Novel Insights into Pontocerebellar Hypoplasia Type 3: Discovery of a New Disease-causing PCLO Variant and Development of a CRISPR-generated Cell Model
DOI:
https://doi.org/10.31661/gmj.v14i.3727Keywords:
PCLO; Piccolo; Pontocerebellar Hypoplasia Type 3; Mutation; CRISPR/Cas9Abstract
Background: Pathogenic variations in the PCLO gene cause Pontocerebellar Hypoplasia type 3 (PCH3), an extremely rare autosomal recessive disease characterized by seizure, intellectual disability, developmental delay, and microcephaly. PCLO encodes the Piccolo protein, which plays a critical role in synaptic function and neurological disorders. To date, only one pathogenic PCLO variant associated with PCH3 has been reported in the literature. While research on PCH3 is ongoing, the rarity of the condition has limited the number of studies. Materials and Methods: A novel homozygous variant in PCLO (NM_033026: c.458T>C, p. Met153Thr) was identified through wholeexome sequencing and confirmed by Sanger sequencing. Functional studies were conducted to assess the pathogenicity of this variant using next-generation sequencing (NGS), in silico analysis, CRISPR-edited cells, and real-time PCR. Results: The proband presented with seizure, microcephaly, mild ataxia, and behavioral issues. Notably, in addition to previously reported symptoms, the patient also exhibited toe-walking, loss of tendon reflexes, and unilateral paralysis. The PCLO knockout cell model and molecular analysis confirmed the loss of function of the Piccolo protein in the homozygous variant. Our findings also demonstrated that Piccolo deficiency may affect the expression of other genes, including CtBp1 and BSN. Conclusion: We identified a novel PCLO variant responsible for PCH3 in a second known family worldwide. Additionally, a CRISPR-based cell model for PCH3 was developed, providing a valuable foundation for further research into the molecular mechanisms underlying Piccolo function and disease pathogenesis.
References
Li P, Lin Z, An Y, Lin J, Zhang A, Wang S et al. Piccolo is essential for the maintenance of mouse retina but not cochlear hair cell function. Aging (Albany NY). 2021;13(8):11678-95.
https://doi.org/10.18632/aging.202861
PMid:33882456 PMCid:PMC8109093
Fenster SD, Garner CC. Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo. Int J Dev Neurosci. 2002;20(3-5):161-71.
https://doi.org/10.1016/S0736-5748(02)00046-1
PMid:12175852
Regus-Leidig H, Ott C, Löhner M, Atorf J, Fuchs M, Sedmak T et al. Identification and immunocytochemical characterization of Piccolino, a novel Piccolo splice variant selectively expressed at sensory ribbon synapses of the eye and ear. PLoS One. 2013;8(8):e70373.
https://doi.org/10.1371/journal.pone.0070373
PMid:23936420 PMCid:PMC3735604
Wagh D, Terry-Lorenzo R, Waites CL, Leal-Ortiz SA, Maas C, Reimer RJ, Garner CC. Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1. PLoS One. 2015;10(4):e0120093.
https://doi.org/10.1371/journal.pone.0120093
PMid:25897839 PMCid:PMC4405365
Ackermann F, Schink KO, Bruns C, Izsvák Z, Hamra FK, Rosenmund C, Garner CC. Critical role for Piccolo in synaptic vesicle retrieval. Elife. 2019;8:e46629.
https://doi.org/10.7554/eLife.46629
PMid:31074746 PMCid:PMC6541439
Waites CL, Leal-Ortiz SA, Okerlund N, Dalke H, Fejtova A, Altrock WD et al. Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation. Embo j. 2013;32(7):954-69.
https://doi.org/10.1038/emboj.2013.27
PMid:23403927 PMCid:PMC3616282
Gundelfinger ED, Reissner C, Garner CC. Role of Bassoon and Piccolo in Assembly and Molecular Organization of the Active Zone. Front Synaptic Neurosci. 2015;7:19.
https://doi.org/10.3389/fnsyn.2015.00019
PMid:26793095 PMCid:PMC4709825
Chen CH, Huang YS, Liao DL, Huang CY, Lin CH, Fang TH. Identification of Rare Mutations of Two Presynaptic Cytomatrix Genes BSN and PCLO in Schizophrenia and Bipolar Disorder. J Pers Med. 2021;11(11):1057.
https://doi.org/10.3390/jpm11111057
PMid:34834409 PMCid:PMC8625612
Liu D, Meyer D, Fennessy B, Feng C, Cheng E, Johnson JS et al. Schizophrenia risk conferred by rare protein-truncating variants is conserved across diverse human populations. Nat Genet. 2023;55(3):369-76.
https://doi.org/10.1038/s41588-023-01305-1
PMid:36914870 PMCid:PMC10011128
Ahmed MY, Chioza BA, Rajab A, Schmitz-Abe K, Al-Khayat A, Al-Turki S et al. Loss of PCLO function underlies pontocerebellar hypoplasia type III. Neurology. 2015;84(17):1745-50.
https://doi.org/10.1212/WNL.0000000000001523
PMid:25832664 PMCid:PMC4424132
Rajab A, Mochida GH, Hill A, Ganesh V, Bodell A, Riaz A et al. A novel form of pontocerebellar hypoplasia maps to chromosome 7q11-21. Neurology. 2003;60(10):1664-7.
