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Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5 , a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.

Adipor1 tm1Dgen and Mfrp rd6 mutant mice share similar eye disease characteristics. Previously, studies established a functional relationship of ADIPOR1 and MFRP proteins in maintaining retinal lipidome homeostasis and visual function. However, the independent and/or interactive contribution of both genes to similar disease phenotypes, including fundus spots, decreased axial length, and photoreceptor degeneration has yet to be examined. We performed a gene-interaction study where homozygous Adipor1 tm1Dgen and Mfrp rd6 mice were bred together and the resulting doubly heterozygous F1 offspring were intercrossed to produce 210 F2 progeny. Four-month-old mice from all nine genotypic combinations obtained in the F2 generation were assessed for white spots by fundus photo documentation, for axial length by caliper measurements, and for photoreceptor degeneration by histology. Two-way factorial ANOVA was performed to study individual as well as gene interaction effects on each phenotype. Here, we report the first observation of reduced axial length in Adipor1 tmlDgen homozygotes. We show that while Adipor1 and Mfrp interact to affect spotting and degeneration, they act independently to control axial length, highlighting the complex functional association between these two genes. Further examination of the molecular basis of this interaction may help in uncovering mechanisms by which these genes perturb ocular homeostasis.

Mouse models provide important resources for many areas of vision research, pertaining to retinal development, retinal function and retinal disease. The Translational Vision Research Models (TVRM) program uses chemical mutagenesis to generate new mouse models for vision research. In this chapter, we report the identification of mouse models for Grm1, Grk1 and Lrit3. Each of these is characterized by a primary defect in the electroretinogram. All are available without restriction to the research community.

Alström Syndrome (ALMS), a recessive, monogenic ciliopathy caused by mutations in ALMS1, is typically characterized by multisystem involvement including early cone-rod retinal dystrophy and blindness, hearing loss, childhood obesity, type 2 diabetes mellitus, cardiomyopathy, fibrosis, and multiple organ failure. The precise function of ALMS1 remains elusive, but roles in endosomal and ciliary transport and cell cycle regulation have been shown. The aim of our study was to further define the spectrum of ALMS1 mutations in patients with clinical features of ALMS. Mutational analysis in a world-wide cohort of 204 families identified 109 novel mutations, extending the number of known ALMS1 mutations to 239 and highlighting the allelic heterogeneity of this disorder. This study represents the most comprehensive mutation analysis in patients with ALMS, identifying the largest number of novel mutations in a single study worldwide. Here, we also provide an overview of all ALMS1 mutations identified to date., (© 2015 WILEY PERIODICALS, INC.)

Purpose: The purpose of this study was to identify the molecular basis and characterize the pathological consequences of a spontaneous mutation named cone photoreceptor function loss 8 (cpfl8) in a mouse model with a significantly reduced cone electroretinography (ERG) response., Methods: The chromosomal position for the recessive cpfl8 mutation was determined by DNA pooling and by subsequent genotyping with simple sequence length polymorphic markers in an F2 intercross phenotyped by ERG. Genes within the candidate region of both mutants and controls were directly sequenced and compared. The effects of the mutation were examined in longitudinal studies by light microscopy, marker analysis, transmission electron microscopy, and ERG., Results: The cpfl8 mutation was mapped to Chromosome 12, and a premature stop codon was identified in the spectrin repeat containing nuclear envelope 2 (Syne2) gene. The reduced cone ERG response was due to a significant reduction in cone photoreceptors. Longitudinal studies of the early postnatal retina indicated that the cone photoreceptors fail to develop properly, rod photoreceptors mislocalize to the inner nuclear layer, and both rods and cones undergo apoptosis prematurely. Moreover, we observed migration defects of secondary neurons and ectopic Müller cell bodies in the outer nuclear layer in early postnatal development., Conclusions: SYNE2 is important for normal retinal development. We have determined that not only is photoreceptor nuclear migration affected, but also the positions of Müller glia and secondary neurons are disturbed early in retinal development. The cpfl8 mouse model will serve as an important resource for further examining the role of nuclear scaffolding and migration in the developing retina.

