Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome

Abstract

Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive ciliopathy characterized by progressive retinal degeneration, obesity, cognitive impairment, polydactyly, and kidney anomalies. The disorder is genetically heterogeneous, with 11 BBS genes identified to date, which account for ~70% of affected families. We have combined single-nucleotide-polymorphism array homozygosity mapping with in silico analysis to identify a new BBS gene, BBS12. Patients from two Gypsy families were homozygous and haploidentical in a 6-Mb region of chromosome 4q27. FLJ35630 was selected as a candidate gene, because it was predicted to encode a protein with similarity to members of the type II chaperonin superfamily, which includes BBS6 and BBS10. We found pathogenic mutations in both Gypsy families, as well as in 14 other families of various ethnic backgrounds, indicating that BBS12 accounts for approximately 5% of all BBS cases. BBS12 is vertebrate specific and, together with BBS6 and BBS10, defines a novel branch of the type II chaperonin superfamily. These three genes are characterized by unusually rapid evolution and are likely to perform ciliary functions specific to vertebrates that are important in the pathophysiology of the syndrome, and together they account for about one-third of the total BBS mutational load. Consistent with this notion, suppression of each family member in zebrafish yielded gastrulation-movement defects characteristic of other BBS morphants, whereas simultaneous suppression of all three members resulted in severely affected embryos, possibly hinting at partial functional redundancy within this protein family.

Figure 1.

Mapping of a candidate BBS region to 4q27. A, Analysis of homozygosity in chromosome 4 in three consanguineous families. The areas of homozygosity with >25 SNPs are black, whereas homozygosity regions defined by 15–25 consecutive SNPs are gray. B, Detailed SNP homozygosity mapping and microsatellite segregation in the region of interest on chromosome 4 in the two Gypsy families. Light- and dark-gray regions represent homozygous SNPs (AA and BB, respectively), whereas white regions indicate heterozygous alleles (AB).

Figure 2.

Schematic of the BBS12 locus and the position and/or nature of mutations. A, BBS12 genomic locus (top), corresponding to FLJ35630 (NCBI [RefSeq] accession number NM_152618). FLJ35630 encodes a predicted protein of 710 aa (UniProt accession number Q6ZW61_HUMAN). The bottom section is a schematic of the protein with the recognized domains in different colors (see key). The insertions in the intermediate and equatorial domains are shown as yellow boxes above the protein. All reported mutant alleles are shown underneath the protein. B, Phylogenetic tree of the BBS family. The tree contains BBS12, BBS10, and BBS6 sequences and representative sequences from all group II chaperonins. BBS12 genes are highlighted with organism abbreviations, and predicted sequences are marked with a black star. XL = X. laevis; GG = G. gallus; MM = M. musculus; RN = R. norvegicus; BT = B. taurus; CF = C. familiaris; HS = H. sapiens; PP = Pongo pygmaeus; DR = D. rerio; TN = T. nigroviridis; TR = T. rubripes. The roots of vertebrate branches are highlighted with gray dots. Bootstrap values are provided for significant nodes when they are >80%. C, Comparison of BBS chaperonin-like domain organization. BBS12, BBS10, and BBS6 are represented with the typical chaperonin group II organization in three domains (equatorial, intermediate, and apical). Specific insertions to the typical chaperonin group II are drawn in yellow and are numbered in order of appearance from N-ter to C-ter part. Points of insertions that appear common to BBS12 and BBS6 and to BBS12 and BBS10 are highlighted with black lines. D, Ribbon drawing of group II chaperonin α subunit (Protein Data Bank 1q2v). Domains are colored according to figure 3A. BBS12 insertions (In1-In5) relative to all group II chaperonin sequences are represented as yellow dashed rectangles.

Figure 3.

Sequence conservation of BBS proteins in vertebrates. The percent identities are given from human to the nematode by use of the human sequence as a reference. HS = H. sapiens; CF = C. familiaris; MM = M. musculus; RN = R. norvegicus; GG = G. gallus; DR = D. rerio; TN = T. nigroviridis; CE = C. elegans. No BBS6, BBS10, and BBS12 orthologues exist in the C. elegans genome.

Figure 4.

CE defects and genetic interaction between the BBS chaperonins in zebrafish. A and B, Side (A) and dorsal (B) views of live embryos injected either with a control MO, or various translational blocking MOs against the chaperonin-like subclass of bbs genes. C, Distribution of phenotypes resulting from suppressing various combinations of bbs6, bbs10, and bbs12. Note the synthetic effect of double suppression and the pronounced effect of the combinatorial suppression of all three genes, with the expansion of the severe class of morphants (class II) being particularly prominent.