rd7/rd7 mice.

The rd7 mouse is a model for hereditary retinal degeneration characterized clinically by retinal spotting throughout the fundus and late onset retinal degeneration, and histologically by retinal dysplasia manifesting as folds and whorls in the photoreceptor layer. This study demonstrates that the rd7 phenotype results from a splicing error created by a genomic deletion of an intron and part of an exon. Hematoxylin/eosin staining of rd7 tissue shows that the whorls in the outer nuclear layer of the retina do not appear during embryonic development but manifest by postnatal day 12.5 (P12.5). Furthermore, in situ hybridization data indicates that the Nr2e3 message is first present at barely discernable levels at embryonic day 18.5, becomes abundant by P2.5, and reaches maximal adult levels by P10.5. Results from these experiments indicate that Nr2e3 message is expressed prior to the development of S-cones. This data coincides with studies in humans showing that mutations in Nr2e3 result in a unique type of retinal degeneration known as enhanced S-cone syndrome, where patients have a 30-fold increase in S-cone sensitivity compared to normal. Immunohistochemical staining of cone cells demonstrates that rd7 retinas have an increased number of cone cells compared to wild-type retinas. Thus, Nr2e3 may function by regulating genes involved in cone cell proliferation, and mutations in this gene lead to retinal dysplasia and degeneration by disrupting normal photoreceptor cell topography as well as cell-cell interactions.

Identification of the gene that, when mutated, causes the human obesity syndrome BBS4.

Bardet-Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease. Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common. We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs. 12,13), mutation of which also causes McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects). MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum. We recently identified a novel gene that causes BBS2. The BBS2 protein has no significant similarity to other chaperonins or known proteins. Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

Positional cloning of a novel gene on chromosome 16q causing Bardet-Biedl syndrome (BBS2).

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder with the primary clinical features of obesity, pigmented retinopathy, polydactyly, hypogenitalism, mental retardation and renal anomalies. Associated features of the disorder include diabetes mellitus, hypertension and congenital heart disease. There are six known BBS loci, mapping to chromosomes 2, 3, 11, 15, 16 and 20. The BBS2 locus was initially mapped to an 18 cM interval on chromosome 16q21 with a large inbred Bedouin kindred. Further analysis of the Bedouin population allowed for the fine mapping of this locus to a 2 cM region distal to marker D16S408. Physical mapping and sequence analysis of this region resulted in the identification of a number of known genes and expressed sequence tag clusters. Mutation screening of a novel gene (BBS2) with a wide pattern of tissue expression revealed homozygous mutations in two inbred pedigrees, including the large Bedouin kindred used to initially identify the BBS2 locus. In addition, mutations were found in three of 18 unrelated BBS probands from small nuclear families.

Rapid communication: the human FEM1B gene maps to chromosome 15q22 and is excluded as the gene for Bardet-Biedl syndrome, type 4.

We have identified a novel human gene, FEM1B, that encodes a protein virtually identical to that encoded by the mouse gene Fem1b. These mammalian proteins are homologs of the FEM-1 protein of Caenorhabditis elegans, which acts as a signal-transduction component within the nematode sex-determination pathway. We report here the mapping of FEM1B to chromosome 15q22, a region that is homologous to the region of mouse chromosome 9, where Fem1b resides. The BBS4 locus, one of the loci causing the autosomal recessive Bardet-Biedl syndrome, maps to this region of chromosome 15. Therefore, we sought to determine whether the FEM1B gene might be involved in this disorder. Radiation hybrid mapping demonstrates that FEM1B does not reside within the interval of chromosome 15 containing the BBS4 locus.

Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate.

