Bardet-Biedl Syndrome Phenotype Research Articles

Ubiquitylation of BBSome is required for ciliary assembly and signaling

Bardet‐Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, renal abnormalities, postaxial polydactyly, and developmental defects. Genes mutated in BBS encode for components and regulators of the BBSome, an octameric complex that controls the trafficking of cargos and receptors within the primary cilium. Although both structure and function of the BBSome have been extensively studied, the impact of ubiquitin signaling on BBSome is largely unknown. We identify the E3 ubiquitin ligase PJA2 as a novel resident of the ciliary compartment and regulator of the BBSome. Upon GPCR‐cAMP stimulation, PJA2 ubiquitylates BBSome subunits. We demonstrate that ubiquitylation of BBS1 at lysine 143 increases the stability of the BBSome and promotes its binding to BBS3, an Arf‐like GTPase protein controlling the targeting of the BBSome to the ciliary membrane. Downregulation of PJA2 or expression of a ubiquitylation‐defective BBS1 mutant (BBS1K143R) affects the trafficking of G‐protein‐coupled receptors (GPCRs) and Shh‐dependent gene transcription. Expression of BBS1K143Rin vivo impairs cilium formation, embryonic development, and photoreceptors' morphogenesis, thus recapitulating the BBS phenotype in the medaka fish model.

Disruption of Dopamine Receptor 1 Localization to Primary Cilia Impairs Signaling in Striatal Neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G-protein-coupled receptors (GPCRs) and their downstream effectors, suggesting that they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding, we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, the loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G-protein-coupled receptors (GPCRs), suggesting that they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either an abnormal accumulation of D1 in cilia or a loss of D1 ciliary localization become obese. In both cases, the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with mild social behavioral phenotypes.

CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.

Characterization of two novel knock-in mouse models of syndromic retinal ciliopathy carrying hypomorphic Sdccag8 mutations.

Mutations in serologically defined colon cancer autoantigen protein 8 (SDCCAG8) were first identified in retinal ciliopathy families a decade ago with unknown function. To investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies in vivo, we employed CRISPR/Cas9-mediated homology-directed recombination (HDR) to generate two knock-in mouse models, Sdccag8Y236X/Y236X andSdccag8E451GfsX467/E451GfsX467, which carry truncating mutations of the mouse Sdccag8, corresponding to mutations that cause Bardet-Biedl syndrome (BBS) and Senior-Løken syndrome (SLS) (c.696T>G p.Y232X and c.1339–1340insG p.E447GfsX463) in humans, respectively. The two mutantSdccag8 knock-in mice faithfully recapitulated human SDCCAG8-associated BBS phenotypes such as rod-cone dystrophy, cystic renal disorder, polydactyly, infertility, and growth retardation, with varied age of onset and severity depending on the hypomorphic strength of the Sdccag8 mutations. To the best of our knowledge, these knock-in mouse lines are the first BBS mouse models to present with the polydactyly phenotype. Major phototransduction protein mislocalization was also observed outside the outer segment after initiation of photoreceptor degeneration. Impaired cilia were observed in the mutant photoreceptors, renal epithelial cells, and mouse embryonic fibroblasts derived from the knock-in mouse embryos, suggesting that SDCCAG8 plays an essential role in ciliogenesis, and cilium defects are a primary driving force of SDCCAG8-associated retinal ciliopathies.

Leucine zipper transcription factor-like 1 (LZTFL1), an intraflagellar transporter protein 27 (IFT27) associated protein, is required for normal sperm function and male fertility

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.

Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model.

Bardet-Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in approximately 25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1M390R/M390R mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1M30R/M390R;BBS1TG+ mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1M300R/M390R;BBS1TG+ mice. The results of this study indicate the male infertility aspect of BBS is an attractive target for gene therapy.

Characteristics of genotype and phenotype in Chinese patients with Bardet-Biedl syndrome.

