Human Molecular Genetics, 2001, Vol. 10, No. 8 865-874
© 2001 Oxford University Press
Positional cloning of a novel gene on chromosome 16q causing Bardet–Biedl syndrome (BBS2)
1Department of Pediatrics and Howard Hughes Medical Institute, University of Iowa, Iowa City, IA 52242, USA, 2Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 3Centre de Genetique Humaine, Institute de Pathologie et de Genetique, Loverval, Belgium, 4Department of Ophthalmology, Children’s Hospital and Harvard Medical School, Boston, MA, USA, 5Bascom Palmer Eye Institute, Miami, FL 33136, USA, 6Valley Children’s Hospital, Madera, CA 93638, USA, 7The Jackson Laboratory, Bar Harbor, ME 04609, USA, 8Medical College of Wisconsin, Milwaukee, WI 53226, USA, 9Chiron Corporation, Emeryville, CA 94608, USA, 10Vision Science Research Program, Toronto Western Research Institute, Toronto, Canada, 11Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38103, USA, 12Department of Medical Genetics, University of Poitiers, Poitiers, France, 13National Human Genome Research Institute, The National Institutes of Health, Bethesda, MD 20892, USA, 14Scheie Eye Institute, Philadelphia, PA 19104, USA, 15Department of Ophthalmology, University of Iowa, Iowa City, IA 52242, USA
Received 29 December 2000; Revised and Accepted 5 February 2001.
DDBJ/EMBL/GenBank accession nos AF342736–AF342739.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bardet–Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder with the primary clinical features of obesity, pigmented retinopathy, polydactyly, hypogenitalism, mental retardation and renal anomalies. Associated features of the disorder include diabetes mellitus, hypertension and congenital heart disease. There are six known BBS loci, mapping to chromosomes 2, 3, 11, 15, 16 and 20. The BBS2 locus was initially mapped to an 18 cM interval on chromosome 16q21 with a large inbred Bedouin kindred. Further analysis of the Bedouin population allowed for the fine mapping of this locus to a 2 cM region distal to marker D16S408. Physical mapping and sequence analysis of this region resulted in the identification of a number of known genes and expressed sequence tag clusters. Mutation screening of a novel gene (BBS2) with a wide pattern of tissue expression revealed homozygous mutations in two inbred pedigrees, including the large Bedouin kindred used to initially identify the BBS2 locus. In addition, mutations were found in three of 18 unrelated BBS probands from small nuclear families.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bardet–Biedl syndrome (BBS) is an autosomal recessive disorder characterized by obesity, pigmentary retinopathy, post-axial polydactyly, mental retardation and hypogonadism (1–4). A high frequency of renal abnormalities is also associated with this disorder (4). Mental retardation in BBS patients is often mild, and some patients have only learning disabilities. Onset of obesity is generally in early infancy and complications including diabetes mellitus and hypertension occur later in life. The associated retinal degeneration is usually severe and most patients become legally blind prior to 20 years of age. A relatively high incidence of BBS is found in the mixed Arab populations of Kuwait and in Bedouin tribes throughout the Middle East, most likely due to the high rate of consanguinity in these populations (5–7) and a founder effect. A relatively high frequency of BBS in Newfoundland has also been reported (4).
BBS has been shown to display a high degree of genetic heterogeneity. This was first demonstrated based on mapping studies performed in large inbred Bedouin kindreds from Israel. The high rate of consanguinity within these groups made it possible to identify inbred kindreds with multiple affected individuals that were large enough for independent linkage analysis. The first BBS locus (now referred to as BBS2) was mapped to chromosome 16 using a large inbred Bedouin kindred (8). Genetic heterogeneity was demonstrated when a second Bedouin BBS kindred did not map to this locus. Subsequent studies in the second Bedouin kindred revealed linkage to chromosome 3 (BBS3) (9). A third Bedouin kindred showed linkage to chromosome 15 (BBS4) (10). To date, studies have demonstrated the existence of six BBS loci (8–14) and a seventh BBS locus has been postulated based on a few small BBS pedigrees that do not appear to map to any of the known loci.
