Abstract

Transforming growth factor (TGF)-β1 (encoded by TGFB1) is the prototypic member of the TGF-β family of 33 proteins that orchestrate embryogenesis, development and tissue homeostasis1,2. Following its discovery3, enormous interest and numerous controversies have emerged about the role of TGF-β in coordinating the balance of pro- and anti-oncogenic properties4,5, pro- and anti-inflammatory effects6, or pro- and anti-fibrinogenic characteristics7. Here we describe three individuals from two pedigrees with biallelic loss-of-function mutations in the TGFB1 gene who presented with severe infantile inflammatory bowel disease (IBD) and central nervous system (CNS) disease associated with epilepsy, brain atrophy and posterior leukoencephalopathy. The proteins encoded by the mutated TGFB1 alleles were characterized by impaired secretion, function or stability of the TGF-β1–LAP complex, which is suggestive of perturbed bioavailability of TGF-β1. Our study shows that TGF-β1 has a critical and nonredundant role in the development and homeostasis of intestinal immunity and the CNS in humans.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Blobe, G. C., Schiemann, W. P. & Lodish, H. F. Role of transforming growth factor-β in human disease. N. Engl. J. Med. 342, 1350–1358 (2000).

  2. 2.

    Wu, M. Y. & Hill, C. S. TGF-β superfamily signaling in embryonic development and homeostasis. Dev. Cell 16, 329–343 (2009).

  3. 3.

    Derynck, R. et al. Human transforming growth factor-β complementary DNA sequence and expression in normal and transformed cells. Nature 316, 701–705 (1985).

  4. 4.

    Principe, D. R. et al. TGF-β: duality of function between tumor prevention and carcinogenesis. J. Natl. Cancer Inst. 106, djt369 (2014).

  5. 5.

    Silberstein, G. B. & Daniel, C. W. Reversible inhibition of mammary gland growth by transforming growth factor-β. Science 237, 291–293 (1987).

  6. 6.

    Li, M. O., Wan, Y. Y., Sanjabi, S., Robertson, A. K. & Flavell, R. A. Transforming growth factor-β regulation of immune responses. Annu. Rev. Immunol. 24, 99–146 (2006).

  7. 7.

    Pohlers, D. et al. TGF-β and fibrosis in different organs—molecular pathway imprints. Biochim. Biophys. Acta 1792, 746–756 (2009).

  8. 8.

    Miyazono, K., Hellman, U., Wernstedt, C. & Heldin, C. H. Latent high-molecular-weight complex of transforming growth factor-β1. Purification from human platelets and structural characterization. J. Biol. Chem. 263, 6407–6415 (1988).

  9. 9.

    Rifkin, D. B. Latent transforming growth factor-β (TGF-β)-binding proteins: orchestrators of TGF-β availability. J. Biol. Chem. 280, 7409–7412 (2005).

  10. 10.

    Li, M. O. & Flavell, R. A. TGF-β: a master of all T cell trades. Cell 134, 392–404 (2008).

  11. 11.

    Janssens, K. et al. Camurati–Engelmann disease: review of the clinical, radiological and molecular data of 24 families and implications for diagnosis and treatment. J. Med. Genet. 43, 1–11 (2006).

  12. 12.

    Loeys, B. L. et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat. Genet. 37, 275–281 (2005).

  13. 13.

    Shi, M. et al. Latent TGF-β structure and activation. Nature 474, 343–349 (2011).

  14. 14.

    Monteleone, G. et al. Blocking SMAD7 restores TGF-β1 signaling in chronic inflammatory bowel disease. J. Clin. Invest. 108, 601–609 (2001).

  15. 15.

    Glocker, E. O. et al. Inflammatory bowel disease and mutations affecting the interleukin-10 receptor. N. Engl. J. Med. 361, 2033–2045 (2009).

  16. 16.

    Kotlarz, D. et al. Loss of interleukin-10 signaling and infantile inflammatory bowel disease: implications for diagnosis and therapy. Gastroenterology 143, 347–355 (2012).

  17. 17.

    Shull, M. M. et al. Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease. Nature 359, 693–699 (1992).

  18. 18.

    Li, M. O., Wan, Y. Y. & Flavell, R. A. T cell–produced transforming growth factor-β1 controls T cell tolerance and regulates TH1- and TH17 cell differentiation. Immunity 26, 579–591 (2007).

