Clin Genet 2010: 78: 424 431 Printed in Singapore. All rights reserved Original Article 2010 John Wiley & Sons A/S CLINICAL GENETICS doi: 10.1111/j.1399-0004.2010.01481.x A founder mutation in BBS2 is responsible for Bardet-Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders Innes AM, Boycott KM, Puffenberger EG, Redl D, MacDonald IM, Chudley AE, Beaulieu C, Perrier R, Gillan T, Wade A, Parboosingh JS. A founder mutation in BBS2 is responsible for Bardet-Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders. Clin Genet 2010: 78: 424 431. John Wiley & Sons A/S, 2010 Bardet-Biedl syndrome (BBS) is a multisystem genetically heterogeneous disorder, the clinical features of which are largely the consequence of ciliary dysfunction. BBS is typically inherited in an autosomal recessive fashion, and mutations in at least 14 genes have been identified. Here, we report the identification of a founder mutation in the BBS2 gene as the cause for the increased incidence of this developmental disorder in the Hutterite population. To ascertain the Hutterite BBS locus, we performed a genome-wide single nucleotide polymorphism (SNP) analysis on a single patient and his three unaffected siblings from a Hutterite family. The analysis identified two large SNP blocks that were homozygous in the patient but not in his unaffected siblings, one of these regions contained the BBS2 gene. Sequence analysis and subsequent RNA studies identified and confirmed a novel splice site mutation, c.472-2a>g, in BBS2. This mutation was also found in homozygous form in three subsequently studied Hutterite BBS patients from two different leuts, confirming that this is a founder mutation in the Hutterite population. Further studies are required to determine the frequency of this mutation and its role, if any, in the expression of other ciliopathies in this population. AM Innes a,kmboycott b, EG Puffenberger c,d,dredl a, IM MacDonald e, AE Chudley f, C Beaulieu a, R Perrier a, T Gillan g,awade h and JS Parboosingh a a Department of Medical Genetics, Alberta Children s Hospital and University of Calgary, Calgary, Alberta, Canada, b Department of Pediatrics, University of Ottawa and Children s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada, c Clinic for Special Children, Strasburg, PA, USA, d Franklin and Marshall College, Lancaster, PA, USA, e Department of Ophthalmology, University of Alberta, Edmonton, Alberta, Canada, f Section of Genetics and Metabolism, Children s Hospital and Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, Canada, g Department of Pathology and Laboratory Medicine, Vancouver General Hospital, University of British Columbia, Vancouver, British Columbia, Canada, and h Department of Pediatrics, Alberta Children s Hospital and University of Calgary, Calgary, Alberta, Canada Key words: Bardet-Biedl syndrome Hutterite identity-by-descent mapping SNP microarray Corresponding author: Dr Kym M Boycott, Department of Genetics, Children s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada. Tel.: 613 737 7600x3223; fax: 613 738 4822; e-mail: kboycott@cheo.on.ca Received 12 March 2010, revised and accepted for publication 28 May 2010 424
BBS2 is the gene responsible for Bardet-Biedl syndrome in the Hutterite population Bardet-Biedl syndrome (BBS; MIM 209900) is a complex autosomal recessive condition characterized by retinal degeneration, polydactyly, renal disease, hypogonadism, obesity, dysmorphic features and variable cognitive impairment [reviewed by Zaghloul and Katsanis (1)]. Although this condition is typically rare in outbred populations, it is seen in some regions, including Kuwait and Newfoundland, with a frequency approaching 1 in 13,500 (2) and 1 in 18,000 (3), respectively. Currently, at least 14 causative genes are associated with BBS, with only mutations in BBS1 and BBS10 occurring in greater than 20% of affected patients. For approximately 20% of well-studied patients with a clinical diagnosis of BBS, the molecular basis is not known. This genetic heterogeneity complicates clinical molecular testing which is critical for early diagnosis, carrier testing and, if requested, prenatal testing. As many mutations in BBS genes are founder mutations, it is important to study the molecular basis of BBS in isolated populations. The Hutterite brethren is an isolated population currently residing on the North American prairies who trace their origins to 16th century Europe (4). The 40,000 modern Hutterites are descendants of fewer than 100 common founders (5). As such, there is an increased frequency of autosomal recessive conditions in this population. In the late 1800s, the Hutterites formed three distinct groups (leuts) which have essentially remained in genetic isolation from each