https://doi.org/10.1212/01.WNL.0000068548.58498.41
PMid:12771259
Zeraatpisheh Z, Sichani AS, Kamal N, Khamirani HJ, Zoghi S, Ehsani E et al. MCM2 mutation causes autosomal dominant nonsyndromic hearing loss (DFNA70): novel variant in the second family. Journal of Genetics. 2022;101(1):1-6.
https://doi.org/10.1007/s12041-022-01364-z
Mohammadi S, Jafari Khamirani H, Baneshi M, Kamal N, Manoocheri J, Saffar M, Dianatpour M, Tabei SM, Dastgheib SA. A novel nonsense variant in the ATL3 gene is associated with disturbed pain sensitivity, numbness of distal limbs and muscle weakness. Annals of Human Genetics. 2023 Jul;87(4):147-57.
https://doi.org/10.1111/ahg.12501
PMid:36856139
Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589-95.
https://doi.org/10.1093/bioinformatics/btp698
PMid:20080505 PMCid:PMC2828108
Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38(16):e164.
https://doi.org/10.1093/nar/gkq603
PMid:20601685 PMCid:PMC2938201
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-24.
https://doi.org/10.1038/gim.2015.30
PMid:25741868 PMCid:PMC4544753
Rychlik W. OLIGO 7 primer analysis software. Methods Mol Biol. 2007;402:35-60.
https://doi.org/10.1007/978-1-59745-528-2_2
PMid:17951789
Consortium TU. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Research. 2022;51(D1):D523-D31.
Fazelzadeh Haghighi M, Jafari Khamirani H, Fallahi J, Monfared AA, Ashrafi Dehkordi K, Tabei SMB. Novel insight into FCSK-congenital disorder of glycosylation through a CRISPR-generated cell model. Molecular Genetics & Genomic Medicine. 2024;12(5):e2445.
https://doi.org/10.1002/mgg3.2445
PMid:38722107 PMCid:PMC11080630
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.
https://doi.org/10.1006/meth.2001.1262
PMid:11846609
UniProt u. the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021;49(1):480-9.
Ren J, Wen L, Gao X, Jin C, Xue Y, Yao X. DOG 1.0: illustrator of protein domain structures. Cell Res. 2009;19(2):271-3.
https://doi.org/10.1038/cr.2009.6
PMid:19153597
Consortium TU. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Research. 2020;49(D1):D480-D9.
Fenster SD, Chung WJ, Zhai R, Cases-Langhoff C, Voss B, Garner AM et al. Piccolo, a presynaptic zinc finger protein structurally related to bassoon. Neuron. 2000;25(1):203-14.
https://doi.org/10.1016/S0896-6273(00)80883-1
PMid:10707984
Fujimoto K, Shibasaki T, Yokoi N, Kashima Y, Matsumoto M, Sasaki T et al. Piccolo, a Ca2+ sensor in pancreatic beta-cells Involvement of cAMP-GEFII Rim2 Piccolo complex in cAMP-dependent exocytosis. J Biol Chem. 2002;277(52):50497-502.
https://doi.org/10.1074/jbc.M210146200
PMid:12401793
Takao-Rikitsu E, Mochida S, Inoue E, Deguchi-Tawarada M, Inoue M, Ohtsuka T, Takai Y. Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release. J Cell Biol. 2004;164(2):301-11.
https://doi.org/10.1083/jcb.200307101
PMid:14734538 PMCid:PMC2172332
Mukherjee K, Yang X, Gerber SH, Kwon HB, Ho A, Castillo PE et al. Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis. Proc Natl Acad Sci U S A. 2010;107(14):6504-9.
https://doi.org/10.1073/pnas.1002307107
PMid:20332206 PMCid:PMC2851964
Falck J, Bruns C, Hoffmann-Conaway S, Straub I, Plautz EJ, Orlando M et al. Loss of Piccolo Function in Rats Induces Cerebellar Network Dysfunction and Pontocerebellar Hypoplasia Type 3-like Phenotypes. J Neurosci. 2020;40(14):2943-59.
https://doi.org/10.1523/JNEUROSCI.2316-19.2020
PMid:32122952 PMCid:PMC7117892
Okerlund ND, Schneider K, Leal-Ortiz S, Montenegro-Venegas C, Kim SA, Garner LC et al. Bassoon Controls Presynaptic Autophagy through Atg5. Neuron. 2017;93(4):897-913.e7.
https://doi.org/10.1016/j.neuron.2017.01.026
PMid:28231469
Dawson WJ. Bassoonists' medical problems-current state of knowledge. Med Probl Perform Art. 2012;27(2):107-12.
https://doi.org/10.21091/mppa.2012.2019
PMid:22739824
Acosta-Baena N, Tejada-Moreno JA, Arcos-Burgos M, Villegas-Lanau CA. CTBP1 and CTBP2 mutations underpinning neurological disorders: a systematic review. Neurogenetics. 2022;23(4):231-40.
https://doi.org/10.1007/s10048-022-00700-w
PMid:36331689 PMCid:PMC9663338

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