Advertisement

Identification of putative second genetic hits in schizophrenia carriers of high-risk copy number variants and resequencing in additional samples

  • Julio Rodríguez-López
  • Beatriz Sobrino
  • Jorge Amigo
  • Noa Carrera
  • Julio Brenlla
  • Santiago Agra
  • Eduardo Paz
  • Ángel Carracedo
  • Mario Páramo
  • Manuel Arrojo
  • Javier Costas
Original Paper

Abstract

Copy number variants (CNVs) conferring risk of schizophrenia present incomplete penetrance, suggesting the existence of second genetic hits. Identification of second hits may help to find genes with rare variants of susceptibility to schizophrenia. The aim of this work was to search for second hits of moderate/high risk in schizophrenia carriers of risk CNVs and resequencing of the relevant genes in additional samples. To this end, ten patients with risk CNVs at cytobands 15q11.2, 15q11.2-13.1, 16p11.2, or 16p13.11, were subjected to whole-exome sequencing. Rare single nucleotide variants, defined as those absent from main public databases, were classified according to bioinformatic prediction of pathogenicity by CADD scores. The average number of rare predicted pathogenic variants per sample was 13.6 (SD 2.01). Two genes, BFAR and SYNJ1, presented rare predicted pathogenic variants in more than one sample. Follow-up resequencing of these genes in 432 additional cases and 432 controls identified a significant excess of rare predicted pathogenic variants in case samples at SYNJ1. Taking into account its function in clathrin-mediated synaptic vesicle endocytosis at presynaptic terminals, our results suggest an impairment of this process in schizophrenia.

Keywords

High-throughput nucleotide sequencing Exome DNA copy number variations Psychosis Rare variant 

Notes

Acknowledgements

This work was supported by Grant CP11/00163 from Instituto de Salud Carlos III, cofounded by FEDER; to JC, by agreement between SERGAS and Fundación Pública Galega de Medicina Xenómica, and by the Innopharma project (USC). The founders had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The genotyping service was carried out at CEGEN-PRB2-ISCIII; it is supported by Grant PT13/0001, ISCIII-SGEFI/FEDER. The authors would like to thank Centro de Supercomputación de Galicia (CESGA) for the use of their computing facilities and the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926), and the Heart GO Sequencing Project (HL-103010).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

406_2017_799_MOESM1_ESM.pdf (156 kb)
Supplementary Figure 1. Schematic representation of the study. Each color represents the different steps carried out in the study. Circles represent the subset of samples of the initial cohort used in each step, hexagons represent the specific assay, and rectangles represent the objective pursued in each step of the study (PDF 155 kb)
406_2017_799_MOESM2_ESM.xlsx (16 kb)
Supplementary Table 1. Main clinical characteristics and genetic findings of the ten schizophrenic CNV carriers (XLSX 15 kb)
406_2017_799_MOESM3_ESM.xlsx (23 kb)
Supplementary Table 2. Putative second hit SNVs identified in the present study (XLSX 23 kb)

