BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1−/−-defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation

Generation of induced pluripotent stem cells, KCi001-A derived from a Bardet-Biedl syndrome patient compound heterozygous for the BBS1 variants c.1169T>G/c.1135G>C

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with a wide range of symptoms including obesity, retinal dystrophy, polycystic kidney disease, polydactyly, hypogonadism and learning difficulties. Here we describe the successful generation of an induced pluripotent stem cell (iPSC) KCi001-A from a BBS patient compound heterozygous for two disease causing BBS1 variants c.1169T>G, p. (Met390Arg)/c.1135G>C, p.(Gly370Arg).Resource tableUnlabelled TableUnique stem cell line identifierKCi001-AAlternative name(s) of stem cell lineBBS1 Clone10InstitutionKennedy Center, RigshospitaletContact information of distributorLisbeth Birk Møller, [email protected] of cell lineInduced pluripotent stem cell line (iPSC)OriginHumanAdditional origin infoFemale, CaucasianCell sourceDermal fibroblastsClonalityClonalMethod of reprogrammingNucleofection with non-integrating episomal plasmids carrying OCT3/4, SOX2, KLF4, L-MYC, LIN28 and shP53Genetic modificationNAType of modificationNAAssociated diseaseAutosomal recessive Bardet-Biedl syndromeGene/locusBBS1, Chr11: g.66293652 T > G, p.(Met390Arg); g.66293618G > C, p.(Gly379Arg); compound heterozygous.Ref sequence: NM_024649.4Method of modificationNAName of transgene or resistanceNAInducible/constitutive systemNADate archived/stock date25-01-2018Cell line repository/bankNAEthical approvalThe study was approved by the regional scientific ethical committee in the Capital Region of Denmark (H-3-2014-140). Written informed consent was obtained from the patients

Generation of induced pluripotent stem cells, KCi002-A derived from a patient with Bardet-Biedl syndrome homozygous for the BBS10 variant c.271insT

Bardet-Biedl syndrome (BBS) is genetically heterogeneous with at least 21 genes involved, and BBS10 encodes, together with BBS6 and BBS12, chaperonin-like proteins which are important for the assembly of the multiprotein complex, the BBSome encoded by other BBS genes. Here we describe the successful generation of an induced pluripotent stem cell (iPSC) line KCi002-A from a male with BBS, homozygous for the disease causing variant c.271insT, p.(Cys91fsX95) in BBS10.Resource tableUnlabelled TableUnique stem cell line identifierKCi002-AAlternative name(s) of stem cell lineBBS10 Clone1AInstitutionRigshospitalet, Kennedy CenterContact information of distributorLisbeth Birk Møller, [email protected] of cell lineinduced pluripotent stem cell (iPSC)OriginHumanAdditional origin infoMale, CaucasianCell sourceDermal fibroblastsClonalityClonalMethod of reprogrammingNucleofection with non-integrating episomal plasmids carrying OCT3/4, SOX2, KLF4, L-MYC, LIN28 and shP53Genetic modificationNAType of modificationNAAssociated diseaseAutosomal recessive Bardet-Biedl syndromeGene/locusBBS10, Chr 12: g.7674149insT, p.(Cys91fsX95), homozygous.Ref sequence: NM_024685.3Method of modificationNAName of transgene or resistanceNAInducible/constitutive systemNADate archived/stock date25-01-2018Cell line repository/bankNAEthical approvalThe study was approved by the regional scientific ethical committee in the Capital Region of Denmark (H-3-2014-140). Written informed consent was obtained from the patients

Mutational analysis of TSC1 and TSC2 in Danish patients with tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in the skin and other organs, including brain, heart, lung, kidney and bones. TSC is caused by mutations in TSC1 and TSC2. Here, we present the TSC1 and TSC2 variants identified in 168 Danish individuals out of a cohort of 327 individu