To investigate complex and different phenotypes in seven Chinese patients diagnosed with Bardet-Biedl syndrome (BBS) and carrying pathogenic mutations. Seven unrelated BBS patients were enrolled. Their medical and ophthalmic histories were reviewed, and comprehensive clinical examinations, such as fundus photography, optical coherence tomography, and medical imaging, were performed. A specific hereditary eye disease enrichment panel based on exome-capture technology was used to collect and amplify the protein-coding regions of 441 targeted hereditary eye disease genes, followed by high-throughput sequencing using the Illumina HiSeq platform. All patients exhibited the primary clinical phenotype of BBS. Seven BBS mutations were found in five patients (BBS7 in two patients, BBS10 in two patients, BBS12 in one patient), for a detection rate of 71% (5/7). The ratio of novel to known BBS mutations was 5:2. This study showed the phenotypic and genotypic spectrum of BBS patients from China, and the findings underscore the importance of obtaining comprehensive clinical observations and molecular analyses for ciliopathies.

Cutaneous findings in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a rare, pleiotropic syndrome and member of a diverse group of disorders known as ciliopathies. Improved understanding of dermatoses in BBS will further understanding of the syndrome and will help define the role of dermatologists in providing care for patients with BBS. The purpose of this study was to describe the cutaneous phenotype of BBS in patients attending a large, multispecialty BBS clinic. All patients attending the multispecialty BBS Clinic at the Marshfield Medical Center over a 12-month period were invited to participate. Complete cutaneous examinations were performed by a board-certified dermatologist, and comprehensive physical examinations were performed by clinic physicians. Molecular genetic results were obtained when available. Comprehensive laboratory studies were performed in each patient including fasting blood sugar and thyroid and renal function. Thirty-one individuals ranging in age between 2 and 69 years (median age, 12 years) participated in the study. Cutaneous findings were present in all subjects. Keratosis pilaris was present in 80.6% of subjects, and seborrheic dermatitis was present in 19.3%. Obesity, a cardinal feature of BBS, was present in the majority of subjects (90.3%) and was accompanied by known obesity-related dermatologic disorders. Cutaneous disorders are common in BBS and suggest disturbance of keratinization and keratinocyte function as well as systemic consequences of BBS on skin health. Increased prevalence of skin barrier dysfunction in this ciliopathy demonstrates the importance of dermatologist contribution to health care in BBS.

Identification of a homozygous BBS7 frameshift mutation in two (related) Chinese Miao families with Bardet-Biedl Syndrome.

Bardet-Biedl Syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder with a wide spectrum of clinical features. To date, mutations in 21 different genes (BBS1-21) have been identified as causing isolated or complex BBS phenotypes. In this report, we present three Chinese Miao ethnic patients who were diagnosed with BBS on the basis of characteristic clinical features and investigated the exsome of these patients. To evaluate disease genes, the Agilent SureSelect system and Illumina HiSeq 2000 platform for whole exome enrichment and sequencing (WES) were used on the proband and her mother. Variants that fit a recessive model of inheritance only were compared and filtered using public databases. Variants detected by exome sequencing were validated by Sanger sequencing. A total of 981 phenotypically normal subjects were enrolled as control data set. A frameshift homozygous germline mutation in BBS7 was detected by WES and identified by Sanger sequencing in affected individuals. This mutation was predicted to result in premature termination of exon5 (c.389_390delAC, p.Asn130ThrfsX3; RefSeq NM_176824.2) and lead to a 133 amino acid truncated protein. The inheritance patterns in the families are consistent with autosomal recessive inheritance, and no such homozygous mutation was found in the other 981 controls. This mutation has not yet been described in any reported literature, and this is the first report on BBS7 mutation in Chinese Miao families with BBS phenotypes.

