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Exome sequencing of Bardet–Biedl syndrome patient identifies a null mutation in the BBSome subunit BBIP1 (BBS18)
  1. Sophie Scheidecker1,
  2. Christelle Etard2,
  3. Nathan W Pierce3,
  4. Véronique Geoffroy4,
  5. Elise Schaefer1,5,
  6. Jean Muller6,7,
  7. Kirsley Chennen1,7,
  8. Elisabeth Flori8,
  9. Valérie Pelletier5,
  10. Olivier Poch7,
  11. Vincent Marion1,
  12. Corinne Stoetzel1,
  13. Uwe Strähle2,
  14. Maxence V Nachury3,
  15. Hélène Dollfus1,5
  1. 1Laboratoire de Génétique Médicale, INSERM U1112, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France
  2. 2Institut für Toxikologie und Genetik Campus Nord, Karlsruher Institut für Technologie, Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen, Germany
  3. 3Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA
  4. 4Plate-forme Bioinformatique de Strasbourg, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), CNRS UMR7104, INSERM U964, Université de Strasbourg, Illkirch, France
  5. 5Service de Génétique Médicale, Centre de Référence pour les Affections Rares en Génétique Ophtalmologique (CARGO), Hôpitaux Universitaires de Strasbourg, Strasbourg, France
  6. 6Laboratoire de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
  7. 7Integrative Genomics and Bioinformatics Laboratory, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), CNRS UMR7104, INSERM U964, ICube UMR 7357, Université de Strasbourg, Illkirch, France
  8. 8Service de Cytogénétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
  1. Correspondence to Professor Helene Dollfus, Faculté de Médecine, Université de Strasbourg, Laboratoire de Génétique Médicale, INSERM 1112, Faculté de Médecine, Bâtiment 3, 11 rue Humann, Strasbourg 67085, France; dollfus{at}


Background Bardet–Biedl syndrome (BBS) is a recessive and genetically heterogeneous ciliopathy characterised by retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioural dysfunction and hypogonadism. 7 of the 17 BBS gene products identified to date assemble together with the protein BBIP1/BBIP10 into the BBSome, a protein complex that ferries signalling receptors to and from cilia.

Methods and results Exome sequencing performed on a sporadic BBS case revealed for the first time a homozygous stop mutation (NM_001195306: c.173T>G, p.Leu58*) in the BBIP1 gene. This mutation is pathogenic since no BBIP1 protein could be detected in fibroblasts from the patient, and BBIP1[Leu58*] is unable to associate with the BBSome subunit BBS4.

Conclusions These findings identify BBIP1 as the 18th BBS gene (BBS18) and suggest that BBSome assembly may represent a unifying pathomechanism for BBS.

  • Clinical Genetics
  • Diagnostics Tests
  • Genetic Screening/Counselling
  • Molecular Genetics
  • Ophthalmology
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Bardet–Biedl syndrome (BBS; MIM 209900) is a canonical ciliopathy characterised by retinitis pigmentosa, obesity, kidney alteration, dystrophic extremities, behavioural dysfunction and hypogonadism.1 Rapidly evolving strategies ranging from traditional homozygosity mapping to next generation sequencing (NGS) approaches have uncovered 17 BBS genes.

Primary cilia dysfunction underlies the pathogenesis of BBS; indeed, eight proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBIP1/BBIP10) have been shown to form the BBSome,2 ,3 a stable complex involved in signalling receptor trafficking to and from cilia. BBS6, BBS10 and BBS12 assemble into a chaperonin complex that mediates BBSome assembly. BBS3/ARL6 recruits the BBSome to membranes, and BBS17/LZTFL1 regulates BBSome entry into cilia. BBSome cargos include Smoothened, a component of the Sonic Hedgehog (Shh) signalling pathway,4 and Somatostatin Receptor 3, a G-protein-coupled receptor.5

Mutations found in the 17 known BBS genes are found in 80% of BBS patients while 20% of them still lack molecular diagnosis.6 Through exome sequencing, we report the first BBS patient carrying a mutation in the BBIP1 gene (also known as BBIP10 standing for the BBSome Interacting Protein 1/of 10 kDa) encoding the eighth subunit of the BBSome.2 Given our prior characterisation of BBIP1 as a BBSome subunit essential for BBSome assembly2 and given the severely reduced levels of BBIP1 in the patient’s fibroblasts, we propose BBIP1 as the 18th BBS gene.