Recently the first BBS gene, MKKS, was identified independently by two groups who hypothesized that mutations in the gene causing McKusick–Kaufman syndome (MKS) would also cause BBS (13,14). MKS is an autosomal recessive disorder characterized by post-axial polydactyly as well as genital and cardiac anomalies. Mutations in MKKS, a putative chaperonin gene on chromosome 20 (15), appear to account for <10% of BBS cases. The mechanism by which mutations in the MKKS gene cause BBS has not been determined.
Interest in the identification of genes causing BBS stems from the pleiotropic nature of the disorder and the hypothesis that identification of BBS genes will provide important insight into biochemical and developmental pathways involved in common complex disorders such as obesity and diabetes mellitus. Since the initial mapping of BBS2 to chromosome 16 in 1993, we have pursued a positional cloning approach aimed at identifying the gene causing this disorder. We now report the identification of a novel gene causing BBS2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clinical data
The clinical features of the large Bedouin kindred (Family 1) have previously been described (8). Briefly, all of the cardinal features of BBS were present in most of the affected members of this family. None of the patients had spastic paraplegia, colobomas or deafness; diagnostic features of Laurence–Moon, Biemond and Alstrom syndromes, respectively. Family 2 consisted of four affected individuals of Kurdish ancestry, all of whom had at least three of the cardinal features of BBS syndrome. Within the two families there was a clear dichotomy between affected and unaffected individuals, in that none of the unaffected individuals had any of the features of BBS. Affected individuals from both families had similar distributions of polydactyly, usually affecting both upper and lower extremities. All but one patient had polydactyly affecting at least three limbs (one individual had two-limb polydactyly). Obesity was more apparent in Family 2 compared with Family 1. Hypogenitalism was apparent in male members of both families. Two patients in Family 1 had unilateral renal hypoplasia. Retinal degeneration was present in all affected members of both families. All affected individuals in the smaller families used in this study met the diagnostic standard of having at least three of the cardinal features of BBS (3,4).
Refinement of the critical interval by genetic analysis
In 1993, linkage studies and haplotype analysis of a large inbred Bedouin kindred mapped the BBS2 locus to an 18 cM region within 16q21 flanked by the markers D16S419 and D16S265 (8). Analysis of additional genetic markers within this region in affected individuals narrowed the critical interval to 
6 cM. The study of unaffected individuals within the pedigree narrowed the critical interval based on the assumption of complete penetrance. One of the unaffected individuals from the large Bedouin family was found to have a recombination event at the distal end of the critical interval that narrowed the distal boundary to a region within the bacterial artificial chromosome (BAC) RP_11-152E5. Analysis of 40 additional DNA samples from unaffected family members identified an unaffected individual who had inherited the affected haplotype in the homozygous state for markers proximal to D16S408. This allowed the exclusion of the region proximal to D16S408. The refined critical interval included an 2 cM region between the markers D16S408 and 152e5-CA.
A second inbred family consisting of four affected individuals was also linked to the BBS2 locus (Fig. 1, Family 2). Genotyping of DNA from three available affected individuals demonstrated that all were homozygous for the same haplotype. This haplotype was not found in the homozygous state in any of the unaffected individuals in the family. Furthermore, the affected haplotype was different to that segregating within the large inbred Bedouin family, suggesting that the mutation in each family is likely to be different. Haplotypes for key individuals from both families are shown in Figure 2.
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Physical mapping
To facilitate the cloning and characterization of the BBS2 gene, we constructed a physical map of the critical interval. An initial physical map that was based on yeast artificial chromosome (YAC) clones allowed for low-resolution localization of genetic markers and candidate genes within the critical interval. Once the genetic interval was refined to the smallest size possible, the physical map was converted to BAC clones. Radiation hybrid mapping using the Stanford G3 mapping panel was used to confirm the order obtained from the BAC-based physical maps and to anchor this region within the Stanford chromosome 16 G3 radiation hybrid map.