Thoraco-Abdominal Abnormalities in Bardet-Biedl Syndrome: Situs Inversus and Heterotaxy

To characterize the diversity and prevalence of thoraco-abdominal abnormalities in Bardet-Biedl syndrome (BBS), a model ciliopathy for understanding the role of cilia in human health. The Clinical Registry Investigating BBS, a worldwide registry exploring the phenotype and natural history of BBS, was used to conduct the study. Protected health information was obtained by subject or family interview and Health Insurance Portability and Accountability Act-approved release of data including imaging studies and genetic testing. Echocardiography and imaging findings were independently confirmed by 2 cardiologists. Thoraco-abdominal abnormalities were identified in 6 of 368 (1.6%) subjects with a minimum prevalence of 1 in 60 Clinical Registry Investigating BBS participants. Diverse laterality defects were observed suggesting that the underlying ciliopathy randomly alters embryonic left-right axis orientation. Congenital heart disease, common in heterotaxy, was present in 2 subjects. Additional defects, uncommonly reported in BBS, were observed in the central nervous, genitourinary, gastrointestinal, and musculoskeletal systems in the subjects. No BBS genotype was favored in the cohort. One subject had genetic and clinical phenotype diagnostic of both primary ciliary dyskinesia and BBS. The variety of thoraco-abdominal abnormalities in BBS suggests the pleiotropic nature of these anomalies is not confined to a single pattern or genotype. Clinicians providing care to individuals with BBS should consider the increased prevalence of thoraco-abdominal anomalies in BBS. Individuals with features suggestive of other ciliopathies, such as primary ciliary dyskinesia, should undergo further evaluation for additional genetic disorders. ClinicalTrials.gov: NCT02329210.

Novel sequence variants in the MKKS gene cause Bardet-Biedl syndrome with intra- and inter-familial variable phenotypes.

