Molecular Genetic Pathogenesis
Defects in cilia or intraflagellar transport (IFT) have been associated with several human disorders including Bardet-Biedl syndrome, Kartagener syndrome, autosomal dominant polycystic kidney disease, and nephronophthisis.
Cilia protrude from almost all vertebrate cells and extend from basal bodies within the cell. Cilia are classified as primary cilia or motile cilia. Primary cilia have a 9+0 axonemal microtubule formation, are usually immotile, lack dynein arms, and are hypothesized to function as sensory organelles [Pazour & Witman 2003]. Motile cilia have a 9+2 axonemal microtubule formation and are usually involved in generating flow or movement. The assembly and maintenance of cilia depend on intraflagellar transport that moves particles from the basal body along the microtubular structure of the ciliary axoneme to the tip.
A significant leap in understanding the molecular pathogenesis of BBS emerged from the discovery of the BBS8 gene, which led to the proposal of ciliary involvement in BBS [Ansley et al 2003]. Compelling evidence was subsequently provided from comparative genomic studies that identified all known BBS orthologues among genes present exclusively in ciliated organisms [Avidor-Reiss et al 2004, Li et al 2004]. All known C.elegans bbs orthologues are exclusively expressed in a subset of ciliated sensory neurons [Ansley et al 2003, Fan et al 2004, Li et al 2004], and bbs-7 and bbs-8 mutants have structural and functional ciliary defects [Blacque et al 2004]. Furthermore, several BBS proteins localize to the centrosome (the "microtubule organizing center" of the cell) and basal body (a product of the centrosome that is positioned at the base of the cilium and required for cilia formation) [Ansley et al 2003, Kim et al 2004, Li et al 2004, Kim et al 2005]. A study of the BBS4 protein suggested that it may act as an adaptor protein facilitating the microtubule-dependent intracellular transport within the cilium or in the cytosol [Kim et al 2004].
Studies of mouse knockouts of Bbs1 [Kulaga et al 2004], Bbs2 [Nishimura et al 2004], Bbs4 [Kulaga et al 2004, Mykytyn et al 2004] and Bbs6 [Fath et al 2005, Ross et al 2005] have provided further support for ciliary involvement in BBS. Mice display sperm flagellation defects, retinal degeneration likely secondary to defective IFT, as well as olfactory dysfunction presenting as partial or complete anosmia with diminution of the ciliated olfactory epithelium. Humans with BBS were subsequently identified with partial or complete anosmia [Kulaga et al 2004, Iannaccone et al 2005]. All of the known BBS genes are down-regulated in the retina of Bbs4-null mice [Nishimura et al 2005].
Figure 1. Schematic of the putative role of BBS proteins. The diagram amalgamates data accrued from several organisms and thus represents an idealized cell system. All BBS proteins have been placed in the transition zone (basal body), centrosome, and/or ciliary axoneme. There is additional evidence for the role of BBS7 and BBS8 in facilitating the selective assembly of IFT proteins into IFT particles. Knock-down of either BBS7 or BBS8 (only BBS7 is illustrated) results in diminished levels of CHE-11 and OSM-5 (polaris) in the ciliary axoneme, culminating in shortening. BBS4 through its direct interaction with p150glued subunit of dynactin probably behaves as an adapter assisting the loading of cargo (such as PCM-1) onto the IFT particles and subsequent transport to the centriolar satellites (in the centrosome and the basal body). Given that the primary structure of BBS6 is similar to the group II chaperonins, we may speculate that its role is to process proteins prior to IFT assembly and loading as well as microtubule-dependent membrane trafficking.
A summary of the localization and putative role of the BBS proteins is illustrated in
Figure 1. For a recent review of the current state of genetics of BBS, see
Beales (2005).
BBS1
Normal allelic variants: BBS1 is composed of 17 exons and encodes a 593-amino acid protein, with the ATG start codon lying within exon 1 [Mykytyn et al 2002, Beales et al 2003, Mykytyn et al 2003].