other to the present day. We have identified over 30 autosomal recessive conditions in this population (6), and new conditions continue to be recognized. Given the characteristics of the Hutterite population, rare alleles causing autosomal recessive conditions were probably introduced into the population by one or at most a few ancestors. This fact has facilitated the mapping and identification of a number of autosomal recessive genes in this population by identity-by-descent mapping [reviewed by Boycott et al. (6)]. This approach can also be used to quickly prioritize the analysis of candidate genes for autosomal recessive conditions which show significant genetic heterogeneity. BBS has been recognized in the Hutterite population; however, the molecular basis of BBS in this population was not known. BBS is the prototypic non-motile ciliopathy (1), a class of genetic conditions in which the disease process affects non-motile, primary cilia. BBS is one of several ciliopathies observed in the Hutterite population; in the course of investigation of this group of conditions in the Hutterites, we ascertained a patient with classic BBS and proceeded to identify the underlying genetic basis using identity-by-descent mapping in a single affected individual to direct the mutation analysis. Materials and methods Patients and samples The study protocol was approved by the University of Calgary Research and Ethics Board, and informed consent was obtained from all participants. The proband is a 19-year-old Hutterite man (Patient 1). He was born to a 31-year-old G5P3 mother after a pregnancy complicated by hypertension. Antenatal ultrasound had revealed intrauterine growth retardation and multicystic kidneys. He was born via emergency C section at 31 weeks for fetal distress. Apgar scores were 3 at 1 min and 7 at 5 min. Birth weight was 1020 g (10th percentile). The Department of Medical Genetics was consulted after delivery for evaluation of congenital anomalies that included postaxial polydactyly of both feet and the left hand, syndactyly of the third and fourth fingers of both hands, enlarged cystic kidneys and hypospadias with chordee. Karyotype was normal 46,XY. Given the presence of polydactyly with renal and genital anomalies in a Hutterite child, a tentative diagnosis of Meckel syndrome [Meckel Gruber syndrome (MKS); MIM 249000] was applied, as other mild or atypical Hutterite children with MKS had been reported (7); however, computed tomography neuroimaging was normal. BBS was considered in the differential diagnosis at 1 month of age when the child s clinical course was somewhat milder than even what was then known of the Hutterite variant of MKS, yet the diagnosis was considered unlikely. The parents were counseled regarding a possible diagnosis of MKS, and the child was not seen by the Genetics service for many years. The family was recontacted when the patient was 14 years old in the context of a study to further characterize the Hutterite MKS, which had been reclassified as a Joubert-syndrome-related disorder (JSRD) and presumed to be a human ciliopathy (8). When reassessed, he clinically had BBS, a different ciliopathy. He had had surgical intervention for his limb and genital anomalies. Outside the newborn period, he had not had ongoing follow-up to monitor his renal function. He had made slow but steady developmental progress; he walked at 2 years of age, had repeated first grade, but at age 14 was in grade 7 and doing grade-level work. His vision had deteriorated since age 12 and he had clinical evidence of night blindness and 425
Innes et al. tunnel vision. Ophthalmologic assessment confirmed findings typical of retinitis pigmentosa (RP). At age 14, he was obese (body mass index of 35.6 kg/m 2 ) with a weight of 96.9 kg (greater than the 95th percentile) and height of 165.0 cm (25th 50th percentile). Physical examination was remarkable for dysmorphism (Fig. 1a) and cutaneous syndactyly between the second and third toes bilaterally (Fig. 1b). The combination of RP, obesity, and limb, genital and renal anomalies provided a firm clinical diagnosis of BBS. Investigation by nephrology identified chronic renal failure, polyuria, polydipsia, hypertension and bilateral renal pelviectasis. By 19 years of age, his visual impairment was significant with only light perception in each eye. He also had myopia with a refraction error of 6.00 + 1.5 90 in the right eye and 5.00 + 1.25 90 in the left eye. The patient s parents are Dariusleut Hutterite. He is the youngest of four living siblings, with three older healthy brothers. His mother had experienced a previous miscarriage at 19 weeks; the fetus had polydactyly of the hands and a normal karyotype. His mother s sister has a son with similar