Bardet-Biedl syndrome (BBS) is a multisystem disorder involving retina, kidney, limbs, and nervous system. Additional features associated with BBS include dental anomalies, cardiovascular defects, hearing loss, and speech impairment (Beales et al. 1999; Ullah et al. 2017). Diagnosis of BBS is based on the presence of four out of six primary or three primary and two secondary phenotypes. Variation in the phenotypes within and among different families has been reported (Beales et al. 1999; Khan et al. 2016). So far, mutations have been reported in 21 disease-causing genes (BBS1–21) (Heon et al. 2016; Suspitsin and Imyanitov, 2016). BBS segregates in autosomal recessive manner. In rare cases, triallelic nature of BBS involving three mutated alleles in two genes has been reported as well. Genes causing BBS phenotypes play important roles in ciliogenesis. Defects in the formation or function of cilia result in syndromic form of BBS. Cilia are cell surface structures involved in cell locomotion, signaling, and chemo/mechano/photo-sensation. Due to localization of cilia in different organs including kidney, liver, pancreas, odontoblasts, retinal photoreceptors, and hypothalamic neurons, ciliary defects cause multisystem disorders. Three BBS proteins including BB6/MKKS, BBS10, and BBS12 form chaperonin complex required for BBsome formation. BBsome is composed of eight BBS proteins including BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBIP10/BBS18. Chaperonin binds to BBS7 and stabilizes it and then interacts with BBS2 and BBS9 to form core of BBsome. Other members bind to BBsome core in order of BBS1, BBS5, BBS8, BBS4, and BBIP10 (Loktev et al. 2008). BBsome participates in the cilia formation. The present study was conducted with the permission of institutional review board of Qauid-i-Azam University Islamabad, Pakistan. Clinical features of affected individuals are presented in Figure 1. Affected members in the two families (A and B) showed previously reported primary features of BBS, which included polydactyly, learning difficulties, retinitis pigmentosa, truncal obesity, and hypogonadism (Khan et al. 2016). In addition, a male affected member of family A showed sparse hairs on scalp and face, and a rare secondary feature of dental anomalies. In the same family, one of the affected members (V-5) had polydactyly in both hands and feet, while the second affected member (V-6) showed this feature only in the right foot. Affected members of family B showed clinodactyly of toes as a secondary feature of BBS (Table S1, Supporting information). DNA from blood was extracted using Sigma GenElute Blood Genomic DNA kit (Sigma Aldrich, St Louis, MO, USA). Homozygosity mapping was performed by genotyping highly polymorphic microsatellite markers which were mapped previously in vicinity of the BBS genes (BBS1–21) ( Fig. S1). This was followed by Sanger sequencing using standard procedure (Ullah et al. 2015), which revealed two novel variants including c.775-775delA in family A and c.287C>T in family B in the MKKS. Mutation effect prediction tools (MutationTaster, SIFT, and Polyphen2) predicted the two novel variants to be deleterious and disease-causing (Table S2). Previously, the MKKS was mapped on chromosome 20p12.2. The gene encodes 570-amino acid long BBS6 protein. It contains three domains including equatorial, intermediate, and apical. The equatorial domain is connected to apical domain by intermediate domain through flexible hinges. The variant (p.Thr259Leufs*21) in MKKS/BBS6, found in family A, is located in the apical domain which is involved in substrate binding. The other variant (p.Ala96Val), found in family B, is located in the most conserved equatorial domain and is highly likely to disrupt its ATPase activity. The BBS6 is a member of chaperonin family, which is involved in the formation of BBsome complex. It is highly likely that sequence variants in the BBS6 would disrupt formation of such a complex leading to buckle cilia formation. We have observed variation in the phenotypes not only in the two families, but also among patients of the same family (Table S1). Position and nature of mutation alter the structure and function of protein differentially. Genetic background and extent of change in the structure and function of protein might be responsible for the intra- and inter-familial variability of phenotypes. In conclusion, we have identified two novel sequence variants in the BBS6 causing BBS phenotypes in two different consanguineous families. This study not only supports the previously reported findings of intra- and inter-familial variations in features of BBS families, but also further extend spectrum of the mutations in the MKKS gene. This study will support genetic testing of BBS patients in Pakistan. We are highly thankful to the family members for their participation in the present research project. MU was supported by Higher Education Commission Islamabad, Pakistan Indigenous PhD fellowship. The authors declare that they have no competing interests. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Whole-exome sequencing identified compound heterozygous variants in MMKS in a Chinese pedigree with Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. At least 21 candidate BBS-associated genes (BBS1-19, NPHP1, and IFT172) have previously been identified, and all of them play important roles in ciliary function. Here, we collected a BBS pedigree with four members and performed whole-exome sequencing on the proband. The variants were analyzed and evaluated to confirm their pathogenicity. We found compound heterozygous variants (c.1192C>T, p.Q398* and c.1175C>T, p.T392M) in MKKS in both the siblings, and these were likely to be pathogenic variants. We also found a missense variant (c.2029G>C, p.E677Q) in NPHP1 and a missense variant (c.2470C>T, p.R824C) in BBS9 in the proband only, which are variants of uncertain significance. The compound heterozygous variants were probably responsible for the BBS phenotype in this Chinese pedigree and the missense mutations in NPHP1 and BBS9 might contribute to the mutation load.

Progress of research on the pathogenesis of obesity in Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disease characterized by retinopathy, obesity, and polydactyly. So far 21 candidate genes have been discovered, and mutations of such genes can all cause the BBS phenotype. As one of the main features of the disease, the obesity in BBS has been associated with leptin resistance and abnormal adipogenesis. However, its molecular etiology is not yet completely clear. Here the molecular mechanism of BBS-associated obesity is reviewed.

Abstract 035: Differential Effects of Bardet-biedl Syndrome 1 Gene Deletion From Agouti-related Peptide Neurons