Alström syndrome is a rare disorder typified by early childhood obesity, neurosensory deficits, cardiomyopathy, progressive renal and hepatic dysfunction, and endocrinological features such as severe insulin resistance, type 2 diabetes, hyperlipidemia, and hypogonadism. Widespread fibrosis leads to multiple organ failure. Mutations in ALMS1 cause Alström syndrome. Two age-matched, unrelated adolescent males of Serbian descent with Alström syndrome underwent an extensive workup of blood chemistries, and ophthalmological, audiological, and genetic evaluations. Although both showed typical features of Alström syndrome in childhood, several differences were observed that have not been reported previously. Patient 1 was first studied at the age of 13 years for multisystemic disease and re-evaluated at the age of 15.5 years. Patient 2 is a 15-year-old boy who presented at birth with epilepsy and psychomotor developmental delay and generalized tonic-clonic seizures with severe cognitive impairment, features not documented previously in this syndrome. Sequencing analysis indicated two novel ALMS1 mutations in exon 8: p.E1055GfsX4 and p.T1386NfsX15. Metabolic and physiological similarities were observed in both patients, including severe insulin resistance, and truncal obesity with fat loss suggestive of partial lipodystrophy, supporting evidence for a role for ALMS1 in adipose tissue function. The unusual phenotypes of clonic-tonic seizures and severe cognitive abnormalities and lipodystrophy-like adiposity pattern have not been documented previously in Alström syndrome and may be an under-reported abnormality.

Purpose: Cilia, complex structures found ubiquitously in most vertebrate cells, serve a variety of functions ranging from cell and fluid movement, cell signaling, tissue homeostasis, to sensory perception. Meckelin is a component of ciliary and cell membranes and is encoded by Tmem67 (Mks3). In this study, the retinal morphology and ciliary function in a mouse model for Meckel Syndrome Type 3 (MKS3) throughout the course of photoreceptor development was examined., Methods: To study the effects of a disruption in the Mks3 gene on the retina, the authors introduced a functional allele of Pde6b into B6C3Fe a/a-bpck/J mice and evaluated their retinas by ophthalmoscopic, histologic, and ultrastructural examination. In addition, immunofluorescence microscopy was used to assess protein trafficking through the connecting cilium and to examine the localization of ciliary and synaptic proteins in Tmem67(bpck) mice and controls., Results: Photoreceptors degenerate early and rapidly in bpck/bpck mutant mice. In addition, phototransduction proteins, such as rhodopsin, arrestin, and transducin, are mislocalized. Ultrastructural examination of photoreceptors reveal morphologically intact connecting cilia but dysmorphic and misoriented outer segment (OS) discs, at the earliest time point examined., Conclusions: These findings underscore the important role for meckelin in intraciliary transport of phototransduction molecules and their effects on subsequent OS morphogenesis and maintenance.

Purpose: To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration., Methods: Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2., Results: Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertk(nmf12). We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertk(nmf12) homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertk(nmf12) homozygotes. Mertk(nmf12) homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes.(2) B6.129P2-Ltb/Tnf/Lta(tm1Dvk)/J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc(-/-) mice. Surprisingly, mice homozygous for both the Mertk(nmf12) and the Ltb/Tnf/Lta(tm1Dvk) allele (Tnfabc(-/-)) demonstrated an increase in the rate of retinal degeneration., Conclusions: These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertk(nmf12) homozygotes from cell death.

Bardet-Biedl syndrome (BBS; OMIM no. 209 900) and Alström syndrome (ALMS; OMIM no. 203 800) are rare, multisystem genetic disorders showing both a highly variable phenotype and considerable phenotypic overlap; they are included in the emerging group of diseases called ciliopathies. The genetic heterogeneity of BBS with 14 causal genes described to date, serves to further complicate mutational analysis. The development of the BBS-ALMS array which detects known mutations in these genes has allowed us to detect at least one mutation in 40.5% of BBS families and in 26.7% of ALMS families validating this as an efficient and cost-effective first pass screening modality. Furthermore, using this method, we found two BBS families segregating three BBS alleles further supporting oligogenicity or modifier roles for additional mutations. We did not observe more than two mutations in any ALMS family.

