TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum

Journal name:
Nature Genetics
Year published:
Published online
Corrected online


Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ~5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.

At a glance


  1. In vivo assay of TTC21B variants in mid-somitic zebrafish embryos.
    Figure 1: In vivo assay of TTC21B variants in mid-somitic zebrafish embryos.

    (a) Lateral and dorsal views of ttc21b morphants. Morpholino (MO)-based suppression of ttc21b results in gastrulation defects. Class I is defined as having a shortened anterior-posterior body axis with small anterior structures and mild somite defects. Class II is defined as having a severely shortened body axis, severely affected anterior structures, broadening and kinking of the notochord, thinning and widening of the somites and tail extension defects. (b) In vivo rescue assay of ttc21b MO with human mRNA. Co-injection of wildtype (WT) human TTC21B with ttc21b translation-blocking MO (tb-MO) results in significant rescue at the ten-somite stage, whereas mRNAs encoding missense alleles result in either partial rescue (p.Pro209Leu, p.Arg411Gly or p.Thr1103Arg) or results that are indistinguishable from MO alone (p.Ile1208Ser). χ2 values for rescue compared to WT are denoted as *P < 0.05 or ***P < 0.0001. (c) Whole embryo flat mounts hybridized in situ with krox20, pax2 and myoD riboprobes. Measurements reflect phenotypes at two different axes: length of the notochord as indicated by adaxial cell labeling (l) and width spanning from the lateral ends of the fifth appreciable somites (w), expressed as a ratio (w/l). (d) Quantitative morphological data for age-matched embryos, measured as indicated in c. The variants shown are significantly different from WT rescue. Student's t-test values for rescue compared to WT are denoted as *P < 0.05. Error bars show s.e.m.

  2. In vitro rescue assay of cilia length defects in mIMCD3-Ttc21b shRNA cells.
    Figure 2: In vitro rescue assay of cilia length defects in mIMCD3-Ttc21b shRNA cells.

    (a) Immunofluorescent staining of mIMCD3-EV or mIMCD3-Ttc21b shRNA cells transfected transiently with plasmids encoding wildtype (WT) or mutant versions of pCAG-V5-TTC21B-IRES-EGFP constructs demonstrate failure to rescue shortened cilia phenotypes. We detected cilia and centrosomes with anti-acetylated α-tubulin and anti-γ-tubulin (red), green signal indicates transfected cells (GFP) and nuclei are stained with Hoechst dye (blue). Asterisks indicate cilia on transiently transfected cells, dashed boxes depict inset. EV, empty vector. Horizontal scale bars, 10 μm; vertical scale bars, 4 μm (insets). (b) Quantification of cilia length measurements. Whereas wildtype TTC21B rescues the cilia length defects induced by Ttc21b shRNA, mutant proteins result in either partial rescue (p.Pro209Leu, p.Arg411Gly or p.Thr1103Arg) or are indistinguishable from shRNA cells alone (p.Ile1208Ser). Student's t-test values for rescue compared to WT are denoted as *P < 0.0001. Green bar, mIMCD3-EV cells; blue bars, mIMCD3-Ttc21b shRNA cells; error bars, s.e.m. (see Supplementary Table 6 for measurement data). (c) Protein stability defects for some TTC21B missense variants. Compared to wildtype, p.Pro209Leu and p.Arg411Gly result in diminished levels of TTC21B; we did not detect the p.Ile1208Ser protein (see Supplementary Table 7 for densitometry data). Na+/K+-ATPase was used as a loading control. NT, not transfected. (d) Transiently transfected pCAG-V5-TTC21B-IRES-EGFP constructs express at similar levels in mIMCD3 cells. RT-PCR data are shown for human TTC21B amplified from complementary DNA (cDNA) generated from total RNA extracted from mIMCD3 cells 72 h post transfection. Murine β-actin was used as a loading control.

  3. TTC21B mutant proteins mislocalize in photoreceptor sensory cilia.
    Figure 3: TTC21B mutant proteins mislocalize in photoreceptor sensory cilia.