Gene | Mutation | Exon | Reference |
---|
BBS1 | p.E549X homozygote | 16 | Mykytyn et al 2002 |
BBS1 | p.M390R homozygote | 12 | Mykytyn et al 2002 |
BBS1 | p.M390R homozygote | 12 | Mykytyn et al 2002 |
BBS1 | p.M390R heterozygote p.E549X heterozygote | 12 16 | Mykytyn et al 2002 |
BBS1 | p.M390R heterozygote p.E549X heterozygote | 12 16 | Mykytyn et al 2002 |
BBS1 | p.E549X heterozygote IVS4+1G>A | 16 4 | Mykytyn et al 2002 |
BBS1 | p.Y284fsX288 homozygote | 10 | Mykytyn et al 2002 |
BBS1 | p.M390R 27 homozygotes | 12 | Mykytyn et al 2002 |
BBS1 | c.(-3)_37del heterozygote p.M390R heterozygote | 1 12 | Mykytyn et al 2003 |
BBS1 | p.Y113X heterozygote p.M390R heterozygote | 4 12 | Mykytyn et al 2003 |
BBS1 | V114fsX150 heterozygote p.L518P heterozygote | 4 12 | Mykytyn et al 2003 |
BBS1 | p.I200_T201del p.M390R heterozygote | 8 12 | Mykytyn et al 2003 |
BBS1 | p.Y284fsX288 heterozygote p.M390R heterozygote | 16 12 | Mykytyn et al 2003 |
BBS1 | p.M347fsX373 heterozygote p.M390R heterozygote | 11 12 | Mykytyn et al 2003 |
BBS1 | p.C377_F378delfsX412 homozygote | 11 | Mykytyn et al 2003 |
BBS1 | p.R440X heterozygote p.M390R heterozygote | 13 12 | Mykytyn et al 2003 |
BBS1 | p.L505fsX556 heterozygote p.M390R heterozygote | 15 12 | Mykytyn et al 2003 |
BBS1 | p.L518P heterozygote p.M390R heterozygote | 15 12 | Mykytyn et al 2003 |
BBS1 | p.H35R, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.K53E, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.L75fsX98, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.Y133X, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.Q128X, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.R146X, 4 mutant alleles | | Beales et al 2003 |
BBS1 | p.D148N, 4 mutant alleles | | Beales et al 2003 |
BBS1 | p.E234K, 1 mutant allele | | Beales et al 2003 |
BBS1 | IVS9-3C>G, 2 mutant alleles | | Beales et al 2003 |
BBS1 | p.Y284fsX288, 3 mutant alleles | | Beales et al 2003 |
BBS1 | p.Q291X, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.G305S, 4 mutant alleles | | Beales et al 2003 |
BBS1 | p.389delI, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.M390R, 74 mutant alleles | | Beales et al 2003 |
BBS1 | p.R429X, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.Y434S, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.R440X, 2 mutant alleles | | Beales et al 2003 |
BBS1 | IVS13-2A>G, 2 mutant alleles | | Beales et al 2003 |
BBS1 | p.R483X, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.L503H, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.L505fsX556, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.L518Q, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.L548fsX579, 1 mutant allele | | Beales et al 2003 |
BBS1 | p.E549X, 1 mutant allele | | Beales et al 2003 |
Pathologic allelic variants: A common p.M390R
mutation within
exon 12 of the
BBS1 gene was shown to be involved in 30% of individuals in a cohort of 129 probands with Bardet-Biedl syndrome [
Mykytyn et al 2003]. In a further study of 259 individuals with Bardet-Biedl syndrome, a total of 74 p.M390R mutant alleles were identified, with M390R contributing to 18% of the cohort and involved in 79% of all families with
BBS1 mutations [
Beales et al 2003]. In addition, frameshift and nonsense mutations have been identified within the
BBS1 coding sequence. See
Table 3. (For more information, see
Genomic Databases table above.)
Normal gene product: The sequence of the protein encoded by BBS1 displays no significant homology to any other known proteins, with the exception of a region near the N terminal shared with BBS2 and BBS7 containing a predicted beta-propeller domain. In C.elegans it is expressed exclusively in ciliated cells and predominantly localizes to the transition zones (akin to basal bodies) as well as moving bidirectionally along the ciliary axoneme [Blacque et al 2004].
Abnormal gene product: Bbs1-null mice display olfactory deficiencies and defects in olfactory structure and function [Kulaga et al 2004].
BBS2
Normal allelic variants: The
BBS2 gene is composed of 17 exons and encodes a 721-amino acid protein; the start
codon lies within
exon 1 [
Nishimura et al 2001] (See
Table 4).