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive human disorder associated with several clinical features including obesity and hypertension. We previously demonstrated that Bbs1 gene deletion from the central nervous system caused obesity and hypertension in mice highlighting the importance of neuronal BBS proteins for energy homeostasis and cardiovascular regulation. Here, we investigated the role of Bbs1 gene in the orexigenic agouti-related peptide (AgRP) neurons. We crossed Bbs1 fl/fl mice with AgRP Cre mice and confirmed Cre recombinase activity in AgRP neurons using td-Tomato reporter mice. Interestingly, female AgRP Cre /Bbs1 fl/fl mice developed profound obesity as indicated by the increased body weight (46.8±2.0 g vs control littermates, 35.5±1.9 g at 25 weeks of age). In contrast, body weight of male AgRP Cre /Bbs1 fl/fl mice (48.5±1.2 g) was similar to the controls (46.7±1.2 g). There was no change in the expression of hypothalamic genes regulating food intake (AgRP, proopiomelanocortin and neuropeptide Y) in AgRP Cre /Bbs1 fl/fl mice relative to controls. Next, we assessed the consequence on arterial pressure (AP) and sympathetic nerve activity (SNA) of ablating the Bbs1 gene from AgRP neurons. Interestingly, deletion of the Bbs1 gene in AgRP neurons did not recapitulate the hypertension phenotype of BBS as indicated by the lack of difference in mean AP (107±5.0 vs 106±5.2 mmHg in controls) and heart rate (597±23.5 vs 589±16.4 in control). However, conscious renal SNA was significantly higher in AgRP Cre /Bbs1 fl/fl mice relative to controls (114±10 vs 78±6 spikes/sec, p<0.05). In addition, the depressor effect of ganglionic blockade (hexamethonium) was higher in AgRP Cre /Bbs1 fl/fl mice (-64.4±10 vs -55.8±8.3 mmHg in control). Finally, we used wire myography to measure mesenteric artery vascular function and found impairment in acetylcholine induced relaxation in AgRP Cre /Bbs1 fl/fl mice (Max. relaxation: 78 ± 6% vs 26 ± 9% in controls; p<0.05), but no changes in the relaxation evoked by sodium nitroprusside, suggesting endothelial, but not smooth muscle, dysfunction. These findings demonstrate that Bbs1 gene in AgRP neurons is critical for energy homeostasis, renal sympathetic outflow and vascular endothelial function.

Depletion of BBS Protein LZTFL1 Affects Growth and Causes Retinal Degeneration in Mice

Bardet-Biedl syndrome (BBS) is a heterogeneous disease characterized by deficiencies in various organs that are caused by defects in genes involved in the genesis, structural maintenance, and protein trafficking of cilia. Leucine zipper transcription factor-like 1 (LZTFL1) has been identified as a BBS protein (BBS17), because patients with mutations in this gene exhibit the common BBS phenotypes. In this study, we generated a knockout mouse model to investigate the effects of LZTFL1 depletion. Lztfl1 knockout mice were born with low birth weight, reached similar weight to those of wild-type mice at 10 weeks of age, and later gained more weight than their wild-type counterparts. LZTFL1 was localized to the primary cilium of kidney cells, and the absence of LZTFL1 increased the ciliary localization of BBS9. Moreover, in the retinas of Lztfl1 knockout mice, the photoreceptor outer segment was shortened, the distal axoneme of photoreceptor connecting cilium was significantly enlarged, and rhodopsin was targeted to the outer nuclear layer. TUNEL assay showed that many of these abnormal photoreceptor cells in Lztfl1 knockout mice underwent apoptosis. Interestingly, the absence of LZTFL1 caused an abnormal increase of the adaptor protein complex 1 (AP1) in some photoreceptor cells. Based on these data, we conclude that LZTFL1 is a cilium protein and regulates animal weight and photoreceptor connecting cilium function probably by controlling microtubule assembly and protein trafficking in cilia.

Genetics of human Bardet-Biedl syndrome, an updates.

Bardet-Biedl syndrome (BBS) is an autosomal recessive multisystemic human genetic disorder characterized by six major defects including obesity, mental retardation, renal anomalies, polydactyly, retinal degeneration and hypogenitalism. In several cases of BBS, few other features such as metabolic defects, cardiovascular anomalies, speech deficits, hearing loss, hypertension, hepatic defects and high incidence of diabetes mellitus have been reported as well. The BBS displays extensive genetic heterogeneity. To date, 19 genes have been mapped on different chromosomes causing BBS phenotypes having varied mutational load of each BBS gene. In this review, we have discussed clinical spectrum and genetics of BBS. This report presents a concise overview of the current knowledge on clinical data and its molecular genetics progress upto date.

