The molecular basis of human retinal and vitreoretinal diseases

During the last two to three decades, a large body of work has revealed the molecular basis of many human disorders, including retinal and vitreoretinal degenerations and dysfunctions. Although belonging to the group of orphan diseases, they affect probably more than two million people worldwide. Most excitingly, treatment of a particular form of congenital retinal degeneration is now possible. A major advantage for treatment is the unique structure and accessibility of the eye and its different components, including the vitreous and retina. Knowledge of the many different eye diseases affecting retinal structure and function (night and color blindness, retinitis pigmentosa, cone and cone rod dystrophies, photoreceptor dysfunctions, as well as vitreoretinal traits) is critical for future therapeutic development. We have attempted to present a comprehensive picture of these disorders, including clinical, genetic and molecular information. The structural organization of the review leads the reader through non-syndromic and syndromic forms of (i) rod dominated diseases, (ii) cone dominated diseases, (iii) generalized retinal degenerations and (iv) vitreoretinal disorders, caused by mutations in more than 165 genes. Clinical variability and genetic heterogeneity have an important impact on genetic testing and counselling of affected families. As phenotypes do not always correlate with the respective genotypes, it is of utmost importance that clinicians, geneticists, counsellors, diagnostic laboratories and basic researchers understand the relationships between phenotypic manifestations and specific genes, as well as mutations and pathophysiologic mechanisms. We discuss future perspectives

Identification of the genetic defect in the original Wagner syndrome family

PURPOSE: The aim of the present study was to determine the genetic defect in Wagner syndrome, a rare disorder belonging to the group of hereditary vitreoretinal degenerations. This disease has been genetically mapped to chromosome 5q14.3. METHODS: Molecular analysis was performed in the progeny of the original pedigree described by Wagner in 1938. We searched for pathogenic mutations and their effects in two candidate genes, CSPG2 and EDIL3, which locate to the critical chromosomal interval. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to investigate potential splice defects of CSPG2 transcripts. RESULTS: While no alterations were detected in the exons of EDIL3, several changes were identified in the CSPG2 gene. Only one of the novel changes, a heterozygous G to A substitution of the first nucleotide in intron 8, cosegregates with the disease phenotype. This change disrupts the highly conserved splice donor sequence. In blood cells of an index patient, we found CSPG2 transcripts with normally spliced exon 8/9 junction but also two additional CSPG2 transcripts, which were not detected in the control. One lacks the entire exon 8, while the other is missing only the last 21 bp of exon 8. CONCLUSIONS: CSPG2 encodes versican, a large proteoglycan, which is an extracellular matrix component of the human vitreous and participates in the formation of the vitreous gel. The splice site mutation described here may lead to a complete lack of exon 8 in CSPG2 transcripts, which shortens the predicted protein by 1754 amino acids and leads to severe reduction of glycosaminoglycan attachment sites

A potential mouse model for the erosive vitreoretinopathy of Wagner disease

Patients with the very rare eye pathology Wagner disease (OMIM #143200) present with an abnormal (empty) vitreous, retinal detachment and altered electroretinogram (ERG). The disease is progressive and can eventually lead to blindness. No therapy can be offered to date. The genetic basis is the presence of mutations in the VCAN gene, encoding the large extracellular matrix molecule versican, which is a component of the vitreous. All identified mutations map to the canonical splice sites flanking exon 8, resulting in low number of aberrant splice products and a severe increase in two (V2, V3) of the four naturally occurring splice variants. The pathomechanism of Wagner's disease is poorly understood and a mouse model may afford further insight. The hdf -/- mice, named for their initial phenotype of heart defects, carry a null allele for Vcan that leads to embryonic lethality when homozygous, but heterozygote animals are viable. Here we investigated a possible eye phenotype in the heterozygous animals. While the overall morphology of retina and ciliary body appears to be normal, older (17 months) mutant animals show a decrease in ERG signaling profiles affecting the a-, b- and c-waves. This aspect of altered ERG profile demonstrates similarities to the human disease manifestation and underlines the suitability of heterozygous hdf+/- mice as a model for Wagner disease

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired conditio

Effect of concurrent vitamin A and iodine deficiencies on the thyroid-pituitary axis in rats

