Abstract

DPH1 variants have been associated with an ultra-rare and severe neurodevelopmental disorder, mainly characterized by variable developmental delay, short stature, dysmorphic features, and sparse hair. We have identified four new patients (from two different families) carrying novel variants in DPH1, enriching the clinical delineation of the DPH1 syndrome. Using a diphtheria toxin ADP-ribosylation assay, we have analyzed the activity of seven identified variants and demonstrated compromised function for five of them [p.(Leu234Pro); p.(Ala411Argfs*91); p.(Leu164Pro); p.(Leu125Pro); and p.(Tyr112Cys)]. We have built a homology model of the human DPH1–DPH2 heterodimer and have performed molecular dynamics simulations to study the effect of these variants on the catalytic sites as well as on the interactions between subunits of the heterodimer. The results show correlation between loss of activity, reduced size of the opening to the catalytic site, and changes in the size of the catalytic site with clinical severity. This is the first report of functional tests of DPH1 variants associated with the DPH1 syndrome. We demonstrate that the in vitro assay for DPH1 protein activity, together with structural modeling, are useful tools for assessing the effect of the variants on DPH1 function and may be used for predicting patient outcomes and prognoses.

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References

  1. 1.

    Collier RJ. Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century. Toxicon. 2001;39:1793–803.

  2. 2.

    Chen CM, Behringer RR. Ovca1 regulates cell proliferation, embryonic development, and tumorigenesis. Genes Dev. 2004;18:320–32.

  3. 3.

    Yu YR, You LR, Yan YT, Chen CM. Role of OVCA1/DPH1 in craniofacial abnormalities of Miller-Dieker syndrome. Hum Mol Genet. 2014;23:5579–96.

  4. 4.

    Alazami AM, Patel N, Shamseldin HE, Anazi S, Al-Dosari MS, Alzahrani F, et al. Accelerating novel candidate gene discovery in neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families. Cell Rep. 2015;10:148–61.

  5. 5.

    Loucks CM, Parboosingh JS, Shaheen R, Bernier FP, McLeod DR, Seidahmed MZ, et al. Matching two independent cohorts validates DPH1 as a gene responsible for autosomal recessive intellectual disability with short stature, craniofacial, and ectodermal anomalies. Hum Mutat. 2015;36:1015–9.

  6. 6.

    Riazuddin S, Hussain M, Razzaq A, Iqbal Z, Shahzad M, Polla DL, et al. Exome sequencing of Pakistani consanguineous families identifies 30 novel candidate genes for recessive intellectual disability. Mol Psychiatry. 2017;22:1604–14.

  7. 7.

    Nakajima J, Oana S, Sakaguchi T, Nakashima M, Numabe H, Kawashima H, et al. Novel compound heterozygous DPH1 mutations in a patient with the unique clinical features of airway obstruction and external genital abnormalities. J Hum Genet. 2018;63:529–32.

  8. 8.

    Sekiguchi F, Nasiri J, Sedghi M, Salehi M, Hosseinzadeh M, Okamoto N, et al. A novel homozygous DPH1 mutation causes intellectual disability and unique craniofacial features. J Hum Genet. 2018;63:487–91.

  9. 9.

    Stahl S, Mueller F, Pastan I, Brinkmann U. Factors that determine sensitivity and resistances of tumor cells towards antibody-targeted protein toxins. In: Verma R, Bonavida B, (eds.). Resistance to Immunotoxins in Cancer Therapy. Resistance to Targeted Anti-Cancer Therapeutics 6. Cham: Springer; 2015. p. 57–73.

  10. 10.

    Mayer K, Schroder A, Schnitger J, Stahl S, Brinkmann U. Influence of DPH1 and DPH5 protein variants on the synthesis of Diphthamide, the target of ADPRibosylating toxins. Toxins. 2017;9:78.

  11. 11.

    Stahl S, da Silva Mateus Seidl AR, Ducret A, Kux van Geijtenbeek S, Michel S, Racek T, et al. Loss of diphthamide pre-activates NF-kappaB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor. Proc Natl Acad Sci USA. 2015;112:10732–7.

  12. 12.

    Zhang Y, Zhu X, Torelli AT, Lee M, Dzikovski B, Koralewski RM, et al. Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme. Nature. 2010;465:891–6.

  13. 13.

    Broderick JB, Duffus BR, Duschene KS, Shepard EM. Radical S-adenosylmethionine enzymes. Chem Rev. 2014;114:4229–317.