Family selection

Among 450 BBS families screened, about 15% were devoid of mutation in known BBS genes. We report herein one of our BBS families analysed by exome sequencing.

Informed consent and ethical approval of the patient and his/her representative were obtained according to the French legislation. The objectives and the aim of the study were clearly explained to the patient.

Whole exome sequencing and SNP calling

Whole exome sequencing was performed by IntegraGen. Exons of DNA samples were captured using the in-solution SureSelect Target Enrichment System (Agilent, Human All Exon Kits v2), followed by a paired-end high-throughput sequencing on reads of 75 bp using the Illumina HiSeq 2000. Image analysis and base calling were performed with default parameters of Illumina RTA v1.14 pipeline. The alignment of clean reads on the human reference genome (hg19/GRCh37) and single nucleotide polymorphism (SNP) calling were performed with CASAVA 1.8 (Illumina).

Variant annotation and ranking with VaRank

VaRank is an in-house pipeline (manuscript in preparation) using Alamut-HT (Interactive Biosoftware) to collect genomic annotations and effect predictions at both nucleotide and protein levels. VaRank gathers variant-specific information such as potential functional effects of amino acid changes on the protein sorting intolerant from tolerant (SIFT),7 PolyPhen28) and splicing effects (Human Splicing Finder,9 MaxEntScan,10 NNSplice11). Known mutations with reported SNPs flagged as ‘probably pathogenic’/‘pathogenic’ in the ‘clinical significance’ field of dbSNP137 are highlighted. From all these information, a score is computed for each SNV/indel. Potential mutations are ranked according to potential pathogenicity. Initial filtering included the removal of variants with <15% of the total coverage or the variants not supported by at least 10×, variants present in dbSNP137, validated by at least two methods or with a minor allele frequency >2%.

Western blotting

Proteins from patient's fibroblasts obtained by skin biopsy (see online supplementary method) were extracted by trichloroacetic acid precipitation and immunoblot analyses performed as previously described.12


The p.Leu58* mutation was introduced by PCR to generate pCS2-6myc-BBIP1[Leu58*]. For co-immunoprecipitation, human embryonic kidney (HEK)293FT cells were transfected with plasmid DNA using X-tremeGENE 9 (Roche). After 48 h, cells were lysed in buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100) containing protease inhibitors (Leupeptin, Bestatin, Chymostatin, E-64, Aprotinin, AEBSF). After centrifugation, lysates were incubated for 1 h with 9E10 anti-myc monoclonal antibody, then 1 h with Protein-G-sepharose. After three washes in lysis buffer, beads were eluted in sodium dodecyl sulfate (SDS) sample buffer and western blotted with anti-BBS4 (as previously described3) or 9E10 antibodies.

Morpholino microinjections in zebrafish

Injections were performed as described previously.13 Morpholinos (Gene Tools, LLC)14 were injected at 12 nL of 0.5 mM stock solution. Phenol red was added to the samples before injection (0.1% final concentration). For detecting situs inversus, morpholinos were injected into the unc45b: green fluorescent protein (GFP) stable line driving GFP expression in cardiac muscles15 (see online supplementary method).

Bbip1 PCR amplification

For rescue experiments, a bbip1 morpholino-resistant fragment was amplified by PCR with primers bbip5′ and bbip3′ (see online supplementary table S1) and cloned into pCs2+GFP.