  19. 19.

    Gorelik, L. & Flavell, R. A. Abrogation of TGF-β signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 12, 171–181 (2000).

  20. 20.

    Boirivant, M. et al. Inhibition of SMAD7 with a specific antisense oligonucleotide facilitates TGF-β1-mediated suppression of colitis. Gastroenterology 131, 1786–1798 (2006).

  21. 21.

    Monteleone, G. et al. Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn’s disease. N. Engl. J. Med. 372, 1104–1113 (2015).

  22. 22.

    Brionne, T. C., Tesseur, I., Masliah, E. & Wyss-Coray, T. Loss of TGF-β1 leads to increased neuronal cell death and microgliosis in mouse brain. Neuron 40, 1133–1145 (2003).

  23. 23.

    Koeglsperger, T. et al. Impaired glutamate recycling and GluN2B-mediated neuronal calcium overload in mice lacking TGF-β1 in the CNS. Glia 61, 985–1002 (2013).

  24. 24.

    De Servi, B., La Porta, C. A., Bontempelli, M. & Comolli, R. Decrease of TGF-β1 plasma levels and increase of nitric oxide synthase activity in leukocytes as potential biomarkers of Alzheimer’s disease. Exp. Gerontol. 37, 813–821 (2002).

  25. 25.

    Tesseur, I. et al. Deficiency in neuronal TGF-β signaling promotes neurodegeneration and Alzheimer’s pathology. J. Clin. Invest. 116, 3060–3069 (2006).

  26. 26.

    Arosio, B. et al. + 10 T/C polymorphisms in the gene of transforming growth factor-β1 are associated with neurodegeneration and its clinical evolution. Mech. Ageing Dev. 128, 553–557 (2007).

  27. 27.

    Amir, E. D. et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat. Biotechnol. 31, 545–552 (2013).

  28. 28.

    Li, H. & Durbin, R. Fast and accurate short-read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  29. 29.

    McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

  30. 30.

    Cingolani, P. et al. A program for annotating and predicting the effects of single-nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w 1118; iso-2; iso-3. Fly 6, 80–92 (2012).

  31. 31.

    McLaren, W. et al. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics 26, 2069–2070 (2010).

  32. 32.

    Vigeland, M. D., Gjøtterud, K. S. & Selmer, K. K. FILTUS: a desktop GUI for fast and efficient detection of disease-causing variants, including a novel autozygosity detector. Bioinformatics 32, 1592–1594 (2016).

  33. 33.

    1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015).

  34. 34.

    Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016).

  35. 35.

    Kumar, P., Henikoff, S. & Ng, P. C. Predicting the effects of coding nonsynonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081 (2009).

  36. 36.

    Adzhubei, I., Jordan, D. M. & Sunyaev, S. R. Predicting functional effect of human missense mutations using PolyPhen-2. Curr. Protoc. Hum. Genet. 76, 7.20 (2013).

  37. 37.

    Kotlarz, D. et al. Loss-of-function mutations in the IL-21 receptor gene cause a primary immunodeficiency syndrome. J. Exp. Med. 210, 433–443 (2013).

  38. 38.

    Dennler, S. et al. Direct binding of SMAD3 and SMAD4 to critical TGF-β-inducible elements in the promoter of human plasminogen activator inhibitor type 1 gene. EMBO J. 17, 3091–3100 (1998).

  39. 39.

    Walton, K. L. et al. Two distinct regions of latency-associated peptide coordinate stability of the latent transforming growth factor-β1 complex. J. Biol. Chem. 285, 17029–17037 (2010).