References

  1. 1.
    Malhotra D, Sebat J (2012) CNVs: harbingers of a rare variant revolution in psychiatric genetics. Cell 148:1223–1241. doi: 10.1016/j.cell.2012.02.039 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Rees E, Walters JT, Georgieva L, Isles AR, Chambert KD, Richards AL, Mahoney-Davies G, Legge SE, Moran JL, McCarroll SA, O’Donovan MC, Owen MJ, Kirov G (2014) Analysis of copy number variations at 15 schizophrenia-associated loci. Br J Psychiatry 204:108–114. doi: 10.1192/bjp.bp.113.131052 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Walsh T, McClellan JM, McCarthy SE, Addington AM, Pierce SB, Cooper GM et al (2008) Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science 320:539–543. doi: 10.1126/science.1155174 CrossRefPubMedGoogle Scholar
  4. 4.
    Girirajan S, Eichler EE (2010) Phenotypic variability and genetic susceptibility to genomic disorders. Hum Mol Genet 19:R176–R187. doi: 10.1093/hmg/ddq366 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Girirajan S, Rosenfeld JA, Cooper GM, Antonacci F, Siswara P, Itsara A et al (2010) A recurrent 16p12.1 microdeletion supports a two-hit model for severe developmental delay. Nat Genet 42:203–209. doi: 10.1038/ng.534 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    O’Roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, Girirajan S et al (2011) Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet 43:585–589. doi: 10.1038/ng.835 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Mitchell KJ, Porteous DJ (2011) Rethinking the genetic architecture of schizophrenia. Psychol Med 41:19–32. doi: 10.1017/S003329171000070X CrossRefPubMedGoogle Scholar
  8. 8.
    Girirajan S, Rosenfeld JA, Coe BP, Parikh S, Friedman N, Goldstein A et al (2012) Phenotypic heterogeneity of genomic disorders and rare copy-number variants. N Engl J Med 367:1321–1331. doi: 10.1056/NEJMoa1200395 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kirov G, Rees E, Walters JT, Escott-Price V, Georgieva L, Richards AL et al (2013) The penetrance of copy number variations for schizophrenia and developmental delay. Biol Psychiatry 75:378–385. doi: 10.1016/j.biopsych.2013.07.022 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Need AC, McEvoy JP, Gennarelli M, Heinzen EL, Ge D, Maia JM et al (2012) Exome sequencing followed by large-scale genotyping suggests a limited role for moderately rare risk factors of strong effect in schizophrenia. Am J Hum Genet 91:303–312. doi: 10.1016/j.ajhg.2012.06.018 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Purcell SM, Moran JL, Fromer M, Ruderfer D, Solovieff N, Roussos P et al (2014) A polygenic burden of rare disruptive mutations in schizophrenia. Nature 506:185–190. doi: 10.1038/nature12975 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Genovese G, Fromer M, Stahl EA, Ruderfer DM, Chambert K, Landén M et al (2016) Increased burden of ultra-rare protein-altering variants among 4,877 individuals with schizophrenia. Nat Neurosci 19:1433–1441. doi: 10.1038/nn.4402 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Xu B, Roos JL, Dexheimer P, Boone B, Plummer B, Levy S et al (2011) Exome sequencing supports a de novo mutational paradigm for schizophrenia. Nat Genet 43:864–868. doi: 10.1038/ng.902 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    McCarthy SE, Gillis J, Kramer M, Lihm J, Yoon S, Berstein Y et al (2014) De novo mutations in schizophrenia implicate chromatin remodeling and support a genetic overlap with autism and intellectual disability. Mol Psychiatry 19:652–658. doi: 10.1038/mp.2014.29 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Fromer M, Pocklington AJ, Kavanagh DH, Williams HJ, Dwyer S, Gormley P et al (2014) De novo mutations in schizophrenia implicate synaptic networks. Nature 506:179–184. doi: 10.1038/nature12929 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Egawa J, Hoya S, Watanabe Y, Nunokawa A, Shibuya M, Ikeda M et al (2016) Rare UNC13B variations and risk of schizophrenia: whole-exome sequencing in a multiplex family and follow-up resequencing and a case-control study. Am J Med Genet B Neuropsychiatr Genet 171:797–805. doi: 10.1002/ajmg.b.32444 CrossRefPubMedGoogle Scholar
  17. 17.
    Timms AE, Dorschner MO, Wechsler J, Choi KY, Kirkwood R, Girirajan S et al (2013) Support for the N-methyl-d-aspartate receptor hypofunction hypothesis of schizophrenia from exome sequencing in multiplex families. JAMA Psychiatry 70:582–590. doi: 10.1001/jamapsychiatry.2013.1195 CrossRefPubMedGoogle Scholar
  18. 18.
    Tiwari AK, Need AC, Lohoff FW, Zai CC, Chowdhury NI, Müller DJ et al (2014) Exome sequence analysis of Finnish patients with clozapine-induced agranulocytosis. Mol Psychiatry 19:403–405. doi: 10.1038/mp.2013.74 CrossRefPubMedGoogle Scholar
  19. 19.
    Balan S, Iwayama Y, Toyota T, Toyoshima M, Maekawa M, Yoshikawa T (2014) 22q11.2 deletion carriers and schizophrenia-associated novel variants. Br J Psychiatry 204:398–399. doi: 10.1192/bjp.bp.113.138420 CrossRefPubMedGoogle Scholar
  20. 20.
    Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J (2014) A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 46:310–315. doi: 10.1038/ng.2892 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Carrera N, Arrojo M, Sanjuán J, Ramos-Ríos R, Paz E et al (2012) Association study of nonsynonymous single nucleotide polymorphisms in schizophrenia. Biol Psychiatry 71:169–177. doi: 10.1016/j.biopsych.2011.09.032 CrossRefPubMedGoogle Scholar
  22. 22.