Three new spontaneous recessive mouse mutations in the leptin receptor gene (Lepr), Lepr(db-rtnd), Lepr(db-dmpg) and Lepr(db-rlpy), originated in the CBA/J (CBA), B10.D2-H8(b)(57N)/Sn (B10) and NU/J strains, respectively. Lepr(db-rtnd) and Lepr(db-dmpg) were maintained on C57BL/6J (B6), resulting in congenic lines of B6.CBA-Lepr(db-rtnd) and B6.B10-Lepr(db-dmpg). Lepr(db-rtnd) was also maintained on CBA post F1 generation of a cross between the B6 and the CBA, generating the congenic line CBA.B6CBA-Lepr(db-rtnd). Lepr(db-rlpy) was maintained as a coisogenic strain. The aims of this study were to determine the molecular bases for these new Lepr mutations and to characterize the new mutant stocks, with respect to obesity and diabetes. Mutations were analyzed by Southern blot analysis, reverse transcriptase-polymerase chain reaction and sequencing. Body weights and plasma glucose and insulin levels were measured, and the histology of the pancreas was carried out. Lepr(db-rtnd) contained one G deletion in exon 4 of Lepr, introducing a frameshift and premature termination. Lepr(db-dmpg) had a deletion in the extracellular domain of LEPR: Lepr(db-rlpy) exhibited a large DNA deletion, leading to a complete lack of LEPR: All three mutations led to morbid obesity and diabetes. It is noteworthy that Lepr(db-rtnd) caused milder hyperglycemia accompanied by higher plasma and pancreatic insulin contents on B6 compared to that on CBA backgrounds. In summary, we discovered three new mutations of Lepr, providing new mouse models for obesity and diabetes. Furthermore, our mutant stocks will be useful in elucidating the effects of the genetic background on the Lepr mutations and in testing the specificity of antibodies to LEPR.

The autosomal recessive mouse mutation retinal degeneration 6 (rd6) causes small, white retinal spots and progressive photoreceptor degeneration similar to that observed in human flecked retinal diseases. Using a positional cloning approach, we determined that rd6 mice carry a splice donor mutation in the mouse homolog of the human membrane-type frizzled-related protein (Mfrp) gene that results in the skipping of exon 4. We found that mRNA of Mfrp is predominantly expressed in the eye, and at a lower level in the brain. To determine where in the eye Mfrp is expressed, in situ hybridization was done and showed that Mfrp is expressed specifically in the retinal pigment epithelium (RPE) and ciliary epithelium of the eye. The deduced amino acid sequence of MFRP contains a region with similarities to the cysteine-rich domain (CRD) of frizzled, a gene originally found in Drosophila that controls tissue polarity. The CRD is essential for Wnt binding and signaling. Wnt signaling has been shown to be involved in the control of gene expression, cell adhesion, planar polarity, proliferation and apoptosis. We also observed the localization of Wnt family proteins in the apical membrane of the RPE. Our results provide genetic evidence for an involvement of the Mfrp gene expressed by RPE in the degeneration of photoreceptors.

Alström syndrome is a homogeneous autosomal recessive disorder that is characterized by childhood obesity associated with hyperinsulinemia, chronic hyperglycemia and neurosensory deficits. The gene involved in Alström syndrome probably interacts with genetic modifiers, as subsets of affected individuals present with additional features such as dilated cardiomyopathy, hepatic dysfunction, hypothyroidism, male hypogonadism, short stature and mild to moderate developmental delay, and with secondary complications normally associated with type 2 diabetes, such as hyperlipidemia and atherosclerosis. Our detection of an uncharacterized transcript, KIAA0328, led us to identify the gene ALMS1, which contains sequence variations, including four frameshift mutations and two nonsense mutations, that segregate with Alström syndrome in six unrelated families. ALMS1 is ubiquitously expressed at low levels and does not share significant sequence homology with other genes reported so far. The identification of ALMS1 provides an entry point into a new pathway leading toward the understanding of both Alström syndrome and the common diseases that characterize it.