Download references

Acknowledgements

We are very grateful to our patients and their parents for allowing us to study their diseases. We thank the medical staff at the Dr. von Hauner Children’s Hospital, Oslo University Hospital and University Malaya Medical Center. In particular, we would like to acknowledge pathologist D. Klotz (Oslo University Hospital) for the histology of colonic biopsies. Whole-exome sequencing of family A was conducted at the Next-Generation Sequencing facility at the Dr. von Hauner Children’s Hospital under the supervision of M. Rohlfs. The sequencing service of family B was provided by the Norwegian Sequencing Centre, a national technology platform supported by the Functional Genomics and Infrastructure Programs of the Research Council of Norway and the Southeastern Regional Health Authorities, and the sequencing data of family B were analyzed by A. Holmgren. We acknowledge the assistance of the Flow Cytometry Core Facility at the Dr. von Hauner Children’s Hospital and of the Harvard Medical School CyTOF Core. Samples from the patient with CED were provided with support of the Oxford Gastrointestinal Illness Biobank and Biomedical Research Center Oxford. We gratefully acknowledge our bioinformatician, J. Puchalka, who died in a tragic accident during the course of the investigations. This work has been supported by The Leona M. and Harry B. Helmsley Charitable Trust, the Collaborative Research Consortium SFB1054 (DFG), PID-NET (BMBF), BioSysNet, the European Research Council, the Gottfried–Wilhelm–Leibniz Program (DFG), the DAAD network on ‘Rare Diseases and Personalized Therapies’, the German Center for Infection Research (DZIF) and the Care-for-Rare Foundation. W.S.L. was partly funded by University Malaya High Impact Research (UM.C/625/HIR/MOHE/CHAN/13/1). D.K. has been a scholar funded by the Else Kröner-Fresenius-Stiftung, the Daimler und Benz Stiftung and the Reinhard Frank-Stiftung.

Author information

Author notes

  1. These authors contributed equally: Daniel Kotlarz and Benjamin Marquardt.

Affiliations

  1. Dr. von Hauner Children’s Hospital, Department of Pediatrics, University Hospital LMU Munich, Munich, Germany

    • Daniel Kotlarz
    • , Benjamin Marquardt
    • , Sebastian Hollizeck
    • , Thomas Magg
    • , Anna S. Lehle
    • , Ingo Borggraefe
    • , Fabian Hauck
    • , Philip Bufler
    • , Raffaele Conca
    •  & Christoph Klein
  2. Department of Medical Genetics, Oslo University Hospital, Oslo, Norway

    • Tuva Barøy
    • , Doriana Misceo
    •  & Eirik Frengen
  3. Faculty of Medicine, University of Oslo, Oslo, Norway

    • Tuva Barøy
    • , Doriana Misceo
    • , Eirik Frengen
    •  & Petter Strømme
  4. Department of Pediatrics, University Malaya Medical Center, Kuala Lumpur, Malaysia

    • Way S. Lee
  5. Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital, Boston, MA, USA

    • Liza Konnikova
    • , Sarah M. Wall
    •  & Scott B. Snapper
  6. Harvard Medical School, Boston, MA, USA

    • Liza Konnikova
    •  & Scott B. Snapper
  7. Department of Pediatric and Newborn Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    • Liza Konnikova
  8. Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany

    • Christoph Walz
  9. Division of Pediatric and Adolescent Medicine, Oslo University Hospital, Oslo, Norway

    • Eva M. Schumacher
    • , Beint S. Bentsen
    •  & Petter Strømme
  10. Department of Pediatric Research, Pediatric Liver Kidney Alimentary Nutrition and Transplantation Research Group, Oslo University Hospital, Oslo, Norway

    • Beint S. Bentsen
  11. Translational Gastroenterology Unit and Department of Pediatrics, University of Oxford, Oxford, UK

    • Holm H. Uhlig
  12. Department of Biochemistry and Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

    • Karl-Peter Hopfner
  13. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada

    • Aleixo M. Muise
  14. Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada

    • Aleixo M. Muise
  15. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    • Aleixo M. Muise
  16. Division of Gastroenterology, Brigham and Women’s Hospital, Boston, MA, USA

