Abstract

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06–1.23 Mb upstream of SOX9, and microdeletions both 1.5 Mb centromeric and 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    A fall of the base of the tongue considered as a new cause of nasopharyngeal respiratory impairment: Pierre Robin sequence, a translation. 1923. Plast. Reconstr. Surg. 93, 1301–1303 (1994).

  2. 2.

    , & Molecular bases of human neurocristopathies. Adv. Exp. Med. Biol. 589, 213–234 (2006).

  3. 3.

    , , , & Genetic and clinical heterogeneity of Stickler syndrome. Am. J. Med. Genet. 41, 44–48 (1991).

  4. 4.

    et al. Isolated Robin sequence associated with a balanced t(2;17) chromosomal translocation. J. Med. Genet. 41, e1 (2004).

  5. 5.

    et al. Pierre Robin sequence may be caused by dysregulation of SOX9 and KCNJ2. J. Med. Genet. 44, 381–386 (2007).

  6. 6.

    , , , & Mapping cis-regulatory domains in the human genome using multi-species conservation of synteny. Hum. Mol. Genet. 14, 3057–3063 (2005).

  7. 7.

    , & Conserved non-genic sequences—an unexpected feature of mammalian genomes. Nat. Rev. Genet. 6, 151–157 (2005).

  8. 8.

    et al. Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol. 3, e7 (2005).

  9. 9.

    et al. Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia. Am. J. Hum. Genet. 76, 652–662 (2005).

  10. 10.

    et al. Gene expression in pharyngeal arch 1 during human embryonic development. Hum. Mol. Genet. 14, 903–912 (2005).

  11. 11.

    & Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348–356 (1994).

  12. 12.

    & In vitro assays fail to predict in vivo effects of regulatory polymorphisms. Hum. Mol. Genet. 16, 1931–1939 (2007).

  13. 13.

    , , & Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation. Development 134, 909–919 (2007).

  14. 14.

    , , , & Comparative genomics of the SOX9 region in human and Fugu rubripes: conservation of short regulatory sequence elements within large intergenic regions. Genomics 78, 73–82 (2001).

  15. 15.

    et al. Long-range upstream and downstream enhancers control distinct subsets of the complex spatiotemporal Sox9 expression pattern. Dev. Biol. 291, 382–397 (2006).

  16. 16.

    , & SOX9cre1, a cis-acting regulatory element located 1.1 Mb upstream of SOX9, mediates its enhancement through the SHH pathway. Hum. Mol. Genet. 16, 1143–1156 (2007).

  17. 17.

    , , , & Sox genes regulate type 2 collagen expression in avian neural crest cells. Dev. Growth Differ. 48, 477–486 (2006).

  18. 18.

    et al. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev. 18, 1072–1087 (2004).

  19. 19.

    , , , & Sox9 is required for cartilage formation. Nat. Genet. 22, 85–89 (1999).

  20. 20.

    et al. Pierre Robin sequence: a series of 117 consecutive cases. J. Pediatr. 139, 588–590 (2001).

  21. 21.

    , , & Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest. Proc. Natl. Acad. Sci. USA 100, 9360–9365 (2003).

  22. 22.

    et al. KCNJ2 mutation results in Andersen syndrome with sex-specific cardiac and skeletal muscle phenotypes. Am. J. Hum. Genet. 71, 663–668 (2002).

  23. 23.

    et al. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79, 1111–1120 (1994).

  24. 24.

    & Long-range control of gene expression: emerging mechanisms and disruption in disease. Am. J. Hum. Genet. 76, 8–32 (2005).

  25. 25.

    et al. A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum. Mol. Genet. 12, 1725–1735 (2003).

  26. 26.

    & Sox proteins and neural crest development. Semin. Cell Dev. Biol. 16, 694–703 (2005).

  27. 27.

    & Importance of SoxE in neural crest development and the evolution of the pharynx. Nature 441, 750–752 (2006).

  28. 28.

    & Raising the estimate of functional human sequences. Genome Res. 17, 1245–1253 (2007).

  29. 29.

    et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545 (2002).

  30. 30.

    et al. Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene 24, 2317–2329 (2005).

Download references

Acknowledgements

We are grateful to the affected individuals and their families who participated in this study, to the Associations Françaises du Syndrome de Robin, to the Centres de Références Anomalies Cranio-Faciales Rares (AP-HP, Necker and Trousseau hospitals), and to C. Ozilou, G. Staub and G. Guédu-Molina for assistance. We thank T. Attié-Bitach, G. Couly and L. Legeai-Mallet (Necker) and V. van Heyningen and R. Hill (MRC HGU) for useful discussion. This study was underwritten by grants from the Agence Nationale de la Recherche (ERARE grant CraniRare), EUROCRAN FP5, the Fondation pour la Recherche Médicale (FRM), the MRC (UK) and the National Health and Medical Research Council (Australia). S.T. was supported in part by grant NS039818 from the US National Institutes of Health and S.B. by the FRM.

Author information

Author notes

    • Sabina Benko
    • , Judy A Fantes
    • , David R FitzPatrick
    •  & Stanislas Lyonnet

    These authors contributed equally to this work.