Abstract 083: Metabolic and Cardiovascular Effects of BBS1 Ablation From the POMC-containing Neurons

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder associated with several features including obesity and hypertension. Deletion of Bbs genes globally, in the nervous system or in the leptin receptor-expressing cells recapitulated many of the BBS phenotypes including obesity and hypertension. Here, we assessed the effect of ablating the Bbs1 gene from the neurons expressing proopiomelanocortin (POMC) neurons. Breeding Bbs1 flox mice with POMC Cre mice created mice deficient in Bbs1 gene only in the POMC-positive neurons (visualized by tdTomato expression). Importantly, POMC Cre /Bbs1 fl/fl mice display an obesity phenotype as indicated by the increased (P<0.05) body weight (48.6±1.7 vs 34.9±0.8 g in controls) and fat pads (3.3±0.3 vs. 0.7±1.1 g for inguinal fat, 2.1±0.2 vs. 0.4±0.05 g for perirenal fat, 3.3±0.3 vs. 0.8±0.3 g for reproduction fat and 0.4±0.02 vs. 0.2±0.02 g for brow fat) associated with increased (P<0.05) food intake (3.79.±0.14 vs. 2.97±0.15 g in controls) in 25 weeks old mice. POMC Cre /Bbs1 fl/fl mice displayed decreased (P<0.05) O 2 consumption (2.6±0.03 vs. 3±0.04 mL O 2 /100g/min in controls) and heat production (8.0±0.09 vs. 9.2±0.13 kcal/kg/hr in controls). These results indicate that hyperphagia and decreased energy expenditure contribute to the development of obesity in POMC Cre /Bbs1 fl/fl mice. Next, we assessed the consequence on arterial pressure (AP) and sympathetic nerve activity (SNA) of ablating the Bbs1 gene from POMC neurons. Interestingly, deletion of the Bbs1 gene in POMC neurons did not recapitulate the hypertension phenotype of BBS as indicated by the slight, but not significant increase in mean AP (116±4.7 vs 109±6.1 mmHg in controls). However, conscious renal SNA was significantly higher in POMC Cre /Bbs1 fl/fl mice relative to controls (132.4±11 vs 74.3±4.2 spikes/sec, P<0.05). Finally, the depressor effect of ganglionic blockade (hexamethonium) was exaggerated in POMC Cre /Bbs1 fl/fl mice (-68.6±4.4 vs -45.2±9.4 mmHg in control, P=0.017). These findings demonstrate that Bbs1 gene in the POMC neurons is critical for energy homeostasis, but not for arterial pressure regulation.

Abstract 016: Bbs1 Gene Deletion From The Leptin Receptor Neurons Causes Obesity And Hypertension In Mice

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder associated with several features including obesity and hypertension. Deletion of BBS genes globally or in the nervous system recapitulated many of the BBS phenotypes including obesity and hypertension. Here, we assessed the effect of ablating the Bbs1 gene from the neurons expressing the long signaling form of the leptin receptor (LepRb). Breeding Bbs1 flox with LepRb Cre mice created mice deficient in the Bbs1 gene only in LepRb-positive neurons (visualized by tdTomato expression) as indicated by loss of leptin activation of Stat3. Importantly, Bbs1 flox /LepR Cre mice display an obesity phenotype as indicated by the increased (P<0.05) body weight (39±2 vs. 30±1 g in controls) and fat mass measured by MRI (14±3 vs. 4±1 g in controls) associated with increased (P<0.05) food intake (3.4±0.1 vs. 2.9±0.1 g in controls) in 25 weeks old mice. However, body weight and fat pads of pair-fed LRb Cre /Bbs1 fl/fl mice remained significantly elevated compared to controls suggesting that LRb Cre /Bbs1 fl/fl mice have reduced energy expenditure. Consistent with this possibility, LRb Cre /Bbs1 fl/fl mice displayed decreased (P<0.05) O 2 consumption (2.6±0.1 vs. 3.1±0.1 mL/100g/min in controls) and heat production (8.1±0.3 vs. 9.6±0.3 kcal/kg/h in controls). These results indicate that hyperphagia and decreased energy expenditure contribute to the development of obesity in Bbs1 flox /LepR Cre mice. Next, we assessed the effect on arterial pressure (AP) and sympathetic nerve activity (SNA) of ablating the Bbs1 gene from the LepR-containing neurons. Interestingly, deletion of the Bbs1 gene in LepR neurons recapitulates the hypertension phenotype of BBS as indicated by elevated mean AP (125±4 vs 109±3 mmHg in controls, P=0.03). Conscious renal SNA was also elevated in LRb Cre /Bbs1 fl/fl mice relative to controls (97±8 vs 62±10 spikes/sec, P<0.05). Finally, the depressor effect of ganglionic blockade (hexamethonium) was exaggerated in Bbs1 flox /LepR Cre mice (-57±5 vs -38±5 mmHg in control, P=0.01). These findings demonstrate that the Bbs1 gene in LepR neurons is critical for energy homeostasis and arterial pressure regulation.

