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Mutation analysis in Bardet–Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals


Bardet–Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II™ platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS.

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Fig. 1



Bardet–Biedl Syndrome


Base pairs


Guanosine triphosphate


Human random control DNA panel-1


Massively parallel resequencing




Polymerase chain reaction


Variants from reference sequence


  1. Ansley SJ, Badano JL, Blacque OE, Hill J, Hoskins BE, Leitch CC, Kim JC, Ross AJ, Eichers ER, Teslovich TM, Mah AK, Johnsen RC, Cavender JC, Lewis RA, Leroux MR, Beales PL, Katsanis N (2003) Basal body dysfunction is a likely cause of pleiotropic Bardet–Biedl syndrome. Nature 425:628–633

  2. Badano JL, Ansley SJ, Leitch CC, Lewis RA, Lupski JR, Katsanis N (2003a) Identification of a novel Bardet–Biedl syndrome protein, BBS7, that shares structural features with BBS1 and BBS2. Am J Hum Genet 72:650–658

  3. Badano JL, Kim JC, Hoskins BE, Lewis RA, Ansley SJ, Cutler DJ, Castellan C, Beales PL, Leroux MR, Katsanis N (2003b) Heterozygous mutations in BBS1, BBS2 and BBS6 have a potential epistatic effect on Bardet–Biedl patients with two mutations at a second BBS locus. Hum Mol Genet 12:1651–1659

  4. Badano JL, Leitch CC, Ansley SJ, May-Simera H, Lawson S, Lewis RA, Beales PL, Dietz HC, Fisher S, Katsanis N (2006) Dissection of epistasis in oligogenic Bardet–Biedl syndrome. Nature 439:326–330

  5. Baker K, Beales PL (2009) Making sense of cilia in disease: the human ciliopathies. Am J Med Genet C Semin Med Genet 151C:281–295

  6. Bardet G (1920) On congenital obesity syndrome with polydactyly and retinitis pigmentosa (a contribution to the study of clinical forms of hypophyseal obesity). Obes Res 3:387–399

  7. Beales PL, Warner AM, Hitman GA, Thakker R, Flinter FA (1997) Bardet–Biedl syndrome: a molecular and phenotypic study of 18 families. J Med Genet 34:92–98

  8. Beales PL, Badano JL, Ross AJ, Ansley SJ, Hoskins BE, Kirsten B, Mein CA, Froguel P, Scambler PJ, Lewis RA, Lupski JR, Katsanis N (2003) Genetic interaction of BBS1 mutations with alleles at other BBS loci can result in non-Mendelian Bardet–Biedl syndrome. Am J Hum Genet 72:1187–1199

  9. Biedl A (1922) A pair of siblings with adiposo-genital dystrophy. Obes Res 3:404

  10. Billingsley G, Bin J, Fieggen KJ, Duncan JL, Gerth C, Ogata K, Wodak SS, Traboulsi EI, Fishman GA, Paterson A, Chitayat D, Knueppel T, Millan JM, Mitchell GA, Deveault C, Heon E (2010) Mutations in chaperonin-like BBS genes are a major contributor to disease development in a multiethnic Bardet–Biedl syndrome patient population. J Med Genet 47:453–463

  11. Chiang AP, Nishimura D, Searby C, Elbedour K, Carmi R, Ferguson AL, Secrist J, Braun T, Casavant T, Stone EM, Sheffield VC (2004) Comparative genomic analysis identifies an ADP-ribosylation factor-like gene as the cause of Bardet–Biedl syndrome (BBS3). Am J Hum Genet 75:475–484

  12. Chiang AP, Beck JS, Yen HJ, Tayeh MK, Scheetz TE, Swiderski RE, Nishimura DY, Braun TA, Kim KY, Huang J, Elbedour K, Carmi R, Slusarski DC, Casavant TL, Stone EM, Sheffield VC (2006) Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet–Biedl syndrome gene (BBS11). Proc Natl Acad Sci USA 103:6287–6292

  13. Farag TI, Teebi AS (1988) Bardet–Biedl and Laurence–Moon syndromes in a mixed Arab population. Clin Genet 33:78–82

  14. Fauser S, Munz M, Besch D (2003) Further support for digenic inheritance in Bardet-Biedl syndrome. J Med Genet 40:e104

  15. Haim M (1992) Prevalence of retinitis pigmentosa and allied disorders in Denmark. II. Systemic involvement and age at onset. Acta Ophthalmol (Copenh) 70:417–426

