Fat aussie--a new Alström syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis

Mutations in the human ALMS1 gene are responsible for Alstr?m syndrome, a disorder in which key metabolic and endocrinological features include childhood-onset obesity, metabolic syndrome, and diabetes, as well as infertility. ALMS1 localizes to the basal bodies of cilia and plays a role in intracellular trafficking, but the biological functions of ALMS1 and how these relate to the pathogenesis of obesity, diabetes, and infertility remain unclear. Here we describe a new mouse model of Alstr?m syndrome, fat aussie, caused by a spontaneous mutation in the Alms1 gene. Fat aussie (Alms1 foz/foz) mice are of normal weight when young but, by 120 d of age, they become obese and hyperinsulinemic. Diabetes develops in Alms1 foz/foz mice accompanied by pancreatic islet hyperplasia and islet cysts. Female mice are fertile before the onset of obesity and metabolic syndrome; however, male fat aussie mice are sterile due to a progressive germ cell loss followed by an almost complete block of development at the round-to-elongating spermatid stage of spermatogenesis. In conclusion, Alms1 foz/foz mouse is a new animal model in which to study the pathogenesis of the metabolic and fertility defects of Alstr?m syndrome, including the role of ALMS1 in appetite regulation, pathogenesis of the metabolic syndrome, pancreatic islet physiology, and spermatogenesis.     

Dietary modification dampens liver inflammation and fibrosis in obesity‐related fatty liver disease

Background: Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High‐fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans. Objective: We sought to establish how dietary composition contributes to NASH pathogenesis. Design and Methods: foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF‐κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined. Result: HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet‐induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF‐κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation. Conclusion: Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF‐κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity‐related NASH.     

Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.     

Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.     

A role for Alström syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence

Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alstr?m syndrome. Alstr?m syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alstr?m syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alstr?m syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alstr?m syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.     

Adaptive failure to high-fat diet characterizes steatohepatitis in Alms1 mutant mice

The biochemical differences between simple steatosis, a benign liver disease, and non-alcoholic steatohepatitis, which leads to cirrhosis, are unclear. Fat aussie is an obese mouse strain with a truncating mutation (foz) in the Alms1 gene. Chow-fed female foz/foz mice develop obesity, diabetes, and simple steatosis. We fed foz/foz and wildtype mice a high-fat diet. Foz/foz mice developed serum ALT elevation and severe steatohepatitis with hepatocyte ballooning, inflammation, and fibrosis; wildtype mice showed simple steatosis. Biochemical pathways favoring hepatocellular lipid accumulation (fatty acid uptake; lipogenesis) and lipid disposal (fatty acid beta-oxidation; triglyceride egress) were both induced by high-fat feeding in wildtype but not foz/foz mice. The resulting extremely high hepatic triglyceride levels were associated with induction of mitochondrial uncoupling protein-2 and adipocyte-specific fatty acid binding protein-2, but not cytochrome P4502e1 or lipid peroxidation. In this model of metabolic syndrome, transition of steatosis to steatohepatitis was associated with hypoadiponectinemia, a mediator of hepatic fatty acid disposal pathways.     

Functional analysis by minigene assay of putative splicing variants found in Bardet–Biedl syndrome patients

Bardet–Biedl syndrome (BBS) and Alström syndrome (ALMS) are rare diseases belonging to the group of ciliopathies. Although mutational screening studies of BBS/ALMS cohorts have been extensively reported, little is known about the functional effect of those changes. Thus, splicing variants are estimated to represent 15% of disease‐causing mutations, and there is growing evidence that many exonic changes are really splicing variants misclassified. In this study, we aimed to analyse for the first time several variants in BBS2, ARL6/BBS3, BBS4 and ALMS1 genes predicted to produce aberrant splicing by minigene assay. We found discordance between bioinformatics analysis and experimental data when comparing wild‐type and mutant constructs. Remarkably, we identified nonsense variants presumably resistant to nonsense‐mediated decay, even when a premature termination codon would be introduced in the second amino acid (p.(G2*) mutation in ARL6/BBS3 gene). As a whole, we report one of the first functional studies of BBS/ALMS1 variants using minigene assay, trying to elucidate their role in disease. Functional studies of variants identified in BBS and ALMS patients are essential for their proper classification and subsequent genetic counselling and could also be the start point for new therapeutic approaches, currently based only on symptomatic treatment.     

Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.     