Pathologic allelic variants: A variety of
nucleotide changes resulting in frameshift, nonsense, and missense mutations have been identified throughout the
BBS2 gene; there is no known
mutation hot spot (See
Table 5) [
Nishimura et al 2001,
Katsanis et al 2000,
Katsanis et al 2001,
Katsanis et al 2002]. (For more information, see
Genomic Databases table above.)
Normal gene product: The sequence of the protein encoded by BBS2 displays no significant homology to any other known proteins, with the exception of a region near the N terminal shared with BBS1 and BBS7 containing a predicted beta-propeller domain. In C.elegans it is expressed exclusively in ciliated cells and predominantly localizes to the transition zones (akin to basal bodies) as well as moving bidirectionally along the ciliary axoneme [Blacque et al 2004].
Abnormal gene product: Bbs2-null mice display obesity, retinal degeneration, renal cysts, male infertility, and olfactory deficiencies [Nishimura et al 2004].
ARL6/BBS3
Normal allelic variants: The ARL6 gene is composed of nine exons and encodes a 186-amino acid protein; the start codon lies within exon 3 [Fan et al 2004, Chiang et al 2004].
Pathologic allelic variants: Mutations in ARL6 account for only a small percentage of BBS (~0.4%). To date, only four homozygous missense mutations and one nonsense mutation have been identified within the coding sequence. (For more information, see Genomic Databases table above.)
Normal gene product: The ARL6 gene encodes an ADP-ribosylation-like factor (ARL) protein that belongs to the Ras superfamily of small GTP-binding proteins essential for various membrane-associated intracellular trafficking events [Fan et al 2004, Chiang et al 2004]. The C.elegans ARL6 orthologue is specifically expressed in ciliated cells and undergoes IFT within the ciliary axoneme [Fan et al 2004].
Abnormal gene product: Protein modeling suggests that the four missense mutations identified so far (p.G169A, p.T31M, p.L170W, and p.T31R) alter residues near to or within the GTP-binding site and are therefore likely to abrogate GTP binding [Fan et al 2004].
BBS4
Normal allelic variants: BBS4 is composed of 16 exons and has an
open reading frame of 519 codons with the start
codon positioned within the first
exon [
Mykytyn et al 2001] (See
Table 6).
Pathologic allelic variants: A variety of
nucleotide changes resulting in frameshift, nonsense, and missense mutations have been identified throughout the
BBS4 gene, as well as one large intragenic
deletion. There is no known
mutation hot spot (See
Table 7) [
Katsanis et al 2001,
Katsanis et al 2002,
Mykytyn et al 2001,
Nishimura et al 2005]. (For more information, see
Genomic Databases table above.)
Normal gene product: The protein encoded by BBS4 contains at least ten TPR domains, which are thought to be involved in protein-protein interactions. It localizes to the basal body and centrosome in cultured cells and may function as an adaptor protein facilitating the loading of cargo onto the dynein-dynactin molecular motor in preparation for microtubule-dependent intracellular transport within the cilium or in the cytosol [Kim et al 2004].
Abnormal gene product: Mice null for Bbs4 are obese and have retinal degeneration, sperm flagellation defects, olfactory deficiencies, and defects in olfactory structure and function [Kulaga et al 2004, Mykytyn & Sheffield 2004]. Silencing of BBS4 in cultured cells leads to de-anchoring of microtubules, arrest of cell division, and apoptopic cell death [Kim et al 2004].
BBS5
Normal allelic variants: BBS5 is composed of 12 exons and has an open reading frame of 342 codons [Li et al 2004].
Pathologic allelic variants: Mutations in BBS5 account for only a small percentage of BBS (~0.4%). To date, one splice donor mutation that leads to a frameshift and a premature termination codon in exon 7, two nonsense mutations [Li et al 2004], and one large intragenic deletion [Nishimura et al 2005] have been identified. (For more information, see Genomic Databases table above.)
Normal gene product: The protein encoded by BBS5 localizes to the basal bodies and faintly within the ciliary axomene in the ependymal cells lining the ventricles of the brain in mouse [Li et al 2004]. In C.elegans bbs-5 is expressed exclusively in ciliated cells and predominantly localizes to the base of the cilia in ciliated head and tail neurons [Li et al 2004].
Abnormal gene product: Silencing of BBS5 in Chlamydomonas results in an aflagellated phenotype [Li et al 2004].
MKKS
Normal allelic variants: The
MKKS gene is composed of six exons and encodes a 570-amino acid protein [
Stone et al 2000]. The start
codon lies within
exon 3 and two alternatively spliced 5' exons are not translated (See
Table 8) [
Stone et al 2000,
Slavotinek et al 2002].