phenotypic features (obesity, polydactyly, visual impairment and learning disability). Despite this family history, DNA for the initial studies was only available from the patient, his three unaffected siblings and his parents. Genotyping and mapping Blood samples were obtained with informed consent from the family. Genomic DNA was purified from peripheral blood leukocytes according to standard techniques. Identity by descent was assumed for the disease-causing mutation, thus a region of homozygosity was expected to surround the disease gene in the patient but not in his unaffected siblings. The patient, his parents and unaffected siblings were assessed for homozygosity at each of the loci reported to be associated with BBS at the beginning of this study (BBS1 11 ). Microsatellite markers from the Linkage Mapping set v2.1 HD5 (Applied Biosystems, Inc., Foster City, CA) flanking each of these loci were amplified according to the manufacturer s recommendations and fragment sizes determined after capillary electrophoresis on an ABI 3130 genetic analyzer using GeneMapper software (Applied Biosystems, Inc., Foster City, CA). To assess for regions of homozygosity, region-specific haplotypes were constructed manually using informative markers, and phase was assigned by minimizing the number of recombinants. Subsequent, genomewide analysis was performed with DNA from the patient and his three siblings using the Human Mapping 10K (Xba 2.0) single nucleotide polymorphism (SNP) array (Affymetrix, Inc., Santa Clara, CA) as described elsewhere (9). Data were analyzed using Microsoft Excel spreadsheets (Microsoft Corporation, Redmond, WA, USA) custom-formatted at the Clinic for Special Children. SNP positions came from Affymetrix genome annotation files, and genotype data came from the Affymetrix GeneChip Human Mapping 10K Xba array. Data analysis was designed to identify genomic regions that were identical by descent in the patient but not in his unaffected siblings. This analysis assumes mutation and locus homogeneity based on the population history of the Hutterites. Mutation identification Primers were designed to assess the coding regions and intron exon boundaries of the prioritized a b c Fig. 1. Clinical features of Hutterite Bardet-Biedl syndrome patients. (a) Facial photograph of Patient 1 at 14 years of age demonstrating round facies, deeply set eyes, short, down-slanting palpebral fissures, bulbous nasal tip, smooth philtrum and thin vermillion of the upper and lower lips. Photographs of the feet of Patient 1 post repair of bilateral postaxial polydactyly (b) demonstrating brachydactyly and cutaneous syndactly between the second and third toes and Patient 2 (c) demonstrating postaxial polydactyly. 426
BBS2 is the gene responsible for Bardet-Biedl syndrome in the Hutterite population gene (BBS2 ; NM 031885.3) using the program Oligo (Molecular Biology Insights, Inc., Cascade, CO). Polymerase chain reaction (PCR) and bidirectional sequencing was performed on DNA samples from the patient, a parent and unaffected control. Primer sequences and reaction conditions are available upon request. Amplified products were sequenced in both directions using the Big Dye Terminator v1.1 cycle sequencing chemistry (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer s recommendation. Sequencing reactions were electrophoresed using an ABI 3130 genetic analyzer (Applied Biosystems, Inc., Foster City, CA) and analysis was performed using Mutation Surveyor (SoftGenetics LLC, State College, PA). RNA analysis RNA was immediately extracted from whole blood using standard lysis buffer and RNeasy mini kit (Qiagen, Inc., Hilden, Germany). Complementary DNA (cdna) was generated using the Super- Script III first-strand synthesis system with an Oligo dt primer (Invitrogen, Inc., Carlsbad, CA), and amplification was performed with primers located within exon 2 (5 -tcttttggcttatgaygtctacaat- 3 ) and at the boundary between exons 7 and 8 (5 -tcacttcgagcatcaaccttcc-3 ) of the BBS2 gene. Agarose gel electrophoresis was used to separate the PCR-amplified fragments according to size. Abnormally sized products were purified from the gel and sequenced using the amplification primers. Results Identity-by-descent mapping Given the characteristics of the Hutterite population, a region of homozygosity surrounding the disease gene was expected to identify the causative locus. Genotyping using microsatellite markers flanking each of the known BBS loci was performed in the affected patient, his parents and each of his three unaffected brothers (results for the closest flanking markers are shown in Table S1). The patient was homozygous for six markers defining an approximate 34 Mb region surrounding BBS3 ; however, two of his unaffected