  16. Harville HM, Held S, Diaz-Font A, Davis EE, Diplas BH, Lewis RA, Borochowitz ZU, Zhou W, Chaki M, MacDonald J, Kayserili H, Beales PL, Katsanis N, Otto E, Hildebrandt F (2010) Identification of 11 novel mutations in eight BBS genes by high-resolution homozygosity mapping. J Med Genet 47:262–267

  17. Hildebrandt F, Heeringa SF, Rüschendorf F, Attanasio M, Nürnberg G, Becker C, Seelow D, Huebner N, Chernin G, Vlangos CN, Zhou W, O’Toole JF, Hoskins BE, Wolf MTF, Hinkes BG, Chaib H, Ashraf S, Allen SJ, Vega-Warner V, Wise E, Harville HM, Lyons RH, Washburn J, MacDonald J, Nürnberg P, Otto EA (2009) A systematic approach to mapping recessive disease genes in individuals from outbred populations. PloS Genet 5:31000353

  18. Hjortshøj TD, Grønskov K, Philp AR, Nishimura DY, Adeyemo A, Rotimi CN, Sheffield VC, Rosenberg T, Brøndum-Nielsen K (2008) Novel mutations in BBS5 highlight the importance of this gene in non-Caucasian Bardet–Biedl syndrome patients. Am J Med Genet 146:517–520

  19. Hjortshoj TD, Gronskov K, Philp AR, Nishimura DY, Riise R, Sheffield VC, Rosenberg T, Brondum-Nielsen K (2010) Bardet–Biedl syndrome in Denmark—report of 13 novel sequence variations in six genes. Hum Mutat 31:429–436

  20. Katsanis N (2004) The oligogenic properties of Bardet–Biedl syndrome. Hum Mol Genet 13(Spec No 1):R65–R71

  21. Katsanis N, Lewis RA, Stockton DW, Mai PM, Baird L, Beales PL, Leppert M, Lupski JR (1999) Delineation of the critical interval of Bardet-Biedl syndrome 1 (BBS1) to a small region of 11q13, through linkage and haplotype analysis of 91 pedigrees. Am J Hum Genet 65:1672–1679

  22. Katsanis N, Beales PL, Woods MO, Lewis RA, Green JS, Parfrey PS, Ansley SJ, Davidson WS, Lupski JR (2000) Mutations in MKKS cause obesity, retinal dystrophy and renal malformations associated with Bardet–Biedl syndrome. Nat Genet 26:67–70

  23. Katsanis N, Ansley SJ, Badano JL, Eichers ER, Lewis RA, Hoskins BE, Scambler PJ, Davidson WS, Beales PL, Lupski JR (2001) Triallelic inheritance in Bardet–Biedl syndrome, a Mendelian recessive disorder. Science 293:2256–2259

  24. Katsanis N, Eichers ER, Ansley SJ, Lewis RA, Kayserili H, Hoskins BE, Scambler PJ, Beales PL, Lupski JR (2002) BBS4 is a minor contributor to Bardet–Biedl syndrome and may also participate in triallelic inheritance. Am J Hum Genet 71:22–29

  25. Khanna H, Davis EE, Murga-Zamalloa CA, Estrada-Cuzcano A, Lopez I, den Hollander AI, Zonneveld MN, Othman MI, Waseem N, Chakarova CF, Maubaret C, Diaz-Font A, Macdonald I, Muzny DM, Wheeler DA, Morgan M, Lewis LR, Logan CV, Tan PL, Beer MA, Inglehearn CF, Lewis RA, Jacobson SG, Bergmann C, Beales PL, Attie-Bitach T, Johnson CA, Otto EA, Bhattacharya SS, Hildebrandt F, Gibbs RA, Koenekoop RK, Swaroop A, Katsanis N (2009) A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies. Nat Genet 41:739–745

  26. Klein D, Ammann F (1969) The syndrome of Laurence–Moon–Bardet–Biedl and allied diseases in Switzerland. Clinical, genetic and epidemiological studies. J Neurol Sci 9:479–513

  27. Lander ES, Botstein D (1987) Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science 236:1567–1570

  28. Leitch CC, Zaghloul NA, Davis EE, Stoetzel C, Diaz-Font A, Rix S, Alfadhel M, Lewis RA, Eyaid W, Banin E, Dollfus H, Beales PL, Badano JL, Katsanis N (2008) Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet–Biedl syndrome. Nat Genet 40:443–448