Mice that carry the recessive mutation tub develop neurosensory defects including retinal and cochlear degeneration, as well as maturity-onset obesity associated with insulin resistance. The biological function of the gene and the mechanism by which it induces its phenotypes are still unclear. In order to elucidate the pathways through which tub functions, in the current study, QTL modifiers were identified in an F2 intercross between (C57BL/6J- tub/tub and AKR/J-+/+) F1 hybrids (AKR intercross). The thickness of the outer nuclear layer of the retina and the number of photoreceptor nuclei were assessed in F2 mice homozygous for the tub mutation. A genome-wide scan revealed a significant linkage on chromosome 11 (named motr1) and two suggestive linkages on chromosomes 2 and 8. Interestingly, the same chromosome 2 region identified for the hearing modifier of tubby, the moth1 locus, showed a peak lod score of 2.3 for protection from retinal degeneration. This result suggests that the gene responsible for the QTL on chromosome 2 might be involved in a common pathway through which retinal and cochlear degeneration are induced in tubby mice., (Copyright 2002 Elsevier Science Ltd.)

Mouse models provide a valuable tool for exploring pathogenic mechanisms underlying inherited human disease. Here, we describe seven mouse models identified through the Translational Vision Research Models (TVRM) program, each carrying a new allele of a gene previously linked to retinal developmental and/or degenerative disease. The mutations include four alleles of three genes linked to human nonsyndromic ocular diseases (Aipl1tvrm119, Aipl1tvrm127, Rpgrip1tvrm111, RhoTvrm334) and three alleles of genes associated with human syndromic diseases that exhibit ocular phentoypes (Alms1tvrm102, Clcn2nmf289, Fkrptvrm53). Phenotypic characterization of each model is provided in the context of existing literature, in some cases refining our current understanding of specific disease attributes. These murine models, on fixed genetic backgrounds, are available for distribution upon request and may be useful for understanding the function of the gene in the retina, the pathological mechanisms induced by its disruption, and for testing experimental approaches to treat the corresponding human ocular diseases. [ABSTRACT FROM AUTHOR]

Alström syndrome is a clinically complex disorder characterized by progressive degeneration of sensory functions, resulting in visual and audiological impairment as well as metabolic disturbances. It is caused by recessively inherited mutations in the ALMS1 gene, which codes for a centrosomal/basal body protein. The purpose of this study was to investigate the genetic and clinical features of two Tunisian affected siblings with Alström syndrome. Detailed clinical examinations were performed including complete ophthalmic examination, serial audiograms and several biochemical and hormonal blood tests. For the molecular study, first genomic DNA was isolated using a standard protocol. Then, linkage analysis with microsatellite markers was performed and DNA array was used to detect known mutations. Subsequently, all ALMS1 exons were simultaneously sequenced for one affected patient with the TaGSCAN targeted sequencing panel. Finally, segregation of the causal variant was performed by Sanger sequencing. Both affected siblings had cone rod dystrophy with impaired visual acuity, sensorineural hearing loss and truncal obesity. One affected individual showed insulin resistance without diabetes mellitus. Other clinical features including cardiac and pulmonary dysfunction, hypothyroidism, hyperlipidemia, acanthosis nigricans, renal and hepatic dysfunction were absent. Genetic analysis showed the presence of a homozygous splice site mutation (c.10388-2A > G) in both affected siblings. Although Alström syndrome is relatively well characterized disease, this syndrome is probably misdiagnosed in Tunisia. Here, we describe the first report of Tunisian patients affected by this syndrome and carrying a homozygous ALMS1 mutation. The diagnosis was suspected after long-term clinical follow-up and confirmed by genetic testing. [ABSTRACT FROM AUTHOR]