Exome sequencing identifies the cause of a mendelian disorder

Journal name:
Nature Genetics
Volume:
42,
Pages:
30–35
Year published:
DOI:
doi:10.1038/ng.499
Received
Accepted
Published online

Abstract

We demonstrate the first successful application of exome sequencing to discover the gene for a rare mendelian disorder of unknown cause, Miller syndrome (MIM%263750). For four affected individuals in three independent kindreds, we captured and sequenced coding regions to a mean coverage of 40× and sufficient depth to call variants at ~97% of each targeted exome. Filtering against public SNP databases and eight HapMap exomes for genes with two previously unknown variants in each of the four individuals identified a single candidate gene, DHODH, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway. Sanger sequencing confirmed the presence of DHODH mutations in three additional families with Miller syndrome. Exome sequencing of a small number of unrelated affected individuals is a powerful, efficient strategy for identifying the genes underlying rare mendelian disorders and will likely transform the genetic analysis of monogenic traits.

At a glance

Figures

  1. Clinical characteristics of an individual with Miller syndrome and an individual with methotrexate embryopathy.
    Figure 1: Clinical characteristics of an individual with Miller syndrome and an individual with methotrexate embryopathy.

    (a,b) A 9-year-old boy with Miller syndrome caused by mutations in DHODH. Facial anomalies (a) include cupped ears, coloboma of the lower eyelids, prominent nose, micrognathia and absence of the fifth digits of the feet (b). (c,d) A 26-year-old man with methotrexate embryopathy. Note the cupped ears, hypertelorism, sparse eyebrows and prominent nose (c) accompanied by absence of the fourth and fifth digits of the feet (d). c and d are reprinted with permission from ref. 30.

  2. Genomic structure of the exons encoding the open reading frame of DHODH.
    Figure 2: Genomic structure of the exons encoding the open reading frame of DHODH.

    DHODH is composed of nine exons that encode untranslated regions (UTR) (orange) and protein coding sequence (blue). Arrows indicate the locations of 11 different mutations found in 6 families with Miller syndrome.

