Neurogenetics J Med Genet: first published as 10.1136/jmedgenet-2020-106987 on 20 August 2020. Downloaded from Original research CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression Xuanye Cao,1 Annika Wolf,2 Sung- Eun Kim,3 Robert M. Cabrera,1 Bogdan J. Wlodarczyk,1 Huiping Zhu,4 Margaret Parker,3 Ying Lin,1 John W. Steele,1,5 Xiao Han,1 Vincent Th Ramaekers ,6 Robert Steinfeld,2,7 Richard H. Finnell,3,8,9 Yunping Lei 1 2 ► Additional material is ABSTRact epilepsy, ataxia and pyramidal tract signs. Folate is published online only. To view Background Cerebral folate deficiency (CFD) absorbed into the bloodstream via the gastrointes- please visit the journal online (http:// dx. doi. org/ 10. 1136/ syndrome is characterised by a low concentration of tinal tract mainly through two uptake systems: the jmedgenet- 2020- 106987). 5- methyltetrahydrofolate in cerebrospinal fluid, while reduced folate carrier 1 (RFC1; SLC19A1) and the folate levels in plasma and red blood cells are in the low proton- coupled folate transporter (PCFT).3 From For numbered affiliations see normal range. Mutations in several folate pathway genes, the bloodstream, folate binds to the folate receptor end of article. including FOLR1 (folate receptor alpha, FRα), DHFR alpha (FRα/FOLR1) on the basolateral endothelial (dihydrofolate reductase) and PCFT (proton coupled surface of the choroid plexus. Through receptor- Correspondence to Dr. Yunping Lei, Baylor College folate transporter) have been previously identified in mediated endocytosis and transcytosis, folate is of Medicine, Houston, Texas, patients with CFD. then transported across the blood- CSF- barrier USA; yunpinglei@ gmail. com Methods In an effort to identify causal mutations for into the CSF.4 FOLR1 is highly expressed in the Dr. Richard H. Finnell; CFD, we performed whole exome sequencing analysis on choroid plexus, and it has a higher affinity to folic richard. finnell@ bcm. edu eight CFD trios and identified eight de novo mutations in Dr. Robert Steinfeld; acid (FA) at neutral pH compared with other folate 5 Robert. Steinfeld@ kispi. uzh. ch seven trios. transporters, such as PCFT and RFC1. These two Results Notably, we found a de novo stop gain characteristics are important factors in building and XC, AW and S- EK contributed mutation in the capicua (CIC) gene. Using 48 sporadic keeping higher folate concentrations in the CSF equally. CFD samples as a validation cohort, we identified than in serum. Previous studies demonstrated that Received 10 March 2020 three additional rare variants in CIC that are putatively dysfunctional FOLR1, such as that which could be Revised 27 May 2020 deleterious mutations. Functional analysis indicates that the result of either FOLR1 mutations6 7 or FOLR1 Accepted 5 June 2020 CIC binds to an octameric sequence in the promoter autoantibodies,8 can result in CFD. Mutations in Published Online First 20 August regions of folate transport genes: FOLR1, PCFT and 9 10 http://jmg.bmj.com/ 2020 other folate related genes, such as DHFR and reduced folate carrier (Slc19A1; RFC1). The CIC nonsense PCFT,11 12 are also reported to be associated with variant (p.R353X) downregulated FOLR1 expression in low 5- MTHF CSF concentrations. We previously HeLa cells as well as in the induced pluripotent stem cell reported a patient carrying a de novo truncating (iPSCs) derived from the original CFD proband. Folate CIC mutation.13 The patient presented with a paedi- binding assay demonstrated that the p.R353X variant atric B- cell acute lymphoblastic leukaemia (B- ALL) decreased cellular binding of folic acid in cells. and was treated with methotrexate (MTX). She Conclusion This study indicates that CIC loss of function on September 29, 2021 by guest. Protected copyright. developed a spectrum of neurobehavioral disor- variants can contribute to the genetic aetiology of ders including signs of autistic spectrum disorder, CFD through regulating FOLR1 expression. Our study seizures, loss of language and motor skills and had described the first mutations in a non- folate pathway CFD, even after cancer remission and the discontin- gene that can contribute to the aetiology of CFD. uation of MTX treatment. The CIC gene was originally discovered in Drosophila, where it was shown to be highly 14 INTRODUCTION expressed in the head and tail region, which Cerebral folate