    • Scott B. Snapper

Authors

  1. Search for Daniel Kotlarz in:

  2. Search for Benjamin Marquardt in:

  3. Search for Tuva Barøy in:

  4. Search for Way S. Lee in:

  5. Search for Liza Konnikova in:

  6. Search for Sebastian Hollizeck in:

  7. Search for Thomas Magg in:

  8. Search for Anna S. Lehle in:

  9. Search for Christoph Walz in:

  10. Search for Ingo Borggraefe in:

  11. Search for Fabian Hauck in:

  12. Search for Philip Bufler in:

  13. Search for Raffaele Conca in:

  14. Search for Sarah M. Wall in:

  15. Search for Eva M. Schumacher in:

  16. Search for Doriana Misceo in:

  17. Search for Eirik Frengen in:

  18. Search for Beint S. Bentsen in:

  19. Search for Holm H. Uhlig in:

  20. Search for Karl-Peter Hopfner in:

  21. Search for Aleixo M. Muise in:

  22. Search for Scott B. Snapper in:

  23. Search for Petter Strømme in:

  24. Search for Christoph Klein in:

Contributions

D.K. and C.K. designed and directed the study, managed recruitment of study participants, obtained clinical samples, supervised B.M. and interpreted the data; B.M. conducted and analyzed functional assays on heterologous cellular models; D.M., E.F. and P.S. supervised T.B. and E.M.S., initiated genetic analysis and drafted the clinical report of P2 and P3, and provided critical revision of the manuscript; T.B. acquired and interpreted genetic data from P2 and P3; R.C. conducted immunophenotypic analysis of PBMCs; T.M. and A.S.L. performed functional immunological assays; S.M.W. performed CyTOF analysis; L.K. supervised S.M.W. and analyzed the CyTOF results; S.H. performed the bioinformatics analysis of sequencing data; K.-P.H. conducted structural analysis of protein variants encoded by the identified TGFB1 mutations; W.S.L., I.B., F.H., P.B., E.M.S. and B.S.B. cared for the patients, collected patient samples and drafted clinical reports; C.W. examined histology; H.H.U. provided clinical information and a specimen from a patient with CED; A.M.M. and S.B.S. screened local cohorts of patients with very early-onset inflammatory bowel disease for mutations in TGFB1 and were instrumental in the interpretation of the human data; C.K. provided laboratory resources; and D.K. and C.K. wrote the manuscript with help from B.M. The manuscript was reviewed and approved by all co-authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Christoph Klein.

Integrated supplementary information

  1. Supplementary Figure 1 CD103 expression in lamina propria T cells from patient 1.

    CyTOF analysis of CD103 expression in colonic lamina propria mononuclear cells derived from patients without IBD (controls: uninflamed; inflamed), a patient with Crohn’s disease and P1. The percentage of CD103-expressing cells is shown for CD3+CD4+ (top), CD3+CD8+ (middle) and CD127lo/–CD25+ (bottom) T cells.

  2. Supplementary Figure 2 Sanger sequencing of patient 1.

    Chromatograms of DNA Sanger sequencing identifying a compound heterozygous mutation in TGBF1 that segregates with the disease phenotype in P1.

  3. Supplementary Figure 3 Clinical phenotype and mutational analysis of TGF-β1 deficiency in patients 2 and 3.

    a, Pedigree of consanguineous Pakistani family B with two affected children. b, Sanger sequencing results confirming segregation of the identified biallelic TGFB1 missense mutation with the disease phenotype in pedigree B. c, Gastrointestinal findings in P3. Colonoscopy (top) revealed extensive colitis, and histology on colonic biopsies (bottom) showed chronic active inflammation accompanied by abscesses and crypt branching. d, Cerebral MRI images of P3 at the age of 2 years displaying gross cortical atrophy with widening of the subarachnoid spaces, delayed myelination and marked thinning of the corpus callosum.

  4. Supplementary Figure 4 Normal STAT6 activity in lamina propria immune cells from patient 1.

    CyTOF analysis of STAT6 phosphorylation (Tyr641; p-STAT6) in lamina propria mononuclear cells derived from patients without IBD (controls: uninflamed, blue; inflamed, orange), a patient with Crohn’s disease (CD) (green) and P1 (black). Histogram plots show baseline p-STAT6 in live cells that were gated on the indicated populations (left), and the heat map representation depicts the corresponding median expression values (MEV) for p-STAT6.

  5. Supplementary Figure 5 Gating strategy for FACS analysis.

    Gating strategy used in Fig. 1d.

  6. Supplementary Figure 6 Gating strategy for CyTOF analysis.

    a,b, Gating strategies used in Supplementary Fig. 1 (a) and in Fig. 2h and Supplementary Fig. 3 (b).

  7. Supplementary Figure 7 Uncropped immunoblots.

    Uncropped original immunoblots of Fig. 2d. The cropped areas are marked in red. Molecular weight markers are indicated in kDa.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1–4

  2. Life Sciences Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41588-018-0063-6