Affiliations

  1. INSERM U-781, Hôpital Necker–Enfants Malades, Paris, France.

    • Sabina Benko
    • , Jeanne Amiel
    • , Sophie Thomas
    • , Christelle Golzio
    • , Michel Vekemans
    • , Arnold Munnich
    • , Heather C Etchevers
    • , Anna Pelet
    •  & Stanislas Lyonnet
  2. Medical Research Council Human Genetics Unit (MRC HGU), Institute of Genetic and Molecular Medicine, Edinburgh EH4 2XU, UK.

    • Judy A Fantes
    • , Dirk-Jan Kleinjan
    • , Jacqueline Ramsay
    • , Abdelkader Essafi
    • , Simon Heaney
    • , David McBride
    • , Malcolm Fisher
    • , Paul Perry
    • , Nicholas D Hastie
    •  & David R FitzPatrick
  3. Assistance Publique–Hôpitaux de Paris (AP-HP), Département de Génétique, Hôpital Necker–Enfants Malades, Paris, France.

    • Jeanne Amiel
    • , Michel Vekemans
    • , Arnold Munnich
    •  & Stanislas Lyonnet
  4. Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia.

    • Negar Jamshidi
    • , Christopher T Gordon
    • , Nicky Kilpatrick
    • , Paul Thomas
    •  & Peter G Farlie
  5. Université Paris Descartes, Faculté de Médecine, Paris, France.

    • Véronique Abadie
    • , Michel Vekemans
    • , Arnold Munnich
    •  & Stanislas Lyonnet
  6. AP-HP, Service de Pédiatrie, Hôpital Necker–Enfants Malades, Paris, France.

    • Véronique Abadie
  7. Fundación Jiménez Díaz, Genética, Ciberer Madrid, Spain.

    • Carmen Ayuso
  8. CHRU de Lille, Hôpital Jeanne de Flandre, Lille, France.

    • Muriel Holder-Espinasse
  9. North Thames Regional Genetics Service, Great Ormond Street Hospital, London, UK.

    • Melissa M Lees
  10. AP-HP, Service de Chirurgie Maxillo-Faciale et Chirurgie Plastique, Hôpital d'Enfants Armand Trousseau, Paris, France.

    • Arnaud Picard
    •  & Marie-Paule Vazquez
  11. Université Pierre et Marie Curie–Paris 6, UFR de Médecine Pierre et Marie Curie, Paris.

    • Arnaud Picard
    •  & Marie-Paule Vazquez
  12. Wessex Clinical Genetics Academic Group, Division of Human Genetics, University of Southampton, Southampton, UK.

    • I Karen Temple
  13. Department of Biology, École Normale Supérieure, CNRS UMR-8541, Paris, France.

    • Hugues Roest Crollius

Authors

  1. Search for Sabina Benko in:

  2. Search for Judy A Fantes in:

  3. Search for Jeanne Amiel in:

  4. Search for Dirk-Jan Kleinjan in:

  5. Search for Sophie Thomas in:

  6. Search for Jacqueline Ramsay in:

  7. Search for Negar Jamshidi in:

  8. Search for Abdelkader Essafi in:

  9. Search for Simon Heaney in:

  10. Search for Christopher T Gordon in:

  11. Search for David McBride in:

  12. Search for Christelle Golzio in:

  13. Search for Malcolm Fisher in:

  14. Search for Paul Perry in:

  15. Search for Véronique Abadie in:

  16. Search for Carmen Ayuso in:

  17. Search for Muriel Holder-Espinasse in:

  18. Search for Nicky Kilpatrick in:

  19. Search for Melissa M Lees in:

  20. Search for Arnaud Picard in:

  21. Search for I Karen Temple in:

  22. Search for Paul Thomas in:

  23. Search for Marie-Paule Vazquez in:

  24. Search for Michel Vekemans in:

  25. Search for Hugues Roest Crollius in:

  26. Search for Nicholas D Hastie in:

  27. Search for Arnold Munnich in:

  28. Search for Heather C Etchevers in:

  29. Search for Anna Pelet in:

  30. Search for Peter G Farlie in:

  31. Search for David R FitzPatrick in:

  32. Search for Stanislas Lyonnet in:

Contributions

S.B., J.A.F. and A.P. performed molecular genetics studies. J.A.F., C.T.G. and N.J. performed chromosomal studies. S.B. and J.R. performed the in vitro enhancer activity experiments. S.B., S.T., C.G., M.V. and H.C.E. performed human expression studies. J.R., S.B. and A.E. performed immunoprecipitation experiments. D.-J.K. performed transgenic assays. J.A.F., S.H., P.P. and D.B. performed the in vivo chromatin compaction studies. M.F. did the OPT image analysis. S.B. and H.R.C. performed the comparative genomic analysis. J.A., V.A., C.A., M.H.-E., N.K., M.M.L., A.P., I.K.T., M.V., P.T., M.-P.V., D.R.F. and S.L. recruited individuals and families affected with PRS. S.L., D.R.F., P.G.F. and J.A. contributed to the concept, strategy, study design and project management. S.B., H.C.E., S.T., P.G.F., D.R.F. and S.L. contributed to the writing of the manuscript. All authors discussed the results.

Corresponding authors

Correspondence to David R FitzPatrick or Stanislas Lyonnet.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5, Supplementary Note, Supplementary Methods and Supplementary Table 1

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ng.329

Further reading