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 (BBS1-BBS14, 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 BBS1-BBS12 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.
Departments of Pediatrics and of Human Genetics, University of Michigan, Ann Arbor, MI 48109-5446, USA.
Sabine
S
Janssen
Gokul
G
Ramaswami
Erica E
EE
Davis
Toby
T
Hurd
Rannar
R
Airik
Jennifer M
JM
Kasanuki
Lauren
L
Van Der Kraak
Susan J
SJ
Allen
Philip L
PL
Beales
Nicholas
N
Katsanis
Edgar A
EA
Otto
Friedhelm
F
Hildebrandt
D000595:Amino Acid Sequence; D020788:Bardet-Biedl Syndrome / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D015331:Cohort Studies; D003241:Consanguinity; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons / Q000235:genetics; D018740:Genetic Heterogeneity; D056426:Genetic Loci / Q000235:genetics; D020022:Genetic Predisposition to Disease; D056385:Group II Chaperonins / Q000235:genetics; D006801:Humans; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011506:Proteins / Q000235:genetics*
C464790:Bbs1 protein, human; C473309:Bbs2 protein, human; D008869:Microtubule-Associated Proteins; D011506:Proteins; D056385:Group II Chaperonins