Identification and functional analysis of the vision-specific BBS3 (ARL6) long isoform

Bardet-Biedl Syndrome (BBS) is a heterogeneous syndromic form of retinal degeneration. We have identified a novel transcript of a known BBS gene, BBS3 (ARL6), which includes an additional exon. This transcript, BBS3L, is evolutionally conserved and is expressed predominantly in the eye, suggesting a specialized role in vision. Using antisense oligonucleotide knockdown in zebrafish, we previously demonstrated that bbs3 knockdown results in the cardinal features of BBS in zebrafish, including defects to the ciliated Kupffer’s Vesicle and delayed retrograde melanosome transport. Unlike bbs3, knockdown of bbs3L does not result in Kupffer’s Vesicle or melanosome transport defects, rather its knockdown leads to impaired visual function and mislocalization of the photopigment green cone opsin. Moreover, BBS3L RNA, but not BBS3 RNA, is sufficient to rescue both the vision defect as well as green opsin localization in the zebrafish retina. In order to demonstrate a role for Bbs3L function in the mammalian eye, we generated a Bbs3L-null mouse that presents with disruption of the normal photoreceptor architecture. Bbs3L-null mice lack key features of previously published Bbs-null mice

A Spectrum of FOXC1 Mutations Suggests Gene Dosage as a Mechanism for Developmental Defects of the Anterior Chamber of the Eye

Mutations in the forkhead transcription–factor gene (FOXC1), have been shown to cause defects of the anterior chamber of the eye that are associated with developmental forms of glaucoma. Discovery of these mutations was greatly facilitated by the cloning and characterization of the 6p25 breakpoint in a patient with both congenital glaucoma and a balanced-translocation event involving chromosomes 6 and 13. Here we describe the identification of novel mutations in the FOXC1 gene in patients with anterior-chamber defects of the eye. We have detected nine new mutations (eight of which are novel) in the FOXC1 gene in patients with anterior-chamber eye defects. Of these mutations, five frameshift mutations predict loss of the forkhead domain, as a result of premature termination of translation. Of particular interest is the fact that two families have a duplication of 6p25, involving the FOXC1 gene. These data suggest that both FOXC1 haploinsufficiency and increased gene dosage can cause anterior-chamber defects of the eye

Comparative Genomics and Gene Expression Analysis Identifies BBS9, a New Bardet-Biedl Syndrome Gene

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies

Leptin resistance contributes to obesity and hypertension in mouse models of Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin’s anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4–/– and Bbs6–/– mice, but not in Bbs2–/– mice. Consequently, Bbs4–/– and Bbs6–/– mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons

Metabolic and proteomic indications of diabetes progression in human aqueous humor

Diabetes mellitus is a multiorgan systemic disease impacting numerous ocular structures that results in significant ocular morbidity and often results in more frequent corneal and glaucoma surgeries for affected individuals. We hypothesize that the systemic metabolic and proteomic derangement observed in the progression of diabetes influences the composition of the aqueous humor (AH), which ultimately impacts the anterior segment health of the eye. To identify changes associated with diabetes progression, we mapped the metabolite profile and proteome of AH samples from patients with varying severities of type II diabetes (T2DM). Patients were classified as nondiabetic (ND or control), non-insulin-dependent diabetic without advanced features of disease (NAD-ni), insulin-dependent diabetic without advanced features (NAD-i), or diabetic with advanced features (AD). AH samples collected from the anterior chamber during elective ophthalmic surgery were evaluated for metabolite and protein expression changes associated with diabetic severity via gas chromatography/mass spectrometry and ultra-high performance liquid chromatography tandem mass spectrometry, respectively. Metabolic and proteomic pathway analyses were conducted utilizing MetaboAnalyst 4.0 and Ingenuity Pathway Analysis. A total of 14 control, 12 NAD-ni, 4 NAD-I, and 14 AD samples were included for analysis. Elevated levels of several branched amino acids (e.g., valine, leucine, isoleucine), and lipid metabolites (e.g., palmitate) were found only with increasing diabetic severity (i.e., the AD group). Similar proteomic trends were noted in amino acid and fatty acid metabolism and the unfolded protein/stress response. These results represent the first report of both metabolomic and proteomic evaluation of aqueous humor. Diabetes results in metabolic and proteomic perturbations detectable in the AH, and unique changes become manifest as T2DM severity worsens. Changes in AH composition may serve as an indicator of disease severity, risk assessment of anterior segment cells and structures, and potential future therapies

BBSome function is required for both the morphogenesis and maintenance of the photoreceptor outer segment

<div><p>Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of <i>Bbs8</i>, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of <i>Bbs8</i> in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting <i>Bbs8</i> gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated.</p></div