John S. Beck

Identification of the gene (BBS1) most commonly involved in Bardet-Biedl syndrome, a complex human obesity syndrome.

Bardet-Biedl syndrome (BBS, OMIM 209900) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation and hypogenitalism. Individuals with BBS are also at increased risk for diabetes mellitus, hypertension and congenital heart disease. What was once thought to be a homogeneous autosomal recessive disorder is now known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13–p12 (BBS3), 15q22.3–q23 (BBS4), 2q31 (BBS5) and 20p12 (BBS6). There has been considerable interest in identifying the genes that underlie BBS, because some components of the phenotype are common. Cases of BBS mapping ro BBS6 are caused by mutations in MKKS; mutations in this gene also cause McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly and congenital heart defects). In addition, we recently used positional cloning to identify the genes underlying BBS2 (ref. 16) and BBS4 (ref. 17). The BBS6 protein has similarity to a Thermoplasma acidophilum chaperonin, whereas BBS2 and BBS4 have no significant similarity to chaperonins. It has recently been suggested that three mutated alleles (two at one locus, and a third at a second locus) may be required for manifestation of BBS (triallelic inheritance). Here we report the identification of the gene BBS1 and show that a missense mutation of this gene is a frequent cause of BBS. In addition, we provide data showing that this common mutation is not involved in triallelic inheritance.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH2-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

A BBSome Subunit Links Ciliogenesis, Microtubule Stability, and Acetylation

Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.

BBS6, BBS10, and BBS12 form a complex with CCT/TRiC family chaperonins and mediate BBSome assembly

Bardet-Biedl syndrome (BBS) is a human genetic disorder resulting in obesity, retinal degeneration, polydactyly, and nephropathy. Recent studies indicate that trafficking defects to the ciliary membrane are involved in this syndrome. Here, we show that a novel complex composed of three chaperonin-like BBS proteins (BBS6, BBS10, and BBS12) and CCT/TRiC family chaperonins mediates BBSome assembly, which transports vesicles to the cilia. Chaperonin-like BBS proteins interact with a subset of BBSome subunits and promote their association with CCT chaperonins. CCT activity is essential for BBSome assembly, and knockdown of CCT chaperonins in zebrafish results in BBS phenotypes. Many disease-causing mutations found in BBS6, BBS10, and BBS12 disrupt interactions among these BBS proteins. Our data demonstrate that BBS6, BBS10, and BBS12 are necessary for BBSome assembly, and that impaired BBSome assembly contributes to the etiology of BBS phenotypes associated with the loss of function of these three BBS genes.

Allelic variants of human melatonin 1A receptor in patients with familial adolescent idiopathic scoliosis.

Study Design. A genetic study of patients with familial adolescent idiopathic scoliosis. Objectives. The purpose of this study was to evaluate the evidence for linkage on chromosome 4q and determine whether mutations in the gene coding for melatonin receptor are present. Summary of Background Data. Adolescent idiopathic scoliosis is the most common spine deformity arising during childhood, but its cause remains unknown. The fact that adolescent idiopathic scoliosis is often seen in several members of the same family strongly suggests a genetic factor. Recent work by Wise et al provides evidence for linkage of adolescent idiopathic scoliosis at several different chromosome sites, including 4q. In addition, there is some evidence that adolescent idiopathic scoliosis may be related to a disturbance in melatonin metabolism, and the human melatonin-1A receptor is known to be located on chromosome 4q. Methods. Probands having clinically relevant idiopathic scoliosis (Cobb angle >30°) and their relatives were identified. Radiographic confirmation was required for a positive diagnosis. Linkage analysis was performed with 15 microsatellite markers of chromosome 4q spaced at approximately 10-cM resolution and 5 microsatellite markers surrounding the site for human melatonin receptor. The gene for human melatonin receptor was screened for mutations in the coding region using genomic DNA samples by single-strand conformational polymorphism analysis. Amplimers showing a band shift were reamplified and sequenced bidirectionally. Results. There was no evidence for linkage at chromosome 4q in this study population. Twenty-nine individuals demonstrated aberrant single-strand conformation polymorphism band patterns, and sequence evaluation demonstrated six genetic polymorphisms for the gene for human melatonin receptor. These genetic variations were found in both affected and nonaffected individuals, and there was no correlation between gene variants and the phenotype for adolescent idiopathic scoliosis. Conclusions. The results of this study demonstrated no evidence of linkage to chromosome 4q and no mutations in the coding region of the gene for human melatonin receptor. The identification of variants in the human melatonin receptor could provide a useful tool for testing the gene in the predisposition to various other melatonin-related disorders and for clarifying the role of melatonin in adolescent idiopathic scoliosis.

The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Lebers congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel–Gruber syndrome, Joubert syndrome, Senor–Loken syndrome and Bardet–Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffers vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290.

A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations.

Abstractp53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay alows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.

Genome-wide analysis of copy number variants in age-related macular degeneration

Age-related macular degeneration (AMD) is a complex genetic disease, with many loci demonstrating appreciable attributable disease risk. Despite significant progress toward understanding the genetic and environmental etiology of AMD, identification of additional risk factors is necessary to fully appreciate and treat AMD pathology. In this study, we investigated copy number variants (CNVs) as potential AMD risk variants in a cohort of 400 AMD patients and 500 AMD-free controls ascertained at the University of Iowa. We used three publicly available copy number programs to analyze signal intensity data from Affymetrix® GeneChip SNP Microarrays. CNVs were ranked based on prevalence in the disease cohort and absence from the control group; high interest CNVs were subsequently confirmed by qPCR. While we did not observe a single-locus “risk CNV” that could account for a major fraction of AMD, we identified several rare and overlapping CNVs containing or flanking compelling candidate genes such as NPHP1 and EFEMP1. These and other candidate genes highlighted by this study deserve further scrutiny as sources of genetic risk for AMD.

Copy Number Variations and Primary Open-Angle Glaucoma

PURPOSE This study sought to investigate the role of rare copy number variation (CNV) in age-related disorders of blindness, with a focus on primary open-angle glaucoma (POAG). Data are reported from a whole-genome copy number screen in a large cohort of 400 individuals with POAG and 500 age-matched glaucoma-free subjects. METHODS DNA samples from patients and controls were tested for CNVs using a combination of two microarray platforms. The signal intensity data generated from these arrays were then analyzed with multiple CNV detection programs including CNAG version 2.0, PennCNV, and dChip. RESULTS A total of 11 validated CNVs were identified as recurrent in the POAG set and absent in the age-matched control set. This set included CNVs on 5q23.1 (DMXL1, DTWD2), 20p12 (PAK7), 12q14 (C12orf56, XPOT, TBK1, and RASSF3), 12p13.33 (TULP3), and 10q34.21 (PAX2), among others. The CNVs presented here are exceedingly rare and are not found in the Database of Genomic Variants. Moreover, expression data from ocular tissue support the role of these CNV-implicated genes in vision-related processes. In addition, CNV locations of DMXL1 and PAK7 overlap previously identified linkage signals for glaucoma on 5p23.1 and 20p12, respectively. CONCLUSIONS The data are consistent with the hypothesis that rare CNV plays a role in the development of POAG.