Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene

Abstract

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.

Figure 1

Pedigrees of BBS families with B1 mutations. Specific mutations detected in each family are indicated below the family identifier. All mutations were found in the homozygous state, with the exception of that in UAP-74. mt = Mutant allele corresponding to the mutation in each family. wt = Wild-type (normal) allele. In UAP-74, mt1 represents the K626fsX647 and mt2 represents the IVS4+1G→C mutation.

Figure 2

Genomic structure of the B1 gene, with exons depicted as boxes. Filled boxes, Coding regions. Open boxes, UTRs. Exon sizes are shown below the exons in the genomic structure. The three isoforms of B1 presented in this study are depicted below the genomic structure. The location of the B1 northern blot probe (hatched box) is shown. Locations of the BBS mutations detected in this study are indicated at the bottom of the figure.

Figure 3

Northern blot analysis of B1 gene expression by northern blot analysis. A, Human B1 probe hybridized to human RNA. B, Mouse B1 probe, corresponding to the same location as the human B1 probe, hybridized to mouse RNA samples.

Figure  4

Intragenic deletions found in BBS4, BBS5, and BBS7. The extent of each deletion is depicted within the partial genomic structure for each gene. The locations of the exons are shown below the line representing the genomic sequence (AC016402, AC093899, and AC079341). Within the genomic sequence, repetitive element family members are indicated as follows: blue, AluS; red, AluJ; green, AluY; yellow, FLAM; and pink, SVA. Translation of the genomic region in the three forward reading frames are shown below each gene figure with start codons (green) and stop codons (red).

Figure 5

Sequence flanking the breakpoints for the BBS4 deletion. The sequences of the 5′ and 3′ Alu elements are shown as well as the sequence of the junction fragment of the BBS4 deletion event in family CAP-1. Box, The region in which the breakpoint has occurred within each Alu element. The 26-bp core sequence within the Alu element identified by Rudiger et al. (1995) is shown at the bottom of the figure, as well as a comparison to the prokaryotic χ-sequence.