Candidate gene identification
The BAC-based physical map was used to select a subset of BACs for sample sequencing at 1x coverage. The sequence information obtained from sample sequencing was combined with that available from the public sequence databases and used for the identification of candidate genes for BBS2. BLASTN analysis was performed against the nr- and dbEST databases that are maintained by NCBI. This allowed us to identify a number of unique genes and Unigene EST clusters. Over 25 unique genes or EST clusters were identified, not including the multiple metallothionein genes that are known to map within the region (Fig. 3). The genes were prioritized for mutation screening based on criteria including (i) availability of known cDNA and/or genomic sequence; (ii) known expression pattern of the gene consistent with the BBS phenotype; and (iii) the availability of any functional information. Attractive candidate genes that mapped within the larger interval defined by an ‘affected-only’ analysis were not excluded, but were deemed to be of lower priority for analysis.
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Mutation screening of candidate genes
The availability of two inbred BBS2 pedigrees that were likely to harbor independent mutations allowed us to conduct a sequencing-based mutation screen of BBS2 candidate genes. PCR amplicons that covered the coding sequence and consensus splice sites for each candidate gene were amplified from genomic DNA from an affected individual of each of the two BBS2 pedigrees and the amplification products were directly sequenced. The DNA sequences generated from the two samples were compared with each other and with sequences in GenBank. Sixteen candidate genes (Fig. 3) were screened without finding any variants that were judged to be pathological.
BBS2 gene structure and expression profile
UniGene EST cluster Hs.24809 was selected for analysis based on its broad expression pattern and on its map position within the smaller candidate interval. The UniGene cluster contained 193 ESTs and six mRNA sequences. When these sequences were assembled into contigs, two distinct, unique contigs were created. Both contigs, each representing a separate gene, mapped to BAC RP_11-5A3 within the BBS2 critical interval.
One of the contigs was found to contain an open reading frame of 1461 bp. A partial gene structure consisting of nine exons was determined. The second contig contained an open reading frame of 2163 bp (721 amino acids). The complete gene structure was ascertained for the second gene, now referred to as BBS2 (GenBank accession no. AF342736). BBS2 was amplified from a human fetal cDNA library and sequenced to confirm the cDNA sequence that was predicted from the EST contig. Of the 193 ESTs from UniGene cluster Hs.24809, 66 were assigned to the BBS2 contig. Comparison of the cDNA sequence with genomic sequence revealed 17 exons (Fig. 4). The tissue distribution of these ESTs suggested that BBS2 was widely expressed. Northern blot analysis confirmed the broad expression pattern of BBS2 and revealed the BBS2 mRNA to be 3.0 kb in size (Fig. 5). This size estimate agrees with the size predicted from the genomic DNA sequence. A minor northern blot band of smaller molecular weight was apparent in tracheal tissue, suggesting possible alternative splicing or cross hybridization. Both genes were screened for mutations. While the mutation screen of the 1461 bp open reading frame of the first gene produced no evidence of pathological variants, a number of mutations were detected in the BBS2 gene.
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BBS2 mutations
Mutation screening of BBS2 revealed candidate mutations in both of the linked BBS2 families that were part of the initial mutation screen. Family 2 was found to harbor a homozygous 1 bp deletion in exon 8 (940delA) in all three affected individuals. This mutation predicts a protein product that is truncated 10 amino acids downstream of the 1 bp deletion at codon 324. The mutation was not found in the homozygous state in unaffected family members. The frameshift has not been detected in any other family or proband, nor in 96 control individuals.
Two sequence variants were detected on the affected chromosome in the large, inbred Bedouin BBS family. An A
G transition at nucleotide position 367 (Ile123Val) was detected in exon 3. Ile123Val is conservative and was therefore not judged to be responsible for the BBS phenotype in the family. A second variant, a TG transversion, was found at nucleotide position 224, predicting a non-conservative valineglycine substitution in exon 2 at residue 75 (Val75Gly). This variant is postulated to be the disease-causing mutation in this family, although we cannot exclude the possibility that both alterations may be necessary to cause the disease. Both DNA sequence variants segregate with the BBS phenotype within the family, in that all affected individuals were homozygous for both sequence variants, all obligate carriers (parents of BBS patients) were heterozygous for both variants, and no unaffected individuals were homozygous for either variant. The Val75Gly mutation was not detected in 50 unrelated Bedouin control samples.