Embryos were fixed in 4% paraformaldehyde/ phosphate-buffered saline (PBS) for 1 h, washed with 1% PBS, blocked with beclomethasone dipropionate (BDP) (5% bovine serum albumin (BSA), 1% dimethyl sulfoxide (DMSO), 1% PBS), incubated overnight at 4°C with monoclonal antibody against acetylated-tubulin (1:1000, Sigma), washed five times and incubated for 1 h with fluorescent secondary antibody (anti-mouse IgG Cy3-conjugated, 1:1000, Sigma). 5 μm sections of the eyes16 were cut with a Leica microtome and stained with toluidine blue.


Clinical studies

Based on four major features (retinitis pigmentosa, obesity, kidney failure, cognitive disability) and one minor feature (brachydactyly), the patient was diagnosed as affected with BBS at 49 years old. He presented an end-stage renal failure 4 years after the diagnosis. He was the only one affected among four siblings born from consanguineous Italian parents.

Clinical examination in our Center for Rare Genetic Ophthalmologic Diseases (CARGO) in Strasbourg showed severe visual impairment (light perception, dense cataracts, retinal dystrophy), obesity (BMI 37.7), behavioural dysfunction, learning difficulties (understood simple orders but never learned to read or write) and brachydactyly (figure 1A).

Figure 1

Identification of the p.[Leu58*];[Leu58*] mutation in the BBIP1 gene. (A) Pictures of the patient depicting an obese phenotype and overall enlarged hands with a shortened aspect of the fingers. (B) Electrophoregrams of a part of BBIP1 exon 3 showing the homozygous mutation in the patient and the heterozygous mutation in the patient's father (mother and sibling's DNA was unavailable). Access the article online to view this figure in colour.

Molecular analysis of known BBS genes and exome sequencing

Prior Sanger sequencing and a preliminary examination of the exome data identified no mutation in any of the 17 known BBS genes. Initial filtering of the exome sequencing data by the VaRank program revealed 7889 variants out of 50 569 (see online supplementary table S2). The 3116 homozygous variants were further filtered against known BBS variations in our in-house database to identify unique homozygous variations. The final data set consisted of 234 variants.

These novel homozygous variants included one nonsense, one frame-shift, two splice mutations, nine missense with pathogenic predictions (SIFT7 or PolyPhen28) and one in-frame deletion. The 47 potentially pathogenic genes were manually screened based on gene function and previous identification of a disease. We were able to focus the subsequent studies on a single homozygous nonsense mutation, c.173T>G, p.Leu58*, in the BBIP1 gene encoding for the eighth BBSome subunit (not yet identified as a disease-causing gene). This mutation was confirmed by Sanger sequencing and found at the heterozygous state in his father's sample (mother and sibling's DNA was unavailable) (figure 1B). This mutation was absent from the EVS database and among 160 exomes performed by IntegraGen.

Depletion of BBIP1 leads to ciliopathy phenotypes in zebrafish

A search in Ensembl (Zv9) revealed in Danio rerio a single 72 amino acids protein (ENSDARP00000095567) exhibiting 69% identity with the human BBIP1. Injection of morpholino oligonucleotide (MO) targeting the start codon of bbip1 (bbip1-mo)2 resulted in bilateral cystic dilations of the pronephros, an equivalent structure to the kidney in zebrafish (figure 2A,B). The effect is specific as it could be rescued by co-injection of a bbip1 mRNA resistant to MO knockdown (figure 2B). Examination of cilia morphology showed that pronephric cilia of morphants failed to maintain a parallel orientation to the anteroposterior axis of the duct and were shorter than in controls (figure 2C–F). It is likely that cysts develop as a consequence of impaired pronephric flow. Kupffer's vesicle (analogous structure of human node) is a ciliated organ that initiates left–right asymmetry of the brain, heart and gut in zebrafish.17 It was shown previously that bbip1 morphants display abnormalities in Kupffer's vesicle.2 Twenty per cent of morphants present with situs inversus compared with three per cent in the wild-type population (figure 2G,H). Bardet–Biedl disease being characterised by retinitis pigmentosa, we also examined sections of eyes from morphant or control embryos at 3 dpf and found that the morphants showed abnormal retinal development. The control larvae developed normal retinal structures with a well-defined ganglion cell layer, inner nuclear and photoreceptor layers (figure 2I,J). The bbip1-mo-treated embryos exhibited a disorganised retina with a lack of separation between the retinal cell layers. Altogether the zebrafish data suggest a role of Bbip1 in cilium function/or assembly.

