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

Abstract

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

Abbreviations

BBS:

Bardet–Biedl Syndrome

bp:

Base pairs

GTP:

Guanosine triphosphate

HRC-1:

Human random control DNA panel-1

MPR:

Massively parallel resequencing

nt:

Nucleotides

PCR:

Polymerase chain reaction

VRS:

Variants from reference sequence

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Acknowledgments

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.

Author information

Correspondence to Friedhelm Hildebrandt.

Additional information

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). https://doi.org/10.1007/s00439-010-0902-8

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Keywords

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