Change history

22 November 2009
In the version of this article initially published online, the panels of Figure 1 were incorrectly labeled. The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. Shendure, J. & Ji, H. Next-generation DNA sequencing. Nat. Biotechnol. 26, 11351145 (2008).
  2. Ng, S.B. et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461, 272276 (2009).
  3. Choi, M. et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc. Natl. Acad. Sci. USA published online, doi:10.1073/pnas.0910672106 (27 October 2009).
  4. Hodges, E. et al. Genome-wide in situ exon capture for selective resequencing. Nat. Genet. 39, 15221527 (2007).
  5. Stenson, P.D. et al. The human gene mutation database: 2008 update. Genome Med 1, 13 (2009).
  6. Kryukov, G.V., Pennacchio, L.A. & Sunyaev, S.R. Most rare missense alleles are deleterious in humans: implications for complex disease and association studies. Am. J. Hum. Genet. 80, 727739 (2007).
  7. Chen, C.T., Wang, J.C. & Cohen, B.A. The strength of selection on ultraconserved elements in the human genome. Am. J. Hum. Genet. 80, 692704 (2007).
  8. Ahituv, N. et al. Deletion of ultraconserved elements yields viable mice. PLoS Biol. 5, e234 (2007).
  9. Pruitt, K.D. et al. The consensus coding sequence (CCDS) project: identifying a common protein-coding gene set for the human and mouse genomes. Genome Res. 19, 13161323 (2009).
  10. Toydemir, R.M. et al. Mutations in embryonic myosin heavy chain (MYH3) cause Freeman-Sheldon syndrome and Sheldon-Hall syndrome. Nat. Genet. 38, 561565 (2006).
  11. Miller. M., Fineman, R. & Smith, D.W. Postaxial acrofacial dysostosis syndrome. J. Pediatr. 95, 970975 (1979).
  12. Splendore, A., Passos-Bueno, M.R., Jabs, E.W., Van Maldergem, L. & Wulfsberg, E.A. TCOF1 mutations excluded from a role in other first and second branchial arch-related disorders. Am. J. Med. Genet. 111, 324327 (2002).
  13. Fineman, R.M. Recurrence of the postaxial acrofacial dysostosis syndrome in a sibship: implications for genetic counseling. J. Pediatr. 98, 8788 (1981).
  14. Oglivy-Stuart, A.L. & Parsons, A.C. Miller syndrome (postaxial acrofacial dysostosis): further evidence for autosomal recessive inheritance and expansion of the phenotype. J. Med. Genet. 28, 695700 (1991).
  15. Donnai, D., Hughes, H.E. & Winter, R.M. Postaxial acrofacial dysostosis (Miller) syndrome. J. Med. Genet. 24, 422425 (1987).
  16. Genée, E. Une forme extensive de dysostose mandibulo-faciale. J. Genet. Hum. 17, 4552 (1969).
  17. Pereira, S.C.S., Rocha, C.M.G., Guion-Almeida, M.L. & Richieri-Costa, A. Postaxial acrofacial dysostosis: report on two patients. Am. J. Med. Genet. 44, 274279 (1992).
  18. Robinow, M., Johnson, G.F. & Apesos, J. Robin sequence and oligodactyly in mother and son. Am. J. Med. Genet. 25, 293297 (1986).
  19. Grabar, P.B., Rozman, B., Logar, D., Praprotnik, S. & Dolzan, V. Dihydroorotate dehydrogenase polymorphism influences the toxicity of leflunomide treatment in patients with rheumatoid arthritis. Ann. Rheum. Dis. 68, 13671368 (2009).
  20. Brosnan, M.E. & Brosnan, J.T. Orotic acid excretion and arginine metabolism. J. Nutr. 137, 1656S1661S (2007).
  21. Breedveld, F.C. & Dayer, J.-M. Leflunomide: mode of action in the treatment of rheumatoid arthritis. Ann. Rheum. Dis. 59, 841849 (2000).
  22. Morgan, T.H. Sex limited inheritance in Drosophila . Science 32, 120122 (1910).
  23. Jarry, B. & Falk, D. Functional diversity within the rudimentary locus of Drosophila melanogaster . Mol. Gen. Genet. 135, 113122 (1974).
  24. Conner, T.W. & Rawls, J.M. Jr. Analysis of the phenotypes exhibited by rudimentary-like mutants of Drosophila melanogaster . Biochem. Genet. 20, 607619 (1982).
  25. Fukushima, R. et al. Teratogenicity study of the dihydroorotate-dehydrogenase inhibitor and protein tyrosine kinase inhibitor Lefunomide in mice. Reprod. Toxicol. 24, 310316 (2007).
  26. Imose, M. et al. Lefunomide protects From T-cell–mediated liver injury in mice through inhibition of nuclear factor κB. Hepatology 40, 11601169 (2004).
  27. Bushdid, P.B., Brantley, D.M. & Yull, F.E. Inhibition of NF-κB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis. Nature 392, 615618 (1998).
  28. Luetteke, N.C. et al. TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73, 263278 (1993).
  29. Chiang, C. et al. Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function. Dev. Biol. 236, 421435 (2001).
  30. Bawle E.V. et al. Teratology 57, 5155 (1978).
  31. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 18511858 (2008).

Download references

Author information

  1. These authors contributed equally to this work.

    • Sarah B Ng &
    • Kati J Buckingham

Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

    • Sarah B Ng,
    • Choli Lee,
    • Deborah A Nickerson,
    • Jay Shendure &
    • Michael J Bamshad
  2. Department of Pediatrics, University of Washington, Seattle, Washington, USA.

    • Kati J Buckingham,
    • Abigail W Bigham,
    • Holly K Tabor &
    • Michael J Bamshad
  3. Treuman Katz Center for Pediatric Bioethics, Seattle Children's Hospital, Seattle, Washington, USA.

    • Holly K Tabor
  4. Department of Pediatrics, University of Utah, Salt Lake City, Utah, USA.

    • Karin M Dent
  5. Department of Human Genetics, University of Utah, Salt Lake City, Utah, USA.

    • Chad D Huff
  6. Institute of Systems Biology, Seattle, Washington, USA.

    • Paul T Shannon
  7. Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, USA.

    • Ethylin Wang Jabs
  8. Department of Pediatrics, Johns Hopkins University, Baltimore, Maryland, USA.

    • Ethylin Wang Jabs
  9. Seattle Children's Hospital, Seattle, Washington, USA.

    • Michael J Bamshad

Contributions

The project was conceived and experiments planned by M.J.B., D.A.N. and J.S. Review of phenotypes and sample collection were performed by E.W.J. and M.J.B. Experiments were performed by S.B.N., K.J.B. and C.L. Genetic counseling and ethical consultation were provided by K.M.D. and H.K.T. Data analysis were performed by S.B.N., K.J.B., A.W.B., C.D.H., P.T.S. and J.S. The manuscript was written by M.J.B., J.S., S.B.N. and K.J.B. All aspects of the study were supervised by M.J.B., D.A.N. and J.S.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (820K)

    Supplementary Figures 1–3

Additional data