brothers were also homozygous for this entire region. Heterozygosity at both flanking markers was detected in the affected patient at the loci for BBS5, 6, 10 and 11 with markers that varied in distance from the causative gene between 1.53 and 9.7 Mb. The patient was also heterozygous at flanking markers 0.15 and 0.45 Mb away from the BBS8 locus but shared homozygosity with all three siblings at an intragenic marker. Homozygosity was detected in one flanking marker for the remaining loci, BBS1, 2, 4, 7 and 9. ForBBS1, 2, 7 and 9, at least one unaffected sibling shared homozygosity for each of the markers homozygous in the patient. No sibling shared homozygosity at the proximal marker for BBS4 ; however, the patient is heterozygous for the opposite flanking marker 0.07 Mb away from BBS4. These results reduced the likelihood that the mutation causing BBS in the Hutterites was in one of the known BBS genes. In the absence of homozygous markers surrounding one of the known loci, genome-wide SNP analysis was performed using a 10K SNP (Xba 2.0) microarray on the patient and his three unaffected siblings. Fourteen regions of contiguous homozygosity of 25 or more SNPs were identified in the patient. All but two regions on chromosomes 1 and 16 were shared by one or more unaffected sibling (Fig. 2). The homozygous region on chromosome 16 is 8.6 Mb, bounded by the SNPs rs1110493 and rs1364153. Integrating the microsatellite data with the SNP data identifies a smaller 1 6-Mb region of homozygosity. This region includes the previously excluded BBS2 gene. In analyzing our microsatellite data, we excluded four loci with shared homozygosity at a single informative flanking marker (Table S1). Shared homozygosity with two unaffected siblings reduces the likelihood that two of these loci, BBS1 and 9, contain the disease-causing gene as two recombination events are less likely than one, leaving BBS2 and 7 to assess. The distance between the homozygous markers shared between the patient and a single sibling and the BBS2 and 7 genes is 0.96 and 5.44 Mb, respectively, and thus, the probability of a recombination event between the marker and the disease gene would be greater for BBS7. Therefore, BBS2 would not have been identified as the most likely candidate based solely upon the microsatellite data due to the presence of a recombination event in the unaffected sibling 207 kb away from the gene (Fig. 3). Mutation analysis Sequence analysis of the coding region and intron exon boundaries of the BBS2 gene in the patient revealed the presence of a homozygous splice variant c.472-2a>g (IVS3-2A>G) (NM 031885.3). Amplification of the patient s cdna using primers complementary to BBS2 exon 2 and to the exon 7 and 8 boundary detected the presence of two abnormally sized products 427
Innes et al. Fig. 2. Genetic mapping studies. A 10K single nucleotide polymorphism microarray was used to identify regions of homozygosity in the patient (red peaks). These results were overlaid with the regions of haplo-identity between the patient and the unaffected siblings (yellow peaks). This analysis yielded two candidate regions: one on chromosome 1 and one on chromosome 16 (arrows). (Fig. 4a). Sequence analysis of these two products confirmed the deletion of exon 4 in one abnormal transcript and of exons 3 and 4 in the other (Fig. 4b). Both mutations result in in-frame deletions in the mrna; translation of these transcripts is predicted to result in smaller proteins excluding amino acids 158 178 (21 amino acids) and 117 178 (63 amino acids), respectively. Sequencing of family members confirmed the expected pattern of heterozygosity in each of the parents and lack of homozygosity for the splice variant in the unaffected siblings (Fig. 3). This change was not seen as a polymorphic variant in dbsnp and was absent in 160 general population control chromosomes. These findings support the pathogenic effect of the c.472-2a>g substitution and extend the spectrum of disease-causing mutations in this gene. The sibling who shared homozygosity for the BBS2 flanking marker D16S3057, 207 kb away, was found to be heterozygous for the mutation, confirming his unaffected status and the recombination event (Fig. 3). Identification of additional patients and haplotype analysis After the identification and confirmation of the mutation in this Dariusleut patient, we had the opportunity to study three additional Hutterite patients with a possible clinical diagnosis of BBS. The first was a 6-month-old male child born to second-cousin Schmiedeleut parents (Patient 2). This was their first pregnancy; a fraternal twin sister is clinically normal. There was a positive family history of learning disability in the family, but no one with similar congenital