    Rodriguez-Lopez J, Carrera N, Arrojo M, Amigo J, Sobrino B, Páramo M et al (2015) An efficient screening method for simultaneous detection of recurrent copy number variants associated with psychiatric disorders. Clin Chim Acta 445:34–40. doi: 10.1016/j.cca.2015.03.013 CrossRefPubMedGoogle Scholar
  23. 23.
    McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A et al (2010) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303. doi: 10.1101/gr.107524.110 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C et al (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491–498. doi: 10.1038/ng.806 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A et al (2013) From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics 43:11.10.1-33. doi: 10.1002/0471250953.bi1110s43 PubMedCrossRefGoogle Scholar
  26. 26.
    Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192. doi: 10.1093/bib/bbs017 CrossRefPubMedGoogle Scholar
  27. 27.
    Fu W, O’Connor TD, Jun G, Kang HM, Abecasis G, Leal SM et al (2013) Analysis of 6,515 exomes reveals the recent origin of most human protein-coding variants. Nature 493:216–220. doi: 10.1038/nature11690 CrossRefPubMedGoogle Scholar
  28. 28.
    The Genomes Project Consortium (2010) A map of human genome variation from population-scale sequencing. Nature 467:1061–1073. doi: 10.1038/nature09534 CrossRefGoogle Scholar
  29. 29.
    Plagnol V, Curtis J, Epstein M, Mok KY, Stebbings E, Grigoriadou S et al (2012) A robust model for read count data in exome sequencing experiments and implications for copy number variant calling. Bioinformatics 28:2747–2754. doi: 10.1093/bioinformatics/bts526 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Kearney HM, Thorland EC, Brown KK, Quintero-Rivera F, South ST, Working Group of the American College of Medical Genetics Laboratory Quality Assurance Committee (2011) American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants. Genet Med 13:680–685. doi: 10.1097/GIM.0b013e3182217a3a CrossRefPubMedGoogle Scholar
  31. 31.
    Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164. doi: 10.1093/nar/gkq603 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    MacArthur DG, North KN (2004) A gene for speed? The evolution and function of alpha-actinin-3. BioEssays 26:786–795CrossRefPubMedGoogle Scholar
  33. 33.
    Cremona O, Di Paolo G, Wenk MR, Lüthi A, Kim WT, Takei K et al (1999) Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99:179–188CrossRefPubMedGoogle Scholar
  34. 34.
    Kononenko NL, Haucke V (2015) Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85:484–496. doi: 10.1016/j.neuron.2014.12.016 CrossRefPubMedGoogle Scholar
  35. 35.
    Schubert KO, Föcking M, Prehn JH, Cotter DR (2012) Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder? Mol Psychiatr 17:669–681. doi: 10.1038/mp.2011.123 CrossRefGoogle Scholar
  36. 36.
    Gong LW, De Camilli P (2008) Regulation of postsynaptic AMPA responses by synaptojanin 1. Proc Natl Acad Sci USA 105:17561–17566. doi: 10.1073/pnas.0809221105 CrossRefPubMedGoogle Scholar
  37. 37.
    Krauss M, Haucke V (2007) Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling. EMBO Rep 8:241–246. doi: 10.1038/sj.embor.7400919 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Quadri M, Fang M, Picillo M, Olgiati S, Breedveld GJ, Graafland J et al (2013) Mutation in the SYNJ1 gene associated with autosomal recessive, early-onset Parkinsonism. Hum Mutat 34:1208–1215. doi: 10.1002/humu.22373 CrossRefPubMedGoogle Scholar
  39. 39.
    Krebs CE, Karkheiran S, Powell JC, Cao M, Makarov V, Darvish H et al (2013) The Sac1 domain of SYNJ1 identified mutated in a family with early-onset progressive Parkinsonism with generalized seizures. Hum Mutat 34:1200–1207. doi: 10.1002/humu.22372 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Olgiati S, De Rosa A, Quadri M, Criscuolo C, Breedveld GJ, Picillo M et al (2014) PARK20 caused by SYNJ1 homozygous Arg258Gln mutation in a new Italian family. Neurogenetics 15:183–188. doi: 10.1007/s10048-014-0406-0 CrossRefPubMedGoogle Scholar
  41. 41.
    Roth W, Kermer P, Krajewska M, Welsh K, Davis S, Krajewski S et al (2003) Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways. Cell Death Differ 10:1178–1187. doi: 10.1038/sj.cdd.4401287 CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang H, Xu Q, Krajewski S, Krajewska M, Xie Z, Fuess S et al (2000) BAR: an apoptosis regulator at the intersection of caspases and Bcl-2 family proteins. Proc Natl Acad Sci USA 97:2597–2602CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Julio Rodríguez-López
    • 1
  • Beatriz Sobrino
    • 1
    • 2
    • 3
  • Jorge Amigo
    • 1
    • 2
    • 3
  • Noa Carrera
    • 1
    • 3
    • 5
  • Julio Brenlla
    • 1
    • 4
  • Santiago Agra
    • 1
    • 4
  • Eduardo Paz
    • 1
    • 4
  • Ángel Carracedo
    • 1
    • 2
    • 3
  • Mario Páramo
    • 1
    • 4
  • Manuel Arrojo
    • 1
    • 4
  • Javier Costas
    • 1
  1. 1.Instituto de Investigación Sanitaria (IDIS) de Santiago de CompostelaComplexo Hospitalario Universitario de Santiago de Compostela (CHUS), Servizo Galego de Saúde (SERGAS)Santiago de CompostelaSpain
  2. 2.Grupo de Medicina XenómicaUniversidade de Santiago de Compostela (USC)Santiago de CompostelaSpain
  3. 3.Fundación Pública Galega de Medicina XenómicaComplexo Hospitalario Universitario de Santiago (CHUS), Servizo Galego de Saúde (SERGAS)Santiago de CompostelaSpain
  4. 4.Servizo de PsiquiatríaComplexo Hospitalario Universitario de Santiago de Compostela, Servizo Galego de Saúde (SERGAS)Santiago de CompostelaSpain
  5. 5.Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical NeurosciencesCardiff UniversityCardiffUK

Personalised recommendations