    We transfected neonatal rat retinas with expression plasmids encoding V5-tagged wildtype (WT) and mutant TTC21B cDNAs and an IRES-EGFP cassette using in vivo electroporation. Four weeks post transfection, the transfected portions of the retinas (EGFP-positive) were stained with V5 antibody (red). The images shown are three dimensional reconstructions of confocal image stacks. Grids are included to show perspective; grid size is 8.2 mm in all images. Wildtype TTC21B localizes to the transition zones of photoreceptor cilia in transfected cells. The p.Arg411Gly mutant protein localized predominantly to the transition zone of transfected cells but more diffusely than the wildtype protein. In contrast, p.Pro209Leu and p.Thr1103Arg mutant proteins were present both in transition zones and inner segments as well as cell bodies of the transfected cells. We did not detect p.Ile1208Ser protein in transfected (GFP-positive) photoreceptor cells. The choroid (Ch) is visible in some images due to detection of mouse immunoglobulin in choroidal blood vessels by the mouse secondary antibody used. White arrows indicate examples. IS, inner segment; ONL, outer nuclear layer.

  4. Summary of all TTC21B variants detected.
    Figure 4: Summary of all TTC21B variants detected.

    (a) Pedigrees of six ciliopathy families in which two TTC21B mutations are sufficient to explain disease. JATD, Jeune asphyxiating thoracic dystrophy; NPHP, nephronophthisis. Filled circles or squares indicate individuals clinically diagnosed with a ciliopathy; unfilled circles or squares indicates phenotypically normal individuals. (b) Schematic of the human TTC21B genomic locus on chromosome 2; black boxes represent the 29 exons. (c) Human TTC21B protein is depicted as a black line, with tetratricopeptide (TPR) domains shown in blue. (d) All previously unreported variants detected by medical resequencing of TTC21B in our cohort of 753 ciliopathy cases and 398 controls are shown with respect to their genomic and protein locations (dashed lines from b and c). Overall pathogenicity of each variant as determined by in vivo functional assay (Supplementary Table 2) is indicated with asterisks. Green, benign; orange, hypomorphic; red, null. Boxes around asterisks indicate that a variant was detected in more than one individual. With the exception of p.Pro209Leu, all alleles were detected in heterozygosity.

  5. In vivo modeling of TTC21B genetic interaction with other ciliopathy loci.
    Figure 5: In vivo modeling of TTC21B genetic interaction with other ciliopathy loci.

    Zebrafish ttc21b interacts genetically with all loci known to contribute to disease in our ciliopathy cohort, including bbs1, bbs2, bbs4, bbs6, bbs7, bbs10, bbs12, tmem216, nphp4, cc2d2a, rpgrip1l, mks1 and cep290 (Table 1). Co-injection of a subeffective dose of ttc21b morpholino with any other single ciliopathy morpholino markedly exacerbates either the overall penetrance of gastrulation phenotypes (in comparison to the same doses injected alone) or a specific severity class.

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NCBI Reference Sequence

Change history

Corrected online 29 March 2011
In the version of this article initially published, the authors should have acknowledged that the work was also funded by a grant from the European Union (EU-SYSCILIA) to E.E.D., C.A.J., P.L.B. and N.K. The error has been corrected in the HTML and PDF versions of the article.


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Author information


  1. Center for Human Disease Modeling, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.

    • Erica E Davis,
    • Bill H Diplas,
    • Lisa M Davey &
    • Nicholas Katsanis
  2. Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA.

    • Erica E Davis &
    • Nicholas Katsanis
  3. F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

    • Qi Zhang,
    • Qin Liu &
    • Eric A Pierce
  4. Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Birmingham, UK.

    • Jane Hartley &
    • Eamonn R Maher
  5. Laboratoire de Génétique Médicale EA3949, Avenir INSERM, Université de Strasbourg, Strasbourg, France.

    • Corinne Stoetzel &
    • Hélène Dollfus
  6. Section of Ophthalmology and Neurosciences, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, UK.