anomalies. This child was born at 35 weeks of gestation with a birth weight of 2550 g (50th percentile) and a head circumference of 32.5 cm (50th percentile). He had bilateral postaxial polydactyly of the hands and feet (Fig. 1c) and in the neonatal period was found to have a small atrial septal defect, a moderatesized unrestrictive membranous ventricular septal defect and pulmonary valve stenosis. An ophthalmologic examination in the immediate newborn period was normal. A working differential diagnosis of McKusick Kaufmann syndrome and BBS was applied. He was found to be homozygous for the Hutterite BBS2 mutation. On follow-up at 6 months of age, length and weight were both at the 90th 97th percentile, his penis was hidden in the suprapubic fat pad and both testes were descended. Renal ultrasound and further ophthalmological assessment are underway. The other two patients are Dariusleut Hutterite brothers assessed in the Department of Ophthalmology at the ages of 6 and 2 years (Patients 3 and 4). The older brother had postaxial polydactyly of each foot and cystic kidney disease. His best corrected visual acuity was 20/200, right eye, and 20/100, left eye, and high myopia ( 11.25 + 5.00 97, right eye, and 11.25 + 5.00 78, left eye) was documented. Visual fields were bilaterally diminished by confrontational field 428
BBS2 is the gene responsible for Bardet-Biedl syndrome in the Hutterite population Fig. 3. Haplotype analysis showed the presence of recombination in a 207-kb region between D16S3057 and the BBS2 gene in an unaffected sibling resulting in shared homozygosity at this locus with the patient. Segregation analysis of the BBS2 c.472-2a>g mutation confirmed the expected pattern of heterozygosity in each of the parents and lack of homozygosity for the splice variant in the unaffected siblings. Marker location is based on build hg18 of the University of California Santa Cruz Genome Browser database (http://genome.ucsc.edu). (a) (b) Fig. 4. RNA analysis reveals the presence of two abnormal transcripts. (a) Reverse transcriptase-polymerase chain reaction in the affected individual identifies two deletion products. Primers located in exon 2 and at the boundary between exons 7 and 8 were used to amplify complementary DNA from the patient. (b) Sequencing of the two transcripts confirms the disruption of splicing resulting in the production of smaller transcripts, one lacking exon 4 (63 bp) and one lacking exons 3 and 4 (198 bp). testing. His electroretinogram showed significant involvement of the rod as well as the cone system. The younger brother could fix and follow test objects, had a fine horizontal jerk nystagmus and was moderately myopic ( 5.00 in both eyes). Both boys were homozygous for the Hutterite BBS2 mutation. The four patients and available family members were analyzed using markers from the region to establish the haplotype surrounding the mutation. A common haplotype shared among the four BBS patients was present as a homozygous block spanning a distance of 1 6 Mb (data not shown). Discussion In this study, we applied whole-genome identityby-descent analysis using SNP microarray technology in a single family with one affected member 429
Innes et al. to rapidly identify the most likely BBS gene for mutation analysis; this after a targeted approach using microsatellite markers was unsuccessful. An 8.6 Mb homozygous block of SNPs that spanned the BBS2 locus on chromosome 16 was recognized and led to the subsequent analysis and identification of a novel splice site mutation in BBS2 that accounts for all known cases of this condition in the Hutterite population to date. A standard clinical molecular approach to the identification of the molecular etiology of BBS in the Hutterite proband of this study would have been challenging, as over 14 genes are known to be associated with BBS [reviewed by Zaghloul and Katsanis (1)]. Sequencing of the two most common genes BBS1 and BBS10 (collectively accounting for 40% of BBS cases) would have been unrevealing. BBS2 is the third most frequent BBS gene, but one of five BBS genes that are each associated with 4 8% of BBS cases (1). As a result, we opted to look for regions of identity by descent to prioritize the analysis. This approach using information from a 10K SNP microarray and only one affected patient was successful, although the importance of his unaffected siblings cannot be overlooked as the additional information from the siblings reduced the candidate regions from 14 to 2. Our initial analysis of this family with microsatellite markers at variable distances from the BBS genes highlights an important point: recombination between a flanking marker and the disease-causing locus leading to marker heterozygosity in the patient