Ciliopathy Is Differentially Distributed in the Brain of a Bardet-Biedl Syndrome Mouse Model

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous inherited human disorder displaying a pleotropic phenotype. Many of the symptoms characterized in the human disease have been reproduced in animal models carrying deletions or knock-in mutations of genes causal for the disorder. Thinning of the cerebral cortex, enlargement of the lateral and third ventricles, and structural changes in cilia are among the pathologies documented in these animal models. Ciliopathy is of particular interest in light of recent studies that have implicated primary neuronal cilia (PNC) in neuronal signal transduction. In the present investigation, we tested the hypothesis that areas of the brain responsible for learning and memory formation would differentially exhibit PNC abnormalities in animals carrying a deletion of the Bbs4 gene (Bbs4-/-). Immunohistochemical localization of adenylyl cyclase-III (ACIII), a marker restricted to PNC, revealed dramatic alterations in PNC morphology and a statistically significant reduction in number of immunopositive cilia in the hippocampus and amygdala of Bbs4-/- mice compared to wild type (WT) littermates. Western blot analysis confirmed the decrease of ACIII levels in the hippocampus and amygdala of Bbs4-/- mice, and electron microscopy demonstrated pathological alterations of PNC in the hippocampus and amygdala. Importantly, no neuronal loss was found within the subregions of amygdala and hippocampus sampled in Bbs4-/- mice and there were no statistically significant alterations of ACIII immunopositive cilia in other areas of the brain not known to contribute to the BBS phenotype. Considered with data documenting a role of cilia in signal transduction these findings support the conclusion that alterations in cilia structure or neurochemical phenotypes may contribute to the cognitive deficits observed in the Bbs4-/- mouse mode.

Bbs1 gene deletion from the leptin receptor‐containing neurons causes obesity (1126.10)

Bardet‐Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder with several features including obesity. Systemic knock‐out mice lacking expression of Bbs2, Bbs4 and Bbs6, or Bbs1M390R knock‐in, recapitulated many BBS phenotypes including obesity. However, the role and contribution of Bbs genes in different tissues to obesity remains unclear. To address this, we generated a new conditional knockout mouse where exon 3 of the Bbs1 gene is floxed. Cre‐mediated recombination causes a frame shift resulting in a premature stop. We assessed whether deletion of the Bbs1 gene in the leptin receptor neurons (LRb) affects body weight. Importantly, Bbs1flox/LRbCre mice display an obesity phenotype as indicated by the increased body weight and fat mass measured by MRI. We found that the obesity phenotype in Bbs1flox/LRbCre mice is due to both an increase in food intake and reduction in energy expenditure as indicated by the decreased O2 consumption and heat production. These results indicate that hyperphagia and low metabolic rate explain the development of obesity in Bbs1flox/LRbCre mice. Finally, in HEK 293 cells, silencing Bbs1 or Bbs2 genes significantly decreased LRb, but not LRa or transferrin receptor, trafficking to the plasma membrane highlighting the important role of Bbs gene in the regulation of LRb signaling. These findings demonstrate that Bbs genes are critical for energy homeostasis.Grant Funding Source: NIH and AHA