    • Katarzyna Szymanska,
    • Clare V Logan &
    • Colin A Johnson
  7. Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA.

    • Gokul Ramaswami,
    • Edgar A Otto &
    • Friedhelm Hildebrandt
  8. Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA.

    • Donna M Muzny,
    • David A Wheeler,
    • Margaret Morgan,
    • Lora R Lewis &
    • Richard A Gibbs
  9. National Institutes of Health Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.

    • $affiliationAuthor,
    • Alice C Young,
    • Pedro Cruz,
    • Praveen Cherukuri,
    • Baishali Maskeri,
    • Nancy F Hansen,
    • James C Mullikin,
    • Robert W Blakesley,
    • Gerard G Bouffard &
    • Eric D Green
  10. Genoscope Centre National de Séquençage, Crémieux, Evry, France.

    • Gabor Gyapay
  11. University Children's Hospital, Heidelberg, Germany.

    • Susanne Rieger &
    • Burkhard Tönshoff
  12. Department of Pediatrics, University Hospital of Geneva, Switzerland.

    • Ilse Kern
  13. Department of Pediatrics, Kasralainy School of Medicine, Cairo University, Cairo, Egypt.

    • Neveen A Soliman
  14. Division of Nephrology, University Children's Hospital Zurich, Zurich, Switzerland.

    • Thomas J Neuhaus
  15. Department of Neurology, University of Utah School of Medicine, Salt Lake City, Utah, USA.

    • Kathryn J Swoboda
  16. Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, USA.

    • Kathryn J Swoboda
  17. Istanbul University, Istanbul Medical Faculty, Medical Genetics, Millet Caddesi, Capa, Fatih, Istanbul, Turkey.

    • Hulya Kayserili
  18. Developmental Pediatrics, University of Hawaii at Manoa, Honolulu, Hawaii, USA.

    • Tomas E Gallagher
  19. Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA.

    • Richard A Lewis
  20. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

    • Richard A Lewis
  21. Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

    • Richard A Lewis
  22. Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.

    • Richard A Lewis
  23. Center for Human Genetics, Bioscientia, Ingelheim, Germany.

    • Carsten Bergmann
  24. Department of Human Genetics, Rheinisch-Westfälische Technische Hochschule (RWTH) University of Aachen, Aachen, Germany.

    • Carsten Bergmann
  25. Inserm U-983, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France.

    • Sophie Saunier
  26. Molecular Medicine Unit, Institute of Child Health, University College London, London, UK.

    • Peter J Scambler &
    • Philip L Beales
  27. Department of Neurosciences, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, USA.

    • Joseph G Gleeson
  28. Département de Génétique et INSERM U-781, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France.

    • Tania Attié-Bitach
  29. Howard Hughes Medical Institute and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA.

    • Friedhelm Hildebrandt
  30. Wilmer Eye Institute and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Nicholas Katsanis


Experiments were designed by E.E.D., E.A.P. and N.K. Mutational screening, analysis and confirmation was conducted by E.E.D., J.H., C.S., K.S., G.R., C.V.L., D.M.M., A.C.Y., D.A.W., P. Cruz., M.M., L.R.L., P. Cherukuri., B.M., N.F.H., J.C.M., R.W.B., G.G.B., the NISC Comparative Sequencing Program, G.G., E.A.O., J.G.G., T.A.-B., C.A.J., E.D.G. and R.A.G. Ciliopathy case samples were provided by J.H., S.R., B.T., I.K., N.A.S., T.J.N., K.J.S., H.K., T.E.G., R.A.L., C.B., S.S., P.J.S., P.L.B., J.G.G., E.R.M., T.A.-B., H.D., C.A.J., F.H. and N.K. In vivo and in vitro functional studies were carried out by E.E.D., Q.Z., Q.L., B.H.D. and L.M.D. The manuscript was written by E.E.D., Q.Z., E.A.P. and N.K. with helpful comments from C.B., J.G.G., E.R.M., T.A.-B., C.A.J. and F.H.

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