OBJECTIVE: Deficiencies of vitamin A and iodine are common in many developing countries. Vitamin A deficiency (VAD) may adversely affect thyroid metabolism. The study aim was to investigate the effects of concurrent vitamin A and iodine deficiencies on the thyroid-pituitary axis in rats. DESIGN: Weanling rats (n = 56) were fed diets deficient in vitamin A (VAD group), iodine (ID group), vitamin A and iodine (VAD + ID group), or sufficient in both vitamin A and iodine (control) for 30 days in a pair-fed design. Serum retinol (SR), thyroid hormones (FT(4), TT(4), FT(3), and TT(3)), serum thyrotropin (TSH), pituitary TSHbeta mRNA expression levels, and thyroid weights were determined at the end of the depletion period. MAIN OUTCOME: Compared to the control and ID groups, SR concentrations were about 35% lower in the VAD and VAD + ID groups (p < 0.001), indicating moderate VA deficiency. Comparing the VAD and control groups, there were no significant differences in TSH, TSHbeta mRNA, thyroid weight, or thyroid hormone levels. Compared to the control group, serum TSH, TSHbeta mRNA, and thyroid weight were higher (p < 0.05), and FT4 and TT4 were lower (p < 0.001), in the VAD + ID and ID groups. Compared to the ID group, TSH, TSHbeta mRNA, and thyroid weight were higher (p < 0.01) and FT(4) and TT(4) were lower (p < 0.001) in the VAD + ID group. There were no significant differences in TT3 or FT3 concentrations among groups. CONCLUSION: Moderate VAD alone has no measurable effect on the pituitary-thyroid axis. Concurrent ID and VAD produce more severe primary hypothyroidism than ID alone

Dysfunctional LAT2 Amino Acid Transporter Is Associated With Cataract in Mouse and Humans.

Cataract, the loss of ocular lens transparency, accounts for ∼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects

Dysfunctional LAT2 amino acid transporter is associated with cataract in mouse and humans

Cataract, the loss of ocular lens transparency, accounts for ∼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects

Regulatory regions of the paraoxonase 1 (PON1) gene are associated with neovascular age-related macular degeneration (AMD)

Physiological stress response and oxidative damage are factors for aging processes and, as such, are thought to contribute to neovascular age-related macular degeneration (AMD). Paraoxonase 1 (PON1) is an enzyme that plays an important role in oxidative stress and aging. We investigated association of DNA sequence variants (SNP) within the upstream regulatory region of the PON1 gene with neovascular AMD in 305 patients and 288 controls. Four of the seven tested SNPs (rs705379, rs705381, rs854573, and rs757158) were more frequently found in AMD patients compared to controls (P = 0.0099, 0.0295, 0.0121, and 0.0256, respectively), and all but one (SNP rs757158) are in linkage disequilibrium. Furthermore, haplotype TGGCCTC conferred protection (odds ratio (OR) = 0.76, (CI) = 0.60-0.97) as it was more frequently found in control individuals, while haplotype CGATGCT increased the risk (OR = 1.55, CI = 1.09-2.21) for AMD. These results were also reflected when haplotypes for the untranscribed and the 5'untranslated regions (5'UTR) were analyzed separately. To assess haplotype correlation with levels of gene expression, the three SNPs within the 5'UTR were tested in a luciferase reporter assay. In retinal pigment epithelium-derived ARPE19 cells, we were able to measure significant differences in reporter levels, while this was not observed in kidney-derived HEK293 cells. The presence of the risk allele A (SNP rs705381) caused an increase in luciferase activity of approximately twofold. Our data support the view that inflammatory reactions mediated through anti-oxidative activity may be relevant to neovascular age-related macular degeneration

Effect of Canagliflozin on Renal Threshold for Glucose, Glycemia, and Body Weight in Normal and Diabetic Animal Models

Background: Canagliflozin is a sodium glucose co-transporter (SGLT) 2 inhibitor in clinical development for the treatment of type 2 diabetes mellitus (T2DM). Methods: 14 C-alpha-methylglucoside uptake in Chinese hamster ovary-K cells expressing human, rat, or mouse SGLT2 or SGLT1; 3 H-2-deoxy-D-glucose uptake in L6 myoblasts; and 2-electrode voltage clamp recording of oocytes expressing human SGLT3 were analyzed. Graded glucose infusions were performed to determine rate of urinary glucose excretion (UGE) at different blood glucose (BG) concentrations and the renal threshold for glucose excretion (RTG) in vehicle or canagliflozin-treated Zucker diabetic fatty (ZDF) rats. This study aimed to characterize the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of T2DM and obesity. Results: Treatment with canagliflozin 1 mg/kg lowered RT G from 415612 mg/dl to 94610 mg/dl in ZDF rats while maintaining a threshold relationship between BG and UGE with virtually no UGE observed when BG was below RTG. Canagliflozin dose-dependently decreased BG concentrations in db/db mice treated acutely. In ZDF rats treated for 4 weeks, canagliflozin decreased glycated hemoglobin (HbA1c) and improved measures of insulin secretion. In obese animal models, canagliflozin increased UGE and decreased BG, body weight gain, epididymal fat, liver weight, and the respiratory exchange ratio