The detection of mutations in the two BBS2 families prompted us to sequence the BBS2 gene in 18 unrelated BBS probands from small nuclear families to identify additional mutations. One proband (Family 3) harbored an exon 8 nonsense mutation at codon 275 (Arg275Ter) in the homozygous state. His parents were found to be carriers and an unaffected sibling inherited two normal alleles. A second exon 8 homozygous nonsense mutation (Arg272Ter) was found in a second proband (Family 4). An unaffected sibling did not inherit the mutation from either parent. Finally, a homozygous 1 bp insertion (1206insA) was observed in exon 10 in a single proband (Family 5). This mutation results in a frameshift that predicts premature termination of translation five amino acids downstream from the insertion. In all, mutations were observed in two of two known BBS2 families, and in three of 18 unrelated BBS probands (Fig. 6).
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Evolutionary conservation
Homology screening of the predicted BBS2 protein against the public sequence databases demonstrates that BBS2 has high similarity to genes from a number of other organisms. Sequence for the mouse ortholog for BBS2 (GenBank accession no. AF342737) was obtained by PCR from a 17-day fetal mouse cDNA library to supplement the sequence that was available from GenBank. The mouse protein is 90% identical and 95% similar (using the BLOSUM62 similarity matrix) to the predicted human BBS2 protein. Sequences for the rat (GenBank accession no. AF342738) and zebrafish (GenBank accession no. AF342739) orthologs of BBS2 were obtained using similar methodology. The rat ortholog was found to be 89% identical and 94% similar at the protein level and the zebrafish ortholog was found to be 74% identical and 84% similar. A comparison of the BBS2 protein sequences between human, mouse, rat and zebrafish is shown in Figure 7. A lower level of similarity was found for Caenorhabditis elegans, Chlamydomonas and Trypanosoma (30–46% identical; 49–57% similar).
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To further investigate the disease-causing nature of the exon 2 Val75Gly variant found in Family 1, sequence was obtained from a number of organisms to determine the level of sequence conservation within this region. Valine was found at this position in human, bovine, rabbit, rat, mouse and zebrafish. In C.elegans, Trypanosoma and Chlamydomonas, the conservative substitution of isoleucine was found at this position. There is a high level of conservation at a number of locations within this region and within the region surrounding the Ile123Val variant in exon 3. However, the isoleucine at codon 123 shows a lower level of conservation, consistent with its postulated assignment as a likely benign sequence variant.
Since the first BBS gene to be identified (MKKS) is a putative chaperonin, we compared BBS2 and known chaperonin or chaperonin-like proteins. No significant similarity was found to any genes with known function by either BLAST analysis or by searching for functional domains within BBS2. We concluded that BBS2 is a novel gene whose function cannot currently be determined by comparison with the primary sequence of other known genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In order to identify the BBS2 gene, we used genetic fine mapping to reduce the size of the BBS2 interval to
6 cM. To further narrow the interval, we searched for unaffected individuals within the extended Bedouin kindred who had the affected haplotype on one chromosome, but were recombinant for the affected haplotype on the homologous chromosome. Two recombinant events in unaffected individuals reduced the candidate interval to 2 cM, facilitating the construction of a BAC contig across the disease region.
The identification of the BBS2 gene was aided by sample sequencing (1x coverage), as well as sequence data from the public Human Genome Project, even though the complete sequence of this interval was not available. Analysis of available sequence resulted in the identification of a number of candidate genes within the narrowest interval. In order to determine which of these genes was the BBS2 gene, we prioritized the genes for mutation screening based on a number of parameters, including sequence homology or putative functional relationship to genes in other known BBS intervals and the pattern of expression. Although this approach yielded a number of high priority candidate genes, none of these genes proved to be mutated in BBS patients. The recent identification of BBS-causing mutations in the MKKS gene provided initial speculation that a chaperonin gene might be found in this interval. A search of the available sequence in the interval failed to identify such a candidate gene. The eventual evaluation of the BBS2 gene was based on the position of this gene within the narrowed disease interval and its broad pattern of expression.