Figure 2

Bbip1 morphants exhibit cilia abnormalities. (A) Kidney tubule dilatation observed in light microscopy in an embryo injected with 0.5 mM bbip1-mo. (B) Embryos were injected with 0.5 mM bbip1-mo or bbip1cont-mo: 37% of them show formation of kidney cysts. Rescue was obtained with 50 ng/μL of bbip1 mRNA. (Numbers indicate the number of embryos examined). (C–F) Anti-acetylated-tubulin staining of wild-type (C), control morphant (D) or bbip1 morphants (E and F (single cilia)). Morphants show shorter and misdirected cilia and pronephric dilation. (G) Graph showing the percentage of morphants exhibiting situs inversus compared with controls and uninjected embryos (Numbers indicate the number of fish examined). (H) Visualisation of situs inversus in morphant in unc45b:GFP reporter line (arrows indicate atrium(a)/ventricle(v) inversion). (I–J) 5 μm section of uninjected (I) or bbip1-mo injected embryos stained with toluidine blue showing a disorganised retina. GCL, ganglion cell layer; INL, internal nuclear layer; ONL, outer nuclear layer. Scale bars: 12 m (C–E), 6 m (F), 0.2 cm (I–J). Age of embryos: 48 hpf (A–F); 120 hpf (G–H); 72 hpf (I–J). Standard deviation is indicated in B and G. Each experiment was done at least three times. Access the article online to view this figure in colour.

BBIP1 is absent from the patient's fibroblasts

To assess the impact of p.Leu58* mutation on the expression of the BBIP1 gene product, we probed patient's fibroblasts protein extracts with a previously characterised antibody against human BBIP1.2 While BBIP1 can be readily detected in extracts from controls or BBS patients with unrelated mutations, it was not detected in the patient's extracts (figure 3A). Although the c.173T>G mutation may lead to nonsense-mediated mRNA decay, it is likely that the BBIP1[Leu58*] is subject to rapid turnover because of low-folding efficiency or poor incorporation into the BBSome. Importantly, since we have previously shown that BBIP1 depletion dramatically affected BBSome assembly, it is likely that BBSome assembly is compromised in the BBIP1[Leu58*] patient.

Figure 3

The p.Leu58* mutation prevents BBIP1 assembly into the BBSome. (A) Immunodetection of BBIP1 in patient's dermal fibroblasts together with a Ponceau S staining of the membrane for protein loading control. (B) Extracts of HEK293 cells transfected with Myc-BBIP1 (WT or Leu58*) were subjected to immunoprecipitation with anti-Myc antibody. Extracts and elutes were immunoprobed for endogenous BBS4 and exogenous Myc-BBIP1. Access the article online to view this figure in colour.

Reduced incorporation of BBIP1[Leu58*] into the BBSome

The impact of p.Leu58* mutation on BBIP1 incorporation into the BBSome was assessed by testing the amounts of the BBS4 subunit that co-assembled with Myc-BBIP1 transiently expressed into HEK cells. While wild-type BBIP1 efficiently captured BBS4, little BBS4 was detected in a complex with BBIP1[Leu58*]. Since BBSome assembly is compromised in cells depleted of BBIP12 and since little BBIP1[Leu58*] becomes incorporated into the BBSome (figure 3B), we conclude that BBSome assembly must be severely affected in the patient.