Hereditary human retinal degenerative diseases usually affect the mature photoreceptor topography by reducing the number of cells through apoptosis, resulting in loss of visual function. Only one inherited retinal disease, the enhanced S-cone syndrome (ESCS), manifests a gain in function of photoreceptors. ESCS is an autosomal recessive retinopathy in which patients have an increased sensitivity to blue light; perception of blue light is mediated by what is normally the least populous cone photoreceptor subtype, the S (short wavelength, blue) cones. People with ESCS also suffer visual loss, with night blindness occurring from early in life, varying degrees of L (long, red)- and M (middle, green)-cone vision, and retinal degeneration. The altered ratio of S- to L/M-cone photoreceptor sensitivity in ESCS may be due to abnormal cone cell fate determination during retinal development. In 94% of a cohort of ESCS probands we found mutations in NR2E3 (also known as PNR), which encodes a retinal nuclear receptor recently discovered to be a ligand-dependent transcription factor. Expression of NR2E3 was limited to the outer nuclear layer of the human retina. Our results suggest that NR2E3 has a role in determining photoreceptor phenotype during human retinogenesis.

Evaluation and molecular characterization of EHD1, a candidate gene for Bardet-Biedl syndrome 1 (BBS1).

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder characterized by developmental abnormalities including mental retardation, obesity, retinitis pigmentosa, polydactyly, short stature, and hypogenitalism. To date, five BBS loci have been identified. BBS1, located on 11q13, is reported to be the most prevalent form of BBS in the Caucasian population. A positional cloning approach is being used to identify the gene responsible for BBS1. EHD1, a new member of the EH-domain containing proteins, was identified in this study as lying within the BBS1 disease interval. RNA analysis of many tissues revealed that expression of EHD1 is ubiquitous, with elevated levels in the testis. The genomic structure of EHD1 was elucidated by direct BAC sequencing. Following identification of the intron/exon boundaries, mutational analysis was performed by single strand conformation polymorphism and direct sequencing of affected individuals from several large kindreds linked to the BBS1 locus, as well as a cohort of unrelated probands. No disease-causing mutations were identified in this analysis, but several polymorphisms were found.

The cloning and developmental expression of unconventional myosin IXA (MYO9A) a gene in the Bardet-Biedl syndrome (BBS4) region at chromosome 15q22-q23.

Bardet-Biedl Syndrome (BBS) is a heterogeneous, autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, syndactyly and/or polydactyly, short stature, and hypogenitalism and is caused by mutations at a number of distinct loci. Using a positional cloning approach for identifying the BBS4 (chromosome 15) gene, we identified and cloned an unconventional myosin gene, myosin IXA (HGMW-approved symbol MYO9A). Since mutations in unconventional myosins are known to cause several human diseases, and since mutations of unconventional myosin VIIa cause retinal degeneration, we evaluated myosin IXA as a candidate for BBS. We exploited PCR-based techniques to clone a 8473-nt cDNA for myosin IXA. A 7644-bp open reading frame predicts a protein with all the hallmarks of class IX unconventional myosins. Human Northern blot analysis and in situ hybridization of mouse embryos reveal that myosin IXA is expressed in many tissues consistent with BBS. Intron/exon boundaries were identified, and myosin IXA DNA and RNA from BBS4 patients were evaluated for mutation.

A Bedouin kindred with infantile nephronophthisis demonstrates linkage to chromosome 9 by homozygosity mapping.

A novel type of infantile nephronophthisis was identified in an extended Bedouin family from Israel. This disease has an autosomal recessive mode of inheritance, with the phenotypic presentation ranging from a Potter-like syndrome to hyperechogenic kidneys, renal insufficiency, hypertension, and hyperkalemia. Affected individuals show rapid deterioration of kidney function, leading to end-stage renal failure within 3 years. Histopathologic examination of renal tissue revealed variable findings, ranging from infantile polycystic kidneys to chronic tubulointerstitial nephritis, fibrosis, and cortical microcysts. A known familial juvenile nephronophthisis locus on chromosome 2q13 and autosomal recessive polycystic kidney disease on chromosome 6p21.1-p12 were excluded by genetic linkage analysis. A genomewide screen for linkage was conducted by searching for a locus inherited by descent in all affected individuals. Pooled DNA samples from parents and unaffected siblings and individual DNA samples from four affected individuals were used as PCR templates with trinucleotide- and tetranucleotide-repeat polymorphic markers. Using this approach, we identified linkage to infantile nephronophthisis for markers on chromosome 9q22-31. The disorder maps to a 12.9-cM region flanked by markers D9S280 and GGAT3G09.