Novel <Emphasis Type="Italic">Alu</Emphasis> retrotransposon insertion leading to Alström syndrome

Alström syndrome is a clinically complex disorder characterized by childhood retinal degeneration leading to blindness, sensorineural hearing loss, obesity, type 2 diabetes mellitus, cardiomyopathy, systemic fibrosis, and pulmonary, hepatic, and renal failure. Alström syndrome is caused by recessively inherited mutations in the ALMS1 gene, which codes for a putative ciliary protein. Alström syndrome is characterized by extensive allelic heterogeneity, however, founder effects have been observed in some populations. To date, more than 100 causative ALMS1 mutations have been identified, mostly frameshift and non-sense alterations resulting in termination signals in ALMS1. Here, we report a complex Turkish kindred in which sequence analysis uncovered an insertion of a novel 333 basepair Alu Ya5 SINE retrotransposon in the ALMS1 coding sequence, a previously unrecognized mechanism underlying the mutations causing Alström syndrome. It is extraordinarily rare to encounter the insertion of an Alu retrotransposon in the coding sequence of a gene. The high frequency of the mutant ALMS1 allele in this isolated population suggests that this recent retrotransposition event spreads quickly, and may be used as a model to study the population dynamics of deleterious alleles in isolated communities.     

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Z _max=2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291–D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Z _max=3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.     

Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling.     

A novel mutation causing mild, atypical fumarylacetoacetase deficiency (Tyrosinemia type I): a case report

Background

A case series of the cardiac magnetic resonance imaging findings in seven adult Alström patients.

Methods

Seven patients from the National Specialist Commissioning Group Centre for Alström Disease, Torbay, England, UK, completed the cardiac magnetic resonance imaging protocol to assess cardiac structure and function in Alström cardiomyopathy.

Results

All patients had some degree of left and right ventricular dysfunction. Patchy mid wall gadolinium delayed enhancement was demonstrated, suggesting an underlying fibrotic process. Some degree of cardiomyopathy was universal. No evidence of myocardial infarction or fatty infiltration was demonstrated, but coronary artery disease cannot be completely excluded. Repeat scanning after 18 months in one subject showed progression of fibrosis and decreased left ventricular function.

Conclusion

Adult Alström cardiomyopathy appears to be a fibrotic process causing impairment of both ventricles. Serial cardiac magnetic resonance scanning has helped clarify the underlying disease progression and responses to treatment. Confirmation of significant mutations in the ALMS1 gene should lead to advice to screen the subject for cardiomyopathy, and metabolic disorders.     

A founder effect in the newfoundland population reduces the Bardet-Biedl syndrome I (BBS1) interval to 1 cM

Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive disorder; major phenotypic findings include dysmorphic extremities, retinal dystrophy, obesity, male hypogenitalism, and renal anomalies. In the majority of northern European families with BBS, the syndrome is linked to a 26-cM region on chromosome 11q13. However, the finding, so far, of five distinct BBS loci (BBS1, 1q; BBS2, 16q; BBS3, 3p; BBS4, 15q; BBS5, 2q) has hampered the positional cloning of these genes. We use linkage disequilibrium (LD) mapping in an isolated founder population in Newfoundland to significantly reduce the BBS1 critical region. Extensive haplotyping in several unrelated BBS families of English descent revealed that the affected members were homozygous for overlapping portions of a rare, disease-associated ancestral haplotype on chromosome 11q13. The LD data suggest that the BBS1 gene lies in a 1-Mb, sequence-ready region on chromosome 11q13, which should enable its identification.     

Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.     

The Centriolar Satellite Protein AZI1 Interacts with BBS4 and Regulates Ciliary Trafficking of the BBSome

Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer''s vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.     

BBS8蛋白参与衣藻鞭毛膜蛋白的运输

真核细胞的纤毛(也称鞭毛)是一种突出于细胞表面的极性细胞器,纤毛不仅参与细胞运动,还参与信号传导等过程,其结构或功能异常引发的一系列人类疾病称为"纤毛相关性疾病"。纤毛相关性疾病巴德-毕德氏综合征(Bardet-Biedl syndrome,简称BBS)由BBS相关基因缺陷导致,为了研究致病基因BBS8的生理作用和功能,构建模式生物莱茵衣藻BBS8基因缺陷突变体,利用性状观测和生化分析检测突变体的表现型和生理功能。免疫荧光表明BBS8蛋白是一种鞭毛蛋白且在基体有特异性定位;bbs8突变体感光极性运动消失,并在解聚诱导实验中鞭毛解聚缓慢;鞭毛的银染和质谱结果表明突变体的鞭毛膜蛋白在鞭毛内异常积累。文中通过实验证据说明BBS8蛋白在参与鞭毛内膜蛋白运输中起到重要作用,并极可能通过介导膜蛋白反向运输发挥生理功能。     