Due to the genetic heterogeneity of BBS, our strategy for mutation screening of candidate genes was to focus the search for mutations by direct sequencing of DNA from an affected individual from each of two inbred families shown to link to the chromosome 16 BBS interval. One of the families was the large Bedouin kindred that allowed the initial mapping and refinement of the 16q21 interval. Focusing the primary mutation search to two inbred BBS2 families conserved resources, but ran the risk of missing the disease-causing gene by too narrowly limiting the mutation search. Nevertheless, sequencing revealed homozygous mutations in the BBS2 gene in each of the two inbred families.
The conclusion that we have correctly identified the BBS2 gene is supported by a number of lines of evidence. First, BBS2 maps to the narrowed disease interval and has a broad pattern of tissue expression, as would be predicted for a gene manifesting pleiotropism. BBS2 was found to have homozygous mutations in the two inbred BBS2 pedigrees, one of which is a frameshift mutation. Each mutation was shown to segregate completely with the disease phenotype in the respective kindred, and neither mutation was found in 96 control individuals. In addition, BBS2 is mutated (both nonsense and frameshift mutations) in three of 18 (17%) of BBS probands from small families, a figure that is consistent with the proportion of BBS2 cases (identified by linkage analysis) reported in the literature (16,17).
The assumption that the Val75Gly variant is the disease-causing mutation in the large Bedouin kindred is supported by the fact that the valine residue at this location is highly conserved across species. It is also the only BBS2 sequence variant identified in this family that is not found in control samples. Of interest is the observation that the Val75Gly mutation is found in complete linkage disequilibrium with the Ile123Val variant in this population. We cannot exclude the possibility that the combination of the Val75Gly and Ile123Val variants in phase is needed for abnormal protein function.
It has been previously hypothesized that the identification of the first BBS gene would lead to the identification of other BBS genes (13,14). In the case of MKKS, this has not yet proven to be the case, as BBS2 has no significant sequence similarity to MKKS and no currently known functional relationship. Despite this fact, we hypothesize that a functional relationship does exist. It is possible that the BBS2 protein plays an unrecognized chaperonin role or is part of a chaperonin complex. Another possibility is that the BBS2 protein is a substrate for MKKS chaperonin function. Additional experiments are needed to test these hypotheses, including the hypothesis that MKKS does indeed function as a chaperonin. Identification of BBS2 as a BBS gene may assist in the identification of other BBS genes. In addition, studies to elucidate the function of BBS2 are expected to help elucidate mechanisms involved in obesity, mental retardation, retinal degeneration and diabetes, as well as limb, kidney and cardiac development.
We have previously sought to characterize phenotypic differences caused by different BBS loci (BBS2, BBS3 and BBS4) based on the phenotype in three large Bedouin kindreds (18). In so doing, we had observed that the BBS2 locus was associated with less severe obesity than other BBS loci. Identification of the BBS2 gene allows us to determine whether the relatively low body mass index in the Bedouin BBS2 kindred was due to the specific Bedouin BBS2 allele (Val75Gly) or is characteristic of patients with mutations in BBS2 in general. Based on the BBS2 family with the 940delA frameshift mutation (Family 2) described in this study, it appears that mutations in BBS2 do not result in milder obesity, but rather that the specific Bedouin allele is a mild allele with respect to the obesity component of the phenotype. The fact that at least some of the phenotypic variability seen in BBS can be explained by allelic heterogeneity is supported by phenotypic differences observed between patients with MKS and BBS due to mutations in the MKKS gene (13–15).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and families
Signed, informed consent was obtained from each patient using protocols approved by the Institutional Review Board at the University of Iowa and collaborating institutions. Genomic DNA was isolated from whole blood according to methods that have been published previously (19).
Physical mapping reagents
YAC DNA was isolated using the DNA-Pure yeast genomic kit (CPG). BAC DNA was prepared via an alkaline lysis protocol using the Wizard Plus Miniprep kit (Promega). The precipitated DNA was washed with 70% EtOH and dried. The DNA pellet was resuspended in 50 µl of ddH20. Plasmid DNA was prepared using a Wizard Plus Miniprep kit (Promega).