Pathologic allelic variants: Nucleotide changes have been identified in all of the coding exons of the
MKKS gene that result in frameshift, nonsense, and missense mutations; there is no known
mutation hot spot. For a number of individuals, only one heterozygous
mutation has been identified; one possible explanation includes triallelic inheritance, as these individuals may harbor mutations at one of the other BBS loci [
Katsanis et al 2001]. See
Table 9. (For more information, see
Genomic Databases table above.)
Normal gene product: The 570-amino acid protein encoded by MKKS [Stone et al 2000], shows strong homology to archeobacterial chaperonins and the eukaryotic T-complex-related-proteins (TCPs), which belong to the type II class of chaperonins [Kim et al 2005]. These proteins are implicated in facilitation of nascent protein folding in an ATP-dependent manner [reviewed by Wickner et al 1999]. MKKS localizes to the pericentriolar material (PCM), a proteinaceous tube surrounding centrioles but during mitosis it is also found at intracellular bridges [Kim et al 2005].
Abnormal gene product: The predicted substrate binding apical domain of the protein encoded by MKKS is sufficient for centrosomal localization, but several patient-derived missense mutations in this domain (p.G52D, p.D285A, p.T325P, and p.G345E) result in the protein mislocalization in cells [Kim et al 2005]. Silencing of MKKS in cultured cells leads to multinucleate and multicentrosomal cells with cytokinesis defects [Kim et al 2005]. Mice null for Mkks/Bbs6 are obese and have retinal degeneration, sperm flagellation defects, olfactory deficiencies, and defects in olfactory structure and function [Fath et al 2005, Ross et al 2005].
BBS7
Normal allelic variants: The BBS7 gene is composed of 19 exons and encodes a 672-amino acid protein [Badano et al 2003]. An alternative isoform results from differential splicing of an alternative exon 18 resulting in an additional 44 residues and a discrete 3' UTR [Badano et al 2003].
Pathologic allelic variants: Only four different pathogenic mutations have been identified in the
BBS7 gene thus far: one that results in a frameshift and the introduction of a premature termination
codon, two missense mutations, and one large intragenic
deletion [
Badano et al 2003,
Nishimura et al 2005]. See
Table 10. (For more information, see
Genomic Databases table above.)
Normal gene product: The sequence of the protein encoded by BBS7 displays no significant homology to any other known proteins, with the exception of a region near the N terminal shared with BBS1 and BBS2 containing a predicted beta-propeller domain. In C.elegans, it is expressed exclusively in ciliated cells and predominantly localizes to the transition zones (akin to basal bodies) as well as moving bidirectionally along the ciliary axoneme [Blacque et al 2004].
Abnormal gene product: C.elegans with mutations with the bbs-7 orthologue have structural and functional ciliary defects and compromised intraflagellar transport [Blacque et al 2004].
TTC8/BBS8
Normal allelic variants: The TTC8 gene is composed of 16 exons and encodes a 531-amino acid protein.
Pathologic allelic variants: Mutations in TTC8 account for only a small percentage of BBS. Two families with identical six base-pair deletions resulting in the deletion of two amino acids and another with a three base-pair deletion abolishing the splice donor site of exon 10 have been identified [Ansley et al 2003]. (For more information, see Genomic Databases table above.)
Normal gene product: BBS8 was identified because of its similarity to the BBS4 protein, containing eight TPR domains possibly involved in protein-protein interactions [Ansley et al 2003]. It also exhibits similarity to a prokaryotic domain pilF involved in twitching mobility and type-IV pilus assembly. The BBS8 protein localizes to the centrosome and basal body of cultured ciliated cells [Ansley et al 2003]. In C.elegans it is expressed exclusively in ciliated cells and predominantly localizes to the transition zones (akin to basal bodies) as well as moving bidirectionally along the ciliary axoneme [Ansley et al 2003, Blacque et al 2004].
Abnormal gene product: C.elegans with mutations with the bbs-8 orthologue have structural and functional ciliary defects and compromised intraflagellar transport [Blacque et al 2004].
B1/BBS9
Normal allelic variants: The parathyroid hormone-responsive gene B1 (B1) was recently identified as BBS9 [Nishimura et al 2005]. It is composed of 25 exons, with all except the first contributing to its various protein isoforms that range between 879-916 amino acids in length.