  29. Li JB, Gerdes JM, Haycraft CJ, Fan Y, Teslovich TM, May-Simera H, Li H, Blacque OE, Li L, Leitch CC, Lewis RA, Green JS, Parfrey PS, Leroux MR, Davidson WS, Beales PL, Guay-Woodford LM, Yoder BK, Stormo GD, Katsanis N, Dutcher SK (2004) Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117:541–552

  30. Moore SJ, Green JS, Fan Y, Bhogal AK, Dicks E, Fernandez BA, Stefanelli M, Murphy C, Cramer BC, Dean JC, Beales PL, Katsanis N, Bassett AS, Davidson WS, Parfrey PS (2005) Clinical and genetic epidemiology of Bardet–Biedl syndrome in Newfoundland: a 22-year prospective, population-based, cohort study. Am J Med Genet A 132:352–360

  31. Muller J, Stoetzel C, Vincent MC, Leitch CC, Laurier V, Danse JM, Helle S, Marion V, Bennouna-Greene V, Vicaire S, Megarbane A, Kaplan J, Drouin-Garraud V, Hamdani M, Sigaudy S, Francannet C, Roume J, Bitoun P, Goldenberg A, Philip N, Odent S, Green J, Cossee M, Davis EE, Katsanis N, Bonneau D, Verloes A, Poch O, Mandel JL, Dollfus H (2010) Identification of 28 novel mutations in the Bardet–Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease. Hum Genet 127:583–593

  32. Mykytyn K, Braun T, Carmi R, Haider NB, Searby CC, Shastri M, Beck G, Wright AF, Iannaccone A, Elbedour K, Riise R, Baldi A, Raas-Rothschild A, Gorman SW, Duhl DM, Jacobson SG, Casavant T, Stone EM, Sheffield VC (2001) Identification of the gene that, when mutated, causes the human obesity syndrome BBS4. Nat Genet 28:188–191

  33. Mykytyn K, Nishimura DY, Searby CC, Shastri M, Yen HJ, Beck JS, Braun T, Streb LM, Cornier AS, Cox GF, Fulton AB, Carmi R, Luleci G, Chandrasekharappa SC, Collins FS, Jacobson SG, Heckenlively JR, Weleber RG, Stone EM, Sheffield VC (2002) Identification of the gene (BBS1) most commonly involved in Bardet–Biedl syndrome, a complex human obesity syndrome. Nat Genet 31:435–438

  34. Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, Merdes A, Slusarski DC, Scheller RH, Bazan JF, Sheffield VC, Jackson PK (2007) A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129:1201–1213

  35. Nishimura DY, Searby CC, Carmi R, Elbedour K, Van Maldergem L, Fulton AB, Lam BL, Powell BR, Swiderski RE, Bugge KE, Haider NB, Kwitek-Black AE, Ying L, Duhl DM, Gorman SW, Heon E, Iannaccone A, Bonneau D, Biesecker LG, Jacobson SG, Stone EM, Sheffield VC (2001) Positional cloning of a novel gene on chromosome 16q causing Bardet–Biedl syndrome (BBS2). Hum Mol Genet 10:865–874

  36. Nishimura DY, Swiderski RE, Searby CC, Berg EM, Ferguson AL, Hennekam R, Merin S, Weleber RG, Biesecker LG, Stone EM, Sheffield VC (2005) Comparative genomics and gene expression analysis identifies BBS9, a new Bardet–Biedl syndrome gene. Am J Hum Genet 77:1021–1033

  37. Otto EA, Ramaswami G, Janssen S, Chaki M, Allen SJ, Zhou W, Airik R, Hurd TW, Ghosh AK, Wolf MT, Hoppe B, Neuhaus TJ, Bockenhauer D, Milford DV, Soliman NA, Antignac C, Saunier S, Johnson CA, Hildebrandt F, the GPN Study Group (2010b) Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. J Med Genet, in press

  38. Otto EA, Helou J, Allen SJ, O’Toole JF, Wise EL, Ashraf S, Attanasio M, Zhou W, Wolf MT, Hildebrandt F (2008) Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing. Hum Mutat 29:418–426