As other ciliopathies, BBS is extremely heterogeneous with 17 BBS genes reported to date. Identifying novel genes remains crucial for a better understanding of the cilia-related pathogenic mechanisms of BBS and to improve the molecular diagnosis and genetic counselling. Identification of novel BBS genes represents a significant challenge as the remaining unidentified genes reside mostly in small-sized families. Herein, we describe a patient with an indisputable diagnosis of BBS. Exome sequencing revealed a nonsense mutation in BBIP1, a gene not yet known to be involved in BBS patient but that we had previously identified as the BBSome subunit playing a central role in BBSome assembly.2

NGS has accelerated discovery of new genes that are very rarely mutated in cohorts of patients for a given disease with sometimes only one family initially identified.12 ,18 Thus, functional analysis remains very important to ascertain the pathogenicity of mutations in a novel gene. Here, we were greatly aided by our prior functional characterisation of the BBSome subunit BBIP1 and its importance in ciliogenesis and BBSome assembly. In addition, the functional consequences of BBIP1[Leu58*] mutation were assessed to validate its pathogenicity. First, as expected for such a nonsense mutation, no protein was detected by western blotting analysis. Second, co-immunoprecipitation assays performed between transfected Myc-BBIP1 and endogenous BBS4 showed a dramatic decrease in the amount of BBS4 captured by BBIP1[Leu58*] compared with BBIP1, thus pointing to a major effect of the mutation on BBSome assembly. Third, the zebrafish morpholino discloses a ciliopathy phenotype as described previously, and, moreover, we show that rescue assays confirm the pathogenicity of the mutation in this model. These three functional assays confirm that this mutation has a major biological effect underlying the phenotype observed in the patient.

Importantly, our data suggest that BBSome assembly may represent a converging pathomechanism of BBS as the vast majority of BBS mutations so far tested result in aberrant BBSome assembly.19 Alternatively, mutations in BBS17/LZTFL1 or BBS3/ARL6 appear to affect BBSome trafficking to cilia without impinging on BBSome assembly.4

It is interesting to note that BBIP1 has been proposed to influence microtubule acetylation independently of the BBSome,2 which suggests that mutations in BBIP1 may lead to further symptoms besides the typical findings in BBS. However, the patient had no specific BBS features that could distinguish him from patients mutated in other BBS genes except for the absence of polydactyly, a feature from about 30% of the BBS patient population.1 Thus, it is likely that BBIP1 functions exclusively within the BBSome.

Targeted high-throughput diagnosis is currently being set for various conditions, including ciliopathies,6 and we suggest that BBIP1 be added to the screening panel as the 18th BBS gene. This patient's mutation adds to the BBS puzzle by pointing to BBIP1 as a rarely mutated BBS gene but encoding for a protein with an important ciliary function.

Web Resources:


Exome Variant Server:



We would like to thank the patient for his participation in the project and the patient's associations, Retina France (100 exomes Program) , Formicoeur, Bardet-Biedl France and Unadev, for their constant and strong support.


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  • SS, CE and NWP contributed equally to this study.

  • Contributors ES and SS provided clinical information. JM, KC, VG and CS analysed the exome sequences. CS tested the mutation in the patient and screened controls. NWP designed and conducted the co-immunoprecipitation experiment. MVN and VM supervised the cell and molecular biology experiments. MVN, SS, CS and HD wrote the manuscript. HD supervised the whole project.

  • Funding This work was funded by Retina France, Unadev, Formicoeur, API program of the Hôpitaux Universitaires de Strasbourg, the AVENIR INSERM program and the French Ministry of Health with the National PHRC program. Research in the MVN lab was supported by NIH (GM089933) and March of Dimes (1-FY11-517). Research in the US lab was supported by European IP ZF-Health and KlausTschira Stiftung.

  • Competing interests None.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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