Pathologic allelic variants: A total of seven B1 mutations, including nonsense, splice site, missense, and frameshift, have been identified [Nishimura et al 2005] (For more information, see Genomic Databases table above.)
Normal gene product: The B1 gene is widely expressed. It has no similarity to other BBS proteins and its specific function is unknown.
Abnormal gene product: The B1 gene is down-regulated in the retina of BBS4-null mice [Nishimura et al 2005].
BBS10
Normal allelic variants: A vertebrate-specific chaperonin-like gene was recently identified as BBS10 [Stoetzel et al 2006]. It is composed of two exons encoding a 723-amino acid protein, with the start codon contained within exon 1.
Pathologic allelic variants: BBS10 is a major locus for BBS, contributing mutant alleles in ~20% of all individuals with BBS. There are numerous missense, frameshifting, and nonsense mutations spread throughout the coding region, with no mutational hot spot [Stoetzel et al 2006].
Normal gene product: BBS10 has a chaperonin domain organization conserved with all three major functional domains — equatorial, intermediate, and apical — and the flexible protrusion region specific to group II chaperonins. The ATP hydrolytic domain is conserved in BBS10, suggesting that it may be an active enzyme, in contrast to BBS6, where this catalytic site is absent.
Abnormal gene product: Suppression of bbs10 expression in zebrafish embryos causes shortening of the body axis and dorsal thinning, broadening and kinking of the notochord, and elongation of the somites [Stoetzel et al 2006].
TRIM32
Normal allelic variants: TRIM32 (BBS11), a ubiquitin ligase, was recently identified [Chiang et al 2006]. It is composed of two exons encoding a 652-amino acid protein, with the ATG start codon in exon 2.
Pathologic allelic variants: The only mutation identified to date in BBS11 associated with BBS is a homozygous missense mutation p.P130S, which lies in the N-terminal B-box domain, in affected individuals in an inbred Bedouin Arab family [Chiang et al 2006]. However, a missense variant, p.D487N in the C-terminal NHL domain of TRIM32, was previously associated with autosomal recessive limb-girdle muscular dystrophy (LGMD) [Frosk et al 2002].
Normal gene product: TRIM32 is a member of the TRIM family that is characterized by a common domain structure composed of a RING finger, B-box, and a coiled-coiled motif. It also contains five C-terminal NHL repeats. TRIM32 is thought to have E3 ubiquitin ligase activity, binds to myosin, and ubiquitinates actin, implicating TRIM32 in regulating components of the cytoskeleton.
Abnormal gene product: Zebrafish embryos with knockdown of BBS11 expression display an abnormal Kuppfer’s vesicle, a transient ciliated organ involved in left-right patterning and a delay in melanosome transport. The p.P130S mutant allele associated with BBS fails to rescue these abnormal phenotypes, in contrast to the p.D487N allele associated with LGMD, suggesting that each mutation disrupts different functions of TRIM32/BBS11 [Chiang et al 2006].
BBS12
Normal allelic variants: BBS12 is a vertebrate-specific predicted chaperonin-like protein [Stoetzel et al 2007]. It is composed of two exons, of which only the second is coding, for a predicted protein of 710 amino acids.
Pathologic allelic variants: BBS12 is mutated in approximately 5% of families affected with [Stoetzel et al 2007]. Mutations identified include frameshifts [one of which, F372X (also known as F372fsX373), is recurrent and present in several families], nonsense, small in-frame deletions, a mutation that is predicted to extend the C-terminus of the protein, and missense alleles.
Normal gene product: BBS12 is related to the group II chaperonins and to a family of vertebrate-specific chaperonin-like sequences encompassing BBS10 and BBS6 [Stoetzel et al 2007]. The classical chaperonin domain architecture (equatorial, intermediate and apical domains) is conserved, but BBS12 has an additional five specific inserted sequences within the intermediate and equatorial domains. However, the functional ATP hydrolysis motif is not conserved in BBS12, as is the case for BBS6.
Abnormal gene product: Injection of bbs12-specific morpholino into zebrafish embryos results in phenotypes consistent with convergence and extension (CE) defects, including shortened body axis, broadened somites, kinked notochord and dorsal thinning [Stoetzel et al 2007]. Simultaneous suppression of bbs12, bbs10, and bbs6 gene expression yielded similar but more severe phenotypes, suggesting a possible partial functional redundancy within this protein family.