  39. Otto EA, Hurd TW, Airik R, Chaki M, Zhou W, Stoetzel C, Patil SB, Levy S, Ghosh AK, Murga-Zamalloa CA, van Reeuwijk J, Letteboer SJ, Sang L, Giles RH, Liu Q, Coene KL, Estrada-Cuzcano A, Collin RW, McLaughlin HM, Held S, Kasanuki JM, Ramaswami G, Conte J, Lopez I, Washburn J, Macdonald J, Hu J, Yamashita Y, Maher ER, Guay-Woodford LM, Neumann HP, Obermüller N, Koenekoop RK, Bergmann C, Bei X, Lewis RA, Katsanis N, Lopes V, Williams DS, Lyons RH, Dang CV, Brito DA, Dias MB, Zhang X, Cavalcoli JD, Nürnberg G, Nürnberg P, Pierce EA, Jackson PK, Antignac C, Saunier S, Roepman R, Dollfus H, Khanna H, Hildebrandt F (2010) Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy. Nat Genet 420:840–850

  40. Ramensky V, Bork P, Sunyaev S (2002) Human non-synonymous SNPs: server and survey. Nucleic Acids Res 30:3894–3900

  41. Slavotinek AM, Stone EM, Mykytyn K, Heckenlively JR, Green JS, Heon E, Musarella MA, Parfrey PS, Sheffield VC, Biesecker LG (2000) Mutations in MKKS cause Bardet–Biedl syndrome. Nat Genet 26:15–16

  42. Stoetzel C, Laurier V, Davis EE, Muller J, Rix S, Badano JL, Leitch CC, Salem N, Chouery E, Corbani S, Jalk N, Vicaire S, Sarda P, Hamel C, Lacombe D, Holder M, Odent S, Holder S, Brooks AS, Elcioglu NH, Silva ED, Rossillion B, Sigaudy S, de Ravel TJ, Lewis RA, Leheup B, Verloes A, Amati-Bonneau P, Megarbane A, Poch O, Bonneau D, Beales PL, Mandel JL, Katsanis N, Dollfus H (2006) BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus. Nat Genet 38:521–524

  43. Stoetzel C, Muller J, Laurier V, Davis EE, Zaghloul NA, Vicaire S, Jacquelin C, Plewniak F, Leitch CC, Sarda P, Hamel C, de Ravel TJ, Lewis RA, Friederich E, Thibault C, Danse JM, Verloes A, Bonneau D, Katsanis N, Poch O, Mandel JL, Dollfus H (2007) Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet–Biedl syndrome. Am J Hum Genet 80:1–11

  44. Stone DL, Slavotinek A, Bouffard GG, Banerjee-Basu S, Baxevanis AD, Barr M, Biesecker LG (2000) Mutation of a gene encoding a putative chaperonin causes McKusick-Kaufman syndrome. Nat Genet 25:79–82

  45. Teebi AS (1994) Autosomal recessive disorders among Arabs: an overview from Kuwait. J Med Genet 31:224–233

  46. Young TL, Penney L, Woods MO, Parfrey PS, Green JS, Hefferton D, Davidson WS (1999) A fifth locus for Bardet–Biedl syndrome maps to chromosome 2q31. Am J Hum Genet 64:900–904

  47. Zaghloul NA, Katsanis N (2009) Mechanistic insights into Bardet–Biedl syndrome, a model ciliopathy. J Clin Invest 119:428–437

  48. Zaghloul NA, Liu Y, Gerdes JM, Gascue C, Oh EC, Leitch CC, Bromberg Y, Binkley J, Leibel RL, Sidow A, Badano JL, Katsanis N (2010) Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet–Biedl syndrome. Proc Natl Acad Sci USA 107:10602–10607

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The authors wish to thank the physicians and families for participating in this study. We would like to thank Dr. E. Héon (Toronto) and Dr. J.L. Duncan (San Francisco) for their contribution of DNA and clinical data from patients. We also thank Robert Lyons and the University of Michigan DNA Sequencing Core for excellent Illumina sequencing. F.H. is an Investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist, and a Frederick G. L. Huetwell Professor. N.K. is a Distinguished George W. Brumley professor. This research was supported by grants from the National Institutes of Health to F.H. (DK1069274, DK1068306, DK064614) and N.K. (HD04260, DK072301, DK075972). P.L.B. is funded by Wellcome Trust.

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Correspondence to Friedhelm Hildebrandt.

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S. Janssen and G. Ramaswami contributed equally to this work.

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Janssen, S., Ramaswami, G., Davis, E.E. et al. Mutation analysis in Bardet–Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals. Hum Genet 129, 79–90 (2011).

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  • Mutation Carrier
  • Homozygosity Mapping
  • Postaxial Polydactyly
  • Biedl Syndrome
  • Direct Sanger Sequencing