  14. 14.

    Liu S, Milne GT, Kuremsky JG, Fink GR, Leppla SH. Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Mol Cell Biol. 2004;24:9487–97.

  15. 15.

    Maio N, Rouault TA. Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery. Biochim Biophys Acta. 2015;1853:1493–512.

  16. 16.

    Dong M, Kathiresan V, Fenwick MK, Torelli AT, Zhang Y, Caranto JD, et al. Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis. Science. 2018;359:1247–50.

  17. 17.

    Uniprot Consortium, T. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017;45:D158–D169.

  18. 18.

    Salamov AA, Nishikawa T, Swindells MB. Assessing protein coding region integrity in cDNA sequencing projects. Bioinformatics. 1998;14:384–90.

  19. 19.

    Wernersson R. Virtual Ribosome--a comprehensive DNA translation tool with support for integration of sequence feature annotation. Nucleic Acids Res. 2006;34:W385–8.

  20. 20.

    Urreizti R, Cueto-Gonzalez AM, Franco-Valls H, Mort-Farre S, Roca-Ayats N, Ponomarenko J, et al. A De novo nonsense mutation in MAGEL2 in a patient initially diagnosed as Opitz-C: similarities between Schaaf-Yang and Opitz-C syndromes. Sci Rep. 2017;7:44138.

  21. 21.

    Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014;42:W252–8.

  22. 22.

    Morris AL, MacArthur MW, Hutchinson EG, Thornton JM. Stereochemical quality of protein structure coordinates. Proteins. 1992;12:345–64.

  23. 23.

    Abraham MJ, Murtola T, Schulz, Páll S, Smith JC, Hess B, et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1-2:19–25.

  24. 24.

    Jorgensen WL, Maxwell DS, Tirado-Rives J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J Am Chem Soc. 1996;118:11225–36.

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Acknowledgements

The authors thank the families for their participation in our research studies. We are also grateful to M. Cozar for technical assistance, and to CNAG for exome sequencing. Funding was from Associació Síndrome Opitz C, Terrassa, Spain; Spanish Ministerio de Economía y Competitividad (SAF2016-75948-R, FECYT, crowdfunding PRECIPITA), Catalan Government (2014SGR932) and from CIBERER (U720), the Mindich Child Health and Development Institute (MCHDI) at the Icahn School of Medicine at Mount Sinai, and the Genetic Disease Foundation (New York, NY).

Author information

Author notes

  1. These authors contributed equally: Roser Urreizti, Klaus Mayer, Gilad D. Evrony

  2. These authors contributed equally: Ulrich Brinkmann, Bryn D. Webb,Susanna Balcells

Affiliations

  1. Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, IBUB, IRSJD, CIBERER, Barcelona, Spain

    • Roser Urreizti
    • , Laura Castilla-Vallmanya
    • , Daniel Grinberg
    •  & Susanna Balcells
  2. Roche Pharma Research and Early Development. Large Molecule Research, Roche Innovation Center, Munich, Nonnenwald 2, 82377, Penzberg, Germany

    • Klaus Mayer
    •  & Ulrich Brinkmann
  3. Center for Human Genetics & Genomics, New York University Langone Health, New York, NY, USA

    • Gilad D. Evrony
  4. Section of Medical Genetics, Mater dei Hospital, Msida, Malta

    • Edith Said
  5. Department of Anatomy and Cell Biology, University of Malta, Msida, Malta

    • Edith Said
  6. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    • Neal A. L. Cody
    • , Bruce D. Gelb
    •  & Bryn D. Webb
  7. Sema4, Stamford, CT, USA

    • Neal A. L. Cody
  8. Lead Molecular Design, S.L, Sant Cugat del Vallés, Spain

    • Guillem Plasencia
  9. Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    • Bruce D. Gelb
    •  & Bryn D. Webb
  10. Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    • Bruce D. Gelb
    •  & Bryn D. Webb

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Conflict of interest

RU, GDE, ES, LCV, BDG, DG, BDW, and SB declare no conflict of interest. KM and UB are employees of Roche. Roche is interested in identifying novel targets and approaches for disease diagnosis and therapy. NC is an employee of Sema4, a for-profit genetic testing laboratory. GP is employed by Lead Molecular Design, SL, a company that develops software and offers modeling services for pharmaceutical industries, but has no competing interests on the results of this article.

Corresponding author

Correspondence to Roser Urreizti.

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DOI

https://doi.org/10.1038/s41431-019-0374-9