Allele-Specific Biased Expression of the CNTN6 Gene in iPS Cell-Derived Neurons from a Patient with Intellectual Disability and 3p26.3 Microduplication Involving the CNTN6 Gene
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Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.
KeywordsCNTN6 gene 3p26.3 microduplication Intellectual disability Induced pluripotent stem cells in vitro neural differentiation Allele-specific expression
The LeGO lentiviral vectors containing the human reprogramming transcription factors OCT4, SOX2, C-MYC, and KLF4 were kindly provided to us by Dr. Sergei L. Kiselev (Moscow). Three lentiviral constructs (FUW-TRE Ngn2/Puro, containing full-length mouse Ngn2 and puromycin-resistance genes, and M2RtTA and FUW-TRE EGFP, containing the GFP gene) were kindly provided to us by Dr. Thomas Südhof (Stanford, CA, USA). Patient's fibroblasts samples were obtained from the collection «Biobank of populations of Northern Eurasia» financed by the Federal Agency for Scientific Organizations of Russia Federation program for supporting the bioresource collections in 2017 (project no. 0550-2017-0019). We thank the family of the patient with 3p26.3 microduplication for their collaboration. We are grateful to the donors of skin fibroblasts and to their parents for their voluntary participation in the research. Cell culture was performed at the Collective Center of ICG SB RAS "Collection of Pluripotent Human and Mammalian Cell Cultures for Biological and Biomedical Research" (state project No. 0324-2016-0002 of Russian Academy of Sciences).
This study was supported by the Russian Science Foundation, grant no. 14-15-00772.
- 3.Luo Y, Zhu D, Du R, Gong Y, Xie C, Xu X, Fan Y, Yu B, Sun X, Chen Y (2015) Uniparental disomy of the entire X chromosome in Turner syndrome patient-specific induced pluripotent stem cells. Cell Discovery, 1, Article number: 15022.Google Scholar
- 4.Hibaoui Y, Grad I, Letourneau A, Sailani MR, Dahoun S, Santori FA, Gimelli S, Guipponi M et al (2014) Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21. EMBO Mol Med 6(2):259–277. https://doi.org/10.1002/emmm.201302848 PubMedCrossRefGoogle Scholar
- 6.Mikhail FM, Lose EJ, Robin NH, Descartes MD, Rutledge KD, Rutledge SL, Korf BR, Carroll AJ (2011) Clinically relevant single gene or intragenic deletions encompassing critical neurodevelopmental genes in patients with developmental delay, mental retardation, and/or autism spectrum disorders. Am J Med Genet A 155A(10):2386–2396. https://doi.org/10.1002/ajmg.a.34177 CrossRefPubMedGoogle Scholar
- 8.Shoukier M, Fuchs S, Schwaibold E, Lingen M, Gärtner J, Brockmann K, Zirn B (2013) Microduplication of 3p26.3 in nonsyndromic intellectual disability indicates an important role of CHL1 for normal cognitive function. Neuropediatrics 44(05):268–271. https://doi.org/10.1055/s-0033-1333874 CrossRefPubMedGoogle Scholar
- 9.Te Weehi L, Maikoo R, Mc Cormack A, Mazzaschi R, Ashton F, Zhang L, George AM, Love DR (2014) Microduplication of 3p26.3 implicated in cognitive development. Case Rep Genet 2014:295359.Google Scholar
- 11.Hu J, Liao J, Sathanoori M, Kochmar S, Sebastian J, Yatsenko SA, Surti U (2015) CNTN6 copy number variations in 14 patients: a possible candidate gene for neurodevelopmental and neuropsychiatric disorders. J Neurodev Disord 7(1):26. https://doi.org/10.1186/s11689-015-9122-9 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Kashevarova AA, Nazarenko LP, Schultz-Pedersen S, Skryabin NA, Salyukova OA, Chechetkina NN, Tolmacheva EN, Rudko AA et al (2014) Single gene microdeletions and microduplication of 3p26.3 in three unrelated families: CNTN6 as a new candidate gene for intellectual disability. Mol Cytogenet 7(1):97. https://doi.org/10.1186/s13039-014-0097-0 CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Mercati O, Huguet G, Danckaert A, André-Leroux G, Maruani A, Bellinzoni M, Rolland T, Gouder L et al (2017) CNTN6 mutations are risk factors for abnormal auditory sensory perception in autism spectrum disorders. Mol Psychiatry 22(4):625–633. https://doi.org/10.1038/mp.2016.61 CrossRefPubMedGoogle Scholar
- 18.Sakurai K, Toyoshima M, Ueda H, Matsubara K, Takeda Y, Karagogeos D, Shimoda Y, Watanabe K (2009) Contribution of the neural cell recognition molecule NB-3 to synapse formation between parallel fibers and Purkinje cells in mouse. Dev Neurobiol 69(12):811–824. https://doi.org/10.1002/dneu.20742 CrossRefPubMedGoogle Scholar
- 20.Moghadasi S, van Haeringen A, Langendonck L, Gijsbers ACJ, Ruivenkamp CAL (2014) A terminal 3p26.3 deletion is not associated with dysmorphic features and intellectual disability in a four-generation family. Am J Med Genet A 164A(11):2863–2868. https://doi.org/10.1002/ajmg.a.36700 CrossRefPubMedGoogle Scholar
- 23.Current protocols in human genetics/editorial board, Nicholas C. Dracopoli et al. 1994. V. 1: 4.6.6.Google Scholar
- 24.Chen X, Schulz-Trieglaff O, Shaw R, Barnes B, Schlesinger F, Källberg M, Cox AJ, Kruglyak S et al (2016) Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics 32(8):1220–1222. https://doi.org/10.1093/bioinformatics/btv710 CrossRefPubMedGoogle Scholar
- 26.Zeitouni B, Boeva V, Janoueix-Lerosey I, Loeillet S, Legoix-né P, Nicolas A, Delattre O, Barillot E (2010) SVDetect: a tool to identify genomic structural variations from paired-end and mate-pair sequencing data. Bioinformatics 26(15):1895–1896. https://doi.org/10.1093/bioinformatics/btq293 CrossRefPubMedPubMedCentralGoogle Scholar
- 39.Koch P, Opitz T, Steinbeck JA, Ladewig J, Bruüstle O (2009) A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Nat Acad Sci USA 106(9):3225–3230. https://doi.org/10.1073/pnas.0808387106 CrossRefPubMedGoogle Scholar
- 41.Yuan SH, Martin J, Elia J, Flippin J, Paramban RI, Hefferan MP, Vidal JG, Mu Y et al (2011) Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6(3):e17540. https://doi.org/10.1371/journal.pone.0017540 CrossRefPubMedPubMedCentralGoogle Scholar
- 42.Kim D-S, Lee DR, Kim H-S, Yoo J-E, Jung SJ, Lim BY, Jang J, Kang H-C et al (2012) Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC derived neural rosettes. PLoS One 7(7):e39715. https://doi.org/10.1371/journal.pone.0039715 CrossRefPubMedPubMedCentralGoogle Scholar