Genotyping
PCR amplification for the analysis of short tandem repeat polymorphisms (STRPs) was performed using 40 ng of genomic DNA in 8.4 µl reactions containing 1.25 µl of 10x PCR buffer [100 mM Tris–HCl pH 8.8, 500 mM KCl, 15 mM MgCl2 and 0.01% gelatin (w/v)], 200 µM each of dATP, dCTP, dGTP and dTTP, 2.5 pmol of each primer and 0.2 U of Taq polymerase (Bioline). Samples were subjected to 35 cycles of 94°C for 30 s, 50, 52, 55 or 57°C as required for 30 s and 72°C for 30 s. Amplification products were electrophoresed on 6% polyacrylamide gels containing 7.7 M urea at 60 W for 2 h. The bands were visualized by silver staining (20).
Marker typing for physical mapping was performed on 2% agarose gels using a PCR reaction size of 10 µl. Reaction conditions were as described above with the following exceptions. For markers that proved difficult to amplify using the standard Taq polymerase, we substituted an equal amount of AmpliTaq (Applied Biosystems) along with an initial incubation of the PCR mixture at 94°C for 10 min. For PCR reactions involving YAC, BAC or plasmid DNA, 1–2 ng of DNA was utilized as a template. For colony PCR, a small number of cells were inoculated into 20 
l of ddH2O. One microliter of this suspension was used as a template for the PCR reaction.
Oligonucleotide primers for the STRPs were obtained as MapPairs (Research Genetics). The custom primers required for this study were designed using the PRIMER 0.5 program and synthesized commercially (Research Genetics or Integrated DNA Technologies).
YAC, BAC and cDNA identification
Initially, YACs were identified by searching the Whitehead Institute/MIT Genome Center database (http://www-genome.wi.mit.edu) (21) with sequence tagged sites (STSs) known to be in the 16q21 region. Subsequently YACs and BACs were identified by a PCR-based screening assay of pooled libraries (Research Genetics or Genome Systems) using various STSs within each region. ESTs were identified by a BLASTN (22) search of the public dbEST database available through a web interface at NCBI.
Gene identification and characterization
Raw SCF files from ABI 377 sequencers were imported directly into the Sequencher version 3.1 program (GeneCodes). Contigs were generated by comparing all fragments in a project with the parameters of a 
50 bp overlap in sequence with 80% level of homology. Genomic sequence of BACs from the 16q21 region was submitted to the BLAST server at NCBI for a BLASTN (22) analysis on both the nr and dbEST databases. Any region which gave a significant score (P < 10–5) was also submitted for a BLASTX screen of the SWISS-PROT database. EST sequence was obtained from GenBank and SCF files from the WashU-Merck ftp site (ftp://genome.wustl.edu).
Sequencing
PCR products for sequencing were amplified in a 50 µl reaction size and purified using the Qiaquick PCR Clean-up kit (Qiagen). Plasmid DNA (150 ng in 4.5 µl) or 4.5 µl of purified PCR product was used as a template for sequencing reactions. One microliter of primer (20 pmoles) and 4.5 µl of terminator sequencing mix (Amersham Pharmacia Biotech and Applied Biosystems) were added for a final reaction volume of 10 µl. Cycling conditions were performed as specified by the manufacturer. The sequencing reactions were precipitated in the presence of linear acrylamide and resuspended in 2 µl of loading buffer. The reactions were analyzed on an ABI 377 using a run time of 3 h.
Mutation detection and confirmation
Mutation detection was performed by direct sequencing of PCR amplification products. The primers used to amplify the BBS2 gene are shown in Table 1. Control samples were assayed by single strand conformation polymorphism (SSCP) analysis. For SSCP, PCR products were electrophoresed on SSCP gels (5 ml of glycerol, 5 ml of 5x TBE, 12.5 ml of 37.5:1 acrylamide/bis and 77.5 ml of ddH2O) for 3–4 h in 0.25x TBE at room temperature. Gels were silver stained as described above. Abnormal variants were sequenced and compared with a control sample to detect any changes from that of the normal sequence.