Ectopic Expression of Human BBS4 Can Rescue Bardet-Biedl Syndrome Phenotypes in Bbs4 Null Mice

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogenitalism and renal defects. Recent findings have associated the etiology of the disease with cilia, and BBS proteins have been implicated in trafficking various ciliary cargo proteins. To date, 17 different genes have been reported for BBS among which BBS1 is the most common cause of the disease followed by BBS10, and BBS4. A murine model of Bbs4 is known to phenocopy most of the human BBS phenotypes, and it is being used as a BBS disease model. To better understand the in vivo localization, cellular function, and interaction of BBS4 with other proteins, we generated a transgenic BBS4 mouse expressing the human BBS4 gene under control of the beta actin promoter. The transgene is expressed in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. These mice were further bred to express the transgene in Bbs4 null mice, and their phenotype was characterized. Here we report that despite tissue specific variable expression of the transgene, human BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. These results provide an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies.     

Antenatal presentation of Bardet-Biedl syndrome may mimic Meckel syndrome

Bardet-Biedl syndrome (BBS) is a multisystemic disorder characterized by postaxial polydactyly, progressive retinal dystrophy, obesity, hypogonadism, renal dysfunction, and learning difficulty. Other manifestations include diabetes mellitus, heart disease, hepatic fibrosis, and neurological features. The condition is genetically heterogeneous, and eight genes (BBS1-BBS8) have been identified to date. A mutation of the BBS1 gene on chromosome 11q13 is observed in 30%-40% of BBS cases. In addition, a complex triallelic inheritance has been established in this disorder--that is, in some families, three mutations at two BBS loci are necessary for the disease to be expressed. The clinical features of BBS that can be observed at birth are polydactyly, kidney anomaly, hepatic fibrosis, and genital and heart malformations. Interestingly, polydactyly, cystic kidneys, and liver anomalies (hepatic fibrosis with bile-duct proliferation) are also observed in Meckel syndrome, along with occipital encephalocele. Therefore, we decided to sequence the eight BBS genes in a series of 13 antenatal cases presenting with cystic kidneys and polydactyly and/or hepatic fibrosis but no encephalocele. These fetuses were mostly diagnosed as having Meckel or "Meckel-like" syndrome. In six cases, we identified a recessive mutation in a BBS gene (three in BBS2, two in BBS4, and one in BBS6). We found a heterozygous BBS6 mutation in three additional cases. No BBS1, BBS3, BBS5, BBS7, or BBS8 mutations were identified in our series. These results suggest that the antenatal presentation of BBS may mimic Meckel syndrome.     

Mutation analysis of the MKKS gene in McKusick-Kaufman syndrome and selected Bardet-Biedl syndrome patients

McKusick-Kaufman syndrome comprises hydrometrocolpos, polydactyly, and congenital heart defects and overlaps with Bardet-Biedl syndrome, comprising retinitis pigmentosa, polydactyly, obesity, mental retardation, and renal and genital anomalies. Bardet-Biedl syndrome is genetically heterogeneous with three cloned genes ( BBS2, BBS4, and MKKS) and at least three other known loci ( BBS1, BBS3, and BBS5). Both McKusick-Kaufman syndrome and Bardet-Biedl syndrome are inherited in an autosomal recessive pattern, and both syndromes are caused by mutations in the MKKS gene. However, mutations in MKKS are found in only 4%-11% of unselected Bardet-Biedl syndrome patients. We hypothesized that an analysis of patients with atypical Bardet-Biedl syndrome and McKusick-Kaufman syndrome (Group I; 15 probands) and patients with Bardet-Biedl syndrome who had linkage results inconsistent with linkage to the other loci (Group II; 12 probands) could increase the MKKS mutation yield. Both mutant alleles were identified in only two families in Group II. Single (heterozygous) sequence variations were found in three Group I families and in two Group II families. Combining these results with previously published data showed that only one mutant allele was detected in nearly half of all patients screened to date, suggesting that unusual mutational mechanisms or patterns of inheritance may be involved. However, sequencing of the BBS2 gene in these patients did not provide any evidence of digenic or "triallelic" inheritance. The frequency of detected mutations in MKKS in Group II patients was 24%, i.e., six times higher than the published rate for unselected BBS patients, suggesting that small-scale linkage analyses may be useful in suitable families.     

Knockdown of Bardet-Biedl syndrome gene BBS9/PTHB1 leads to cilia defects

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals.