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On the Origin of Pontocerebellar Hypoplasia: Finding Genes for a Rare Disease

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On the Origin of Pontocerebellar Hypoplasia: Finding Genes for a Rare Disease UvA-DARE (Digital Academic Repository) On the origin of pontocerebellar hypoplasia: Finding genes for a rare disease Eggens, V.R.C. Publication date 2016 Document Version Final published version Link to publication Citation for published version (APA): Eggens, V. R. C. (2016). On the origin of pontocerebellar hypoplasia: Finding genes for a rare disease. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:06 Oct 2021 Al ben ik meer dan eens de weg kwijt En rijd ik hele einden om Ik ben op weg om je te vinden Dus wees gerust, ik kom - Boudewijn de Groot TOE1 mutatiOns cause pontOcerebellar hypoplasia and disOrders Of sex deVelOPment Veerle rC eggens, david Chitayat, Hülya Kayserili, nicola foulds, Tessa van dijk, Bart appelhof, Kazuhiro Muramatsu, Kimberly a aldinger, William B dobyns, david Manchester, Linda de Meirleir, Mary Louise freckmann, Linda Warwick, Chistina fagerberg, Maria Kibaek, Marie-Cecile nassogne, Justin H davis, Umut altunoglu, Hirotomo saitsu, Masaaki shiina, Kazuhiro ogata, Kanako Kurata, Peter g Barth, naomichi Matsumoto, frank Baas Manuscript in preparation2 AbsTract Pontocerebellar hypoplasia type 7 (PCH7) – also described as pontocerebellar hypo- plasia and disorders of sex development (PCH and DSD) - is a rare disease in which both brain and genital development are affected. only four cases have been described in literature displaying this rare combination of symptoms. Until know it was uncer- tain whether the combination of symptoms in these patients occurred coincidentally or not. Here, we show that the presence of PCH and DSD is a single gene syndrome. We collected a cohort of ten patients (eight families) presenting PCH and DSD, and identified recessive mutations in the TOE1 gene in all of them. Brain MRI of the pa- tients revealed a small pons and cerebellum and enlarged ventricles. all eight 46, Xy patients present some degree of undervirilised genitals. 28 inTroduction Pontocerebellar hypoplasia (PCH) represents a group of autosomal recessive neu- rodegenerative disorders with prenatal onset. Patients show variable hypoplasia of pons and cerebellum and severe motor and mental impairments. nowadays, ten PCH subtypes have been described (PCH1-10) with each subtype having its characteristic Chapter clinical and/or genetic features in addition to a hypoplastic pons and cerebellum. PCH7 is characterised by PCH plus disorders of sex development (DSD). DSD is a 2 broad group of conditions where defects of gonadal development occur, leading to ambiguous genitals or complete sex reversal. The frequency of 46, Xy DSD is 1:20,000 T [1]. in literature, four cases of PCH plus DSD have been described [2-5]. These patients oe had a small pons and cerebellum, enlarged ventricles and a 46,Xy karyotype with 1 mutations in PCH7 feminizing genitalia. so far, it was unclear whether the combination of PCH and geni- tal anomalies was fortuitous or a distinct syndrome. in this study we report eight families (ten patients) with PCH and DSD, and present the target of egr1 (TOE1) gene as locus for this disease in all described families. TOE1 is a nuclear protein that can bind RNA, has deadenylation activity [6] and can have an inhibitory effect on viral replication [7]. furthermore, it is essential for Cajal body maintenance, and thus potentially involved in mRNA splicing [8]. The gene has previously been associated with cerebellar abiotrophy in arabian horses [9]. With the identification of TOE1 mutations in ten patients with PCH and DSD, we confirm the presence of a single gene syndrome causing both brain and genital abnormalities. MaTeriaLs and Methods exome capture and sequencing family 1 was analysed at the academic Medical Center, genomics core-facility in am- sterdam and sequenced on a SOLid4 platform according to manufacturer’s protocols. fragment libraries were prepared followed by a nimblegens eZexome v2.0 sequence capture. reads were aligned against hg19 with Bioscope1.3. genomic DNA for patient 7 and both parents were sequenced by the University of Washington Center for Men- delian genomics. DNA was captured using the roche nimblegen seqCap eZ Human exome Library v.2.0 library and sequenced with paired-end 50 bp reads on an illumina Hiseq sequencer. reads were aligned against hg19 using the Burrows-Wheeler aligner v.0.6.2. for patient 8, genomic DNA of patient and parental samples was captured using the sureselect Human all exon v5 Kit (agilent Technologies) and sequenced on an illumina Hiseq2000 (illumina) with 101 bp paired-end reads. reads were aligned to GRCh37 with novoalign (novocraft Technologies). single nucleotide variants (SNVs) 29 were called with the genome analysis Toolkit’s (GATK) Unifiedgenotyper. rare (<1% in the nHLBi exome sequencing Project exome Variant server), deleterious variants were analysed under de novo and recessive mutation models. SNVs in TOE1 were confirmed by sanger sequencing. sanger sequencing Variants in TOE1 in families 2,3,4,5 and 6 were identified by sanger sequencing. sanger sequencing of PCr amplified DNA was performed using BigdyeTerminator chemistry (applied Biosystems) and analysed on an aBi3730xl sequencer. sequences were analysed using CodonCode aligner software 3.6.1. Primer sequences are outlined in supplementary Table 1. CLK2 knockout mouse CLK2 whole body knockout mice were bred from Clk2flox/flox mice and zp3-Cre trans- genic mice on a C57BL/6 background. Littermates carrying the floxed allele but not Cre were used as controls. Mice were bred at Puigserver Laboratory (dept. of Can- cer Biology, Harvard Medical school, Boston, USA) who kindly provided fixed brain samples. standard hematoxylin and eosin staining was performed on sagittal slides. morpholino injections in zebrafish Morpholino (Mo) antisense oligonucleotides (gene Tools) were designed to target zebrafish clk2 mRNA (nM_001076751.1) either upstream of the aTg startcodon (5’-CCGGTgCgTTTgTCCCaCAGAAAAT-3’) or at the exon 5 splice donor site (5’-CaTCAAT- GAACAGCTCTTaCTTCTT-3). standard control Mos were provided by gene Tools. Mos were injected in 1-2 cell stage embryos using glass needles and a microinjector. fish injected with splice clk2 Mo were checked for alternative splicing: total RNA was ex- tracted with TRIzol (Thermofisher)/chloroform, cDNA was synthesized using dT-oligos and superscript III first strand synthesis system (invitrogen) and PCr was performed with primers 5’-AGAGCCGGTCCaTaTCaTCa-3’ and 5’-CCGAAATCCaCAATCCTCaC-3’. mRNA transcription and injections in zebrafish Human CLK2 cDNA cloned in a poTB7 vector was obtained from the iMAGE Consortium. The CLK2 insert was subcloned into a pCs2+ vector using primers with BamHi and Xbai restriction sites (5’- TCgTaCAGGCTaCCTGGATCCgCCaCCaTgCCgCaTCCTCGAA-3’ and 5’- gTgTCaCTGACTgCaCCgTgTCTAGATGGGGGTCAAATGAAG-3’ respectively, restric- tion sites in bold). The construct was linearized with noti. for zebrafish clk2 cDNA (nM_001044879.2), a vector with clk2 insert was synthesised at Life Technologies and cloned into a pcDNA3 vector using primers with HindIII (5’- TAAGCaAAGCTTaTgC- CaCaCTCCAGGCGGTa -3’) and Xhoi (5’-TgCTTaCTCGAGTCaCCGGCTGATgTCaCGGTT-3’) 30 restriction sites. The construct was linearized with stui. In vitro transcription was performed using the T7 mMESSAGE mMaCHINE Kit (ambion, Warrington, UK). in situ hybridisation Plasmids with fgf8 insert were linearised with ecorV and antisense digoxigenin (DIG)-RNA labelled probes were synthesized using a sP6 polymerase RNA labelling Chapter kit (roche). Zebrafish embryos were dechorionated, and fixed overnight in formalin. Whole mount in situ hybridisation procedures were adapted from [10]. Preceding 2 ISH, fish were bleached (10% H2o2; 5% formamide; 2,5% 20x SSC in water) for a few minutes until pigmentation had vanished. fish were permeabilised using 10 µg/ml T proteinase K in PBs, and incubated over night with 1 ng/µl fgf8 DIG-RNA probe. stain- oe ing was visualised using a nBT/BCiP solution (roche). 1 mutations in PCH7 TUNEL assay Zebrafish embryos were fixated, bleached and permeabilised as described above. TUNEL staining was performed using TdT buffer (roche), TdT enzyme (roche) and DIG-UTP (roche). fish were incubated over night with anti-DIG-aP antibody (roche). staining was visualised using a nBT/BCiP solution. recombinant CLK2 protein Murine flag-clk2 cDNA in a pcDNA3 vector was kindly provided by P. Puigserver (dept. of Cancer Biology, Harvard Medical school, Boston, USA). Mutant flag-clk2 was gener- ated using site directed Mutagenesis (stratagene) and primers 5’-aTGACAACAGAGA- gCaTCTatCCaTGATGGAAAGGATC-3’ and 5’-GATCCTTTCCaTCaTGGaTAGATgCTCTCTgTT- gTCaT-3, the mutation site indicated in bold. Wild type and mutant flag-clk2
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