or homozygosity in an unaffected sibling can result in the inappropriate exclusion of loci. Therefore, the density and proximity of the markers to the disease locus is the key, and the rapidity and ease with which microarray technology can provide this information suggests that this is the platform of choice. This inexpensive approach has been successfully applied to the clinical molecular diagnosis of known genetically heterogeneous conditions in other isolated populations such as the Amish (10 12) and Mennonites (13) and more recently successfully applied to consanguineous families (14, 15). The identified mutation produced two abnormal mrna transcripts: one lacking exon 4 and the second lacking exons 3 and 4. The transcript lacking exon 4 could be predicted from the location of the mutation in the splice acceptor site of intron 3. However, the transcript lacking exons 3 4 was not predicted. Splicing is a highly complex nonlinear process; there are many different sequence elements that define the splicing code (16), and weak splice sites that lead to leaky splicing and alternate transcripts are well recognized (17). Our results suggest that the abnormal splicing of intron 3 may have a more global effect on processing of the BBS2 mutant transcript. In this report, we have identified four patients homozygous for a BBS2 mutation and a surrounding common haplotype, confirming that this is a founder mutation in the Hutterite population. Because the BBS2 mutation is present in at least two of the three Hutterite leuts, the age of this mutation is more than 135 years based on the date of the establishment of the endogamous leuts in North America (4). The proband has a similarly affected cousin, and we have seen at least one other classically affected Schmiedeleut patient from whom DNA is not currently available. This suggests that BBS is not rare in the Hutterite population and with at least six known affected patients in a population of approximately 40,000, it is possible that the frequency of BBS among the Hutterites is among the highest of any population. Additional support for the high frequency of BBS in the Hutterite population comes from the identification of one carrier in a cohort of 22 unrelated healthy Schmiedeleut individuals from South Dakota, suggesting a carrier frequency of approximately 4.5% (Carole Ober, personal communication). For Hutterite patients with clinical features suggestive of BBS, a simple mutation test is now clinically available to facilitate timely confirmation of diagnosis, anticipatory care and genetic counseling for families. In the three Hutterite patients who have had detailed ophthalmological evaluation, myopia is a consistent clinical feature. This is in distinction to a previous report showing no association between BBS2 and myopia (18). However, that paper studied 10 individuals from a single family with a p.ile234val BBS2 mutation, ranging in age from 4 to 37, and as the authors indicate, it would be important to analyze other families before making genotype phenotype correlations. BBS is one of the several known or presumed ciliopathies we have seen in the Hutterite population. The proband was initially ascertained as part of a clinical and genetic study on a Hutterite JSRD, a condition that overlaps significantly with MKS (8), and studies are ongoing to identify the molecular basis of this syndrome. Several Hutterite patients are also known with the classic autosomal recessive renal ciliopathy juvenile nephronophthisis (JNPHP). Some of these patients are homozygous for the known recurrent deletion of NPHP1 and others are not, supporting genetic heterogeneity of JNPHP in the Hutterites (6). In addition, we have seen three Hutterite patients with a rare recessive condition known as cranioectodermal dysplasia (19), a disorder of unknown etiology that 430
BBS2 is the gene responsible for Bardet-Biedl syndrome in the Hutterite population has many features including retinal degeneration, hepatorenal disease and skeletal anomalies in common with other ciliopathies. Usher syndrome, characterized by retinal disease and hearing loss, is also seen in the Hutterite population and is genetically heterogeneous with some cases due to mutations in PCDH15 (20). Finally, a gene, TRIM32, identified in the Hutterite brethren as causative of a mild autosomal recessive muscular dystrophy (LGMD2H) has also been associated with a single family with BBS (21), suggesting that TRIM32 may interact with other ciliary genes. It is becoming increasingly clear that alleles at different ciliary loci can interact to modify the phenotypes of these disorders (22, 23). As the primary mutations underlying all the known ciliopathies in the Hutterite population are identified, it will be of interest to determine their distribution among affected Hutterite patients and the Hutterite population in general. Supporting Information The following Supporting information is available for this article: Table S1. Exclusion of known BBS loci by microsatellite marker analysis. Additional Supporting information may be found in the online version of this article. Please note: Wiley-Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Acknowledgements We thank the families for their enthusiastic participation in this study. We would like to acknowledge Linda MacLaren for clinical support. This work was supported by an Alberta Children s Hospital Foundation Grant. K. B. is supported by a Canadian Institutes of Health Research (CIHR), Institute of Genetics Clinical Investigatorship. C. B. and D. R. were supported by the CIHR Training Program in Genetics, Child Development, and Health at the University of Calgary. Conflict of interest The authors have no conflict of interest to declare. References 1. Zaghloul NA, Katsanis N. Mechanistic insights into Bardet- Biedl syndrome, a model ciliopathy. J Clin Invest 2009: 119: 428 437. 2. Farag TI, Teebi AS. High incidence of Bardet Biedl syndrome among the Bedouin. Clin Genet 1989: 36: 463 464. 3. Moore SJ, Green JS, Fan Y et al. Clinical and genetic epidemiology of Bardet-Biedl syndrome in Newfoundland: a 22- year prospective, population-based, cohort study. Am J Med Genet A 2005: 132: 352 360. 4. Hostetler J. History and relevance of the Hutterite population for genetic studies. Am J Med Genet 1985: 22: 453 462. 5. Nimgaonkar VL, Fujiwara TM, Dutta M et al. Low prevalence of psychoses among the Hutterites, an isolated religious community. Am J Psychiatry 2000: 157: 1065 1070. 6. Boycott KM, Parboosingh JS, Chodirker BN et al. Clinical genetics and the Hutterite population: a review of Mendelian disorders. Am J Med Genet A 2008: 146A: 1088 1098. 7. Schurig V, Bowen P, Harley F et al. The Meckel syndrome in the Hutterites. Am J Med Genet 1980: 5: 373 381. 8. Boycott KM, Parboosingh JS, Scott JN et al. Meckel syndrome in the Hutterite population is actually a Joubert-related cerebello-oculo-renal syndrome. Am J Med Genet A 2007: 143A: 1715 1725. 9. Puffenberger EG, Hu-Lince D, Parod JM et al. Mapping of sudden infant death with dysgenesis of the testes syndrome (SIDDT) by a SNP genome scan and identification of TSPYL loss of function. Proc Natl Acad Sci U S A 2004: 101: 11689 11694. 10. Xin B, Puffenberger E, Tumbush J et al. Homozygosity for a novel splice site mutation in the cardiac myosin-binding protein C gene causes severe neonatal hypertrophic cardiomyopathy. Am J Med Genet A 2007: 143A: 2662 2667. 11. Strauss KA, Puffenberger EG, Bunin N et al. Clinical application of DNA microarrays: molecular diagnosis and HLA matching of an Amish child with severe combined immune deficiency. Clin Immunol 2008: 128: 31 38. 12. Xin B, Puffenberger E, Nye L et al. A novel mutation in the GDAP1 gene is associated with autosomal recessive Charcot- Marie-Tooth disease in an Amish family. Clin Genet 2008: 74: 274 278. 13. Strauss KA, Puffenberger EG, Craig DW et al. Genome-wide SNP arrays as a diagnostic tool: clinical description, genetic mapping, and molecular characterization of Salla disease in an Old Order Mennonite population. Am J Med Genet A 2005: 138A: 262 267. 14. Safieh LA, Aldahmesh M, Shamseldin H et al. Clinical and molecular characterization of Bardet-Biedl syndrome in consanguineous populations: the power of homozygosity mapping. J Med Genet 2010: 47: 236 241. 15. Alkuraya FS. Homozygosity mapping: one more tool in the clinical geneticist s toolbox. Genet Med 2010: 12: 236 239. 16. Barash Y, Calarco JS, Gao W et al. Deciphering the splicing code. Nature 2010: 465: 53 59. 17. Chu C-S, Trapnell BC, Curristin S et al. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mrna. Nat Genet 1993: 3: 151 156. 18. Heon E, Westall C, Carmi R et al. Ocular phenotypes of three genetic variants of Bardet-Biedl syndrome. Am J Med Genet A 2005: 132A: 283 287. 19. Innes A, Thomas M. Cranioectodermal dysplasia: another human ciliopathy? A review with focus on heterogeneity and mechanism. Proceed Greenwood Genet Cen 2009: 28: 92. 20. Alagramam K, Yuan H, Kuehn M et al. Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F. Hum Mol Genet 2001: 10: 1709 1718. 21. Chiang AP, Beck JS, Yen HJ et al. Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11 ). Proc Natl Acad Sci U S A 2006: 103: 6287 6292. 22. Badano JL, Leitch CC, Ansley SJ et al. Dissection of epistasis in oligogenic Bardet-Biedl syndrome. Nature 2006: 439: 326 330. 23. Louie CM, Caridi G, Lopes VS et al. AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis. Nat Genet 2010: 42: 175 180. 431