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Northern blot analysis
Human multiple tissue northern (MTN) blots I and III and human fetal MTN blot II were obtained from Clontech. The blots were hybridized with a 300 bp DNA probe derived from the 3' untranslated region (UTR) of the human BBS2 gene. The probe was amplified by PCR using the BBS2-forward (5'-AATAACCTTGGTGAGTTGTAC-3') and BBS2-reverse (5'-ATACAAATGGGCAATTCTGAT-3') primers. The probe was labeled with 32P-dCTP using Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech). Hybridization and autoradiography were performed as described previously (23). The blots were stripped of radioactivity and re-hybridized with a cDNA probe for ß-actin (Clontech) to assess equal loading of the RNA.
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ACKNOWLEDGEMENTS |
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We are grateful to the patients and their families for their participation in this study. We also thank Michael Andrews, Gretel Beck and Christine Hartzler for technical assistance, Terry Braun, Kirk Mykytyn and Mythrei Shastri for helpful discussions and Denise Crouch for administrative assistance. This work was supported by the following grants and organizations: NIH grant R01-EY-11298 (E.M.S. and V.C.S.), Foundation Fighting Blindness (S.G.J., E.M.S. and V.C.S.), Carver Endowment for Molecular Ophthalmology (E.M.S. and V.C.S.) and Research to Prevent Blindness, New York, NY (unrestricted grant to the Department of Ophthalmology, University of Tennesse, Memphis). V.C.S. is an Associate Investigator for the Howard Hughes Medical Institute.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 319 335 6898; Fax: +1 319 335 7588; Email: val-sheffield@uiowa.edu
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REFERENCES |
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1 Bardet, G. (1920) Sur un syndrome d’obesite infantile avec polydactylie et retinite pigmentaire (contribution a l’etude des formes cliniques de l’obesite hypophysaire). PhD Thesis, Paris, France.
2 Biedl, A. (1922) Ein Geschwisterpaar mit adipose-genitaler Dystrophie. Dtsch. Med. Woschenschr., 48, 1630.
3 Schachat, A.P. and Maumenee, I.H. (1982) Bardet-Biedl syndrome and related disorders. Arch. Ophthalmol., 100, 285–288.
4 Green, J.S., Parfrey, P.S., Harnett, J.D., Farid, N.R., Cramer, B.C., Johnson, G., Heath, O., McManamon, P.J., O’Leary, E. and Pryse-Phillips, W. (1989) The cardinal manifestations of Bardet-Biedl syndrome, a form of Laurence-Moon-Biedl syndrome. New Engl. J. Med., 321, 1002–1009.[Abstract]
5 Farag, T.I. and Teebi, A.S. (1988) Bardet-Biedl and Laurence-Moon syndromes in a mixed Arab population. Clin. Genet., 33, 78–82.[Web of Science][Medline]
6 Farag, T.I. and Teebi, A.S. (1989) High incidence of Bardet-Biedl syndrome among the Bedouin [letter]. Clin. Genet., 36, 463–464.[Web of Science][Medline]
7 Teebi, A.S. (1994) Autosomal recessive disorders among Arabs: an overview from Kuwait. J. Med. Genet., 31, 224–233.
8 Kwitek-Black, A.E., Carmi, R., Duyk, G.M., Buetow, K.H., Elbedour, K., Parvari, R., Yandava, C.N., Stone, E.M. and Sheffield, V.C. (1993) Linkage of Bardet-Biedl syndrome to chromosome 16q and evidence for non-allelic genetic heterogeneity. Nature Genet., 5, 392–396.[Web of Science][Medline]
9 Sheffield, V.C., Carmi, R., Kwitek-Black, A., Rokhlina, T., Nishimura, D., Duyk, G.M., Elbedour, K., Sunden, S.L. and Stone, E.M. (1994) Identification of a Bardet-Biedl syndrome locus on chromosome 3 and evaluation of an efficient approach to homozygosity mapping. Hum. Mol. Genet., 3, 1331–1335.
10 Carmi, R., Rokhlina, T., Kwitek-Black, A.E., Elbedour, K., Nishimura, D., Stone, E.M. and Sheffield, V.C. (1995) Use of a DNA pooling strategy to identify a human obesity syndrome locus on chromosome 15. Hum. Mol. Genet., 4, 9–13.
11 Leppert, M., Baird, L., Anderson, K.L., Otterud, B., Lupski, J.R. and Lewis, R.A. (1994) Bardet-Biedl syndrome is linked to DNA markers on chromosome 11q and is genetically heterogeneous. Nature Genet., 7, 108–112.[Web of Science][Medline]
12 Young, T.L., Penney, L., Woods, M.O., Parfrey, P.S., Green, J.S., Hefferton, D. and Davidson, W.S. (1999) A fifth locus for Bardet-Biedl syndrome maps to chromosome 2q31. Am. J. Hum. Genet., 64, 900–904.[Web of Science][Medline]
13 Katsanis, N., Beales, P.L., Woods, M.O., Lewis, R.A., Green, J.S., Parfrey, P.S., Ansley, S.J., Davidson, W.S. and Lupski, J.R. (2000) Mutations in MKKS cause obesity, retinal dystrophy and renal malformations associated with Bardet-Biedl syndrome. Nature Genet., 26, 67–70.[Web of Science][Medline]
14 Slavotinek, A.M., Stone, E.M., Mykytyn, K., Heckenlively, J.R., Green, J.S., Heon, E., Musarella, M.A., Parfrey, P.S., Sheffield, V.C. and Biesecker, L.G. (2000) Mutations in MKKS cause Bardet-Biedl syndrome. Nature Genet., 26, 15–16.[Web of Science][Medline]
15 Stone, D.L., Slavotinek, A., Bouffard, G.G., Banerjee-Basu, S., Baxevanis, A.D., Barr, M. and Biesecker, L.G. (2000) Mutation of a gene encoding a putative chaperonin causes McKusick-Kaufman syndrome. Nature Genet., 25, 79–82.[Web of Science][Medline]
16 Bruford, E.A., Riise, R., Teague, P.W., Porter, K., Thomson, K.L., Moore, A.T., Jay, M., Warburg, M., Schinzel, A., Tommerup, N. et al. (1997) Linkage mapping in 29 Bardet-Biedl syndrome families confirms loci in chromosomal regions 11q13, 15q22.3-q23, and 16q21. Genomics, 41, 93–99.[Web of Science][Medline]
17 Woods, M.O., Young, T.L., Parfrey, P.S., Hefferton, D., Green, J.S. and Davidson, W.S. (1999) Genetic heterogeneity of Bardet-Biedl syndrome in a distinct Canadian population: evidence for a fifth locus. Genomics, 55, 2–9.[Web of Science][Medline]
18 Carmi, R., Elbedour, K., Stone, E.M. and Sheffield, V.C. (1995) Phenotypic differences among patients with Bardet-Biedl syndrome linked to three different chromosome loci. Am. J. Med. Genet., 59, 199–203.[Web of Science][Medline]
19 Nishimura, D.Y., Swiderski, R.E., Alward, W.L., Searby, C.C., Patil, S.R., Bennet, S.R., Kanis, A.B., Gastier, J.M., Stone, E.M. and Sheffield, V.C. (1998) The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nature Genet., 19, 140–147.[Web of Science][Medline]
20 Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem., 196, 80–83.[Web of Science][Medline]
21 Hudson, T.J., Stein, L.D., Gerety, S.S., Ma, J., Castle, A.B., Silva, J., Slonim, D.K., Baptista, R., Kruglyak, L., Xu, S.H. et al. (1995) An STS-based map of the human genome. Science, 270, 1945–1954.[Abstract]
22 Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215, 403–410.[Web of Science][Medline]
23 Swiderski, R.E., Ying, L., Cassell, M.D., Alward, W.L., Stone, E.M. and Sheffield, V.C. (1999) Expression pattern and in situ localization of the mouse homolog of the human MYOC (GLC1A) gene in adult brain. Brain Res. Mol. Brain Res., 68, 64–72.[Medline]
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|
|
|
|
|
|
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|
|
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|
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|
|
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|
|
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