deficiency (CFD) is defined as resulted in the naming of it ‘head and tail’ or any neurological syndrome associated with low ‘capicua’ in Catalan. It is a transcriptional repressor 5- methyltetrahydrofolate (5- MTHF) concentra- of the high mobility group (HMG)- box family, tions in cerebrospinal fluid (CSF), while folate which binds a specific octameric DNA sequence, © Author(s) (or their T(G/C)AATG(A/G)A, in the genome via their employer(s)) 2021. Re-use concentrations in serum are typically in the low 1 15 permitted under CC BY-NC . No normal range. In humans, the folate concentration HMG- box and C1 domain. CIC is known to be commercial re-use . See rights in the CSF is normally ≥1.5- fold higher than in transported into the nucleus by KPNA3, a nuclear and permissions. Published serum. As folate is essential for normal development importin protein that can interact with CIC at its by BMJ. and production of the biogenic amines and pterins C- terminal nuclear localisation signal domain.16 To cite: Cao X, Wolf A, in the central nervous system, prenatal and post- It was found to function downstream of the EGF- Kim S- E, et al. J Med Genet natal folate deficiency produces a variety of neuro- RTK- RAS- ERK- MAPK signalling pathway to 2021;58:484–494. logical symptoms, including intellectual disability, control embryonic pattern formation.14 Recently, 484 Cao X, et al. J Med Genet 2021;58:484–494. doi:10.1136/jmedgenet-2020-106987 Neurogenetics J Med Genet: first published as 10.1136/jmedgenet-2020-106987 on 20 August 2020. Downloaded from studies also indicated CIC influences Toll/Interleukin-1 signal- Sequencing analysis of DNA samples ling.17 The most well- known CIC downstream target genes DNA sequencing libraries were built using NEBNext Ultra include polyomavirus enhancer activator 3 family members DNA Library Prep Kit (New England Biolabs) following proto- genes, ETV1, ETV4 and ETV5 as well as matrix metalloproteinase cols as described by the manufacturer (V.3.0). The Agilent Sure- 9 (MMP9)18 and MMP24.19 In the absence of EGFR-ERK signal- Select Human All Exon V5 (Agilent Technologies) was used for ling, CIC binds to and represses those genes, while activation of exome enrichment. Libraries were sequenced by the Genome the pathway results in inactivation of CIC through phosphor- Sequencing and Analysis Facility at University of Texas at ylation induced degradation or relocalisation from the nucleus Austin using an Illumina Hi- Seq 2000 platform (Illumina). to the cytoplasm.20–23 In mammals, CIC forms a transcriptional Next generation sequencing data were analysed following repressor complex with ataxin-1, a gene that contributes to the GATK Best Practices (https:// software. broadinstitute. org/ gatk/ pathogenesis of spinocerebellar ataxia type 1.24 Conventional best- practices/). Briefly, FASTQ format data were mapped to 29 Cic knockout (KO) mice present with omphalocele, abnormally hg19 using BWA alignment software, sorted and indexed by 30 31 small litter size and lung differentiation defects that result in SAMtools, base recalibrated by GATK and duplicate reads perinatal lethality.18 25 In early development, mice with a condi- removed by Picard software (https:// broadinstitute. github. tional CIC KO display impaired forebrain neurodevelopment, io/ picard/). Variants were called using GATK Haplotype- resulting in hyperactivity, decreased learning ability and impaired Caller methods. De novo variant analysis was performed by 32 memory.13 In adult mice, systemic conditional CIC inactivation TrioDeNovo software. Variants annotation was performed 33 induces T- cell acute lymphoblastic lymphoma. CIC was also with Annovar. Sanger sequencing was performed following 34 reported to modify peripheral immune tolerance by suppressing previously published primers and protocols. CIC sequencing aberrant activation of adaptive immunity.26 In humans, CIC primers are in online supplementary table 1. Polyphen2, SIFT mutations have been associated with several diseases, including and CADD scores were used for predicting variants’ func- 35–37 neurobehavioral phenotypes,13 oligodendroglioma,27 lung carci- tion. The gnomAD database was used for examining the 38 noma19 and prostate cancer.28 allele frequency of each mutants. Although studies have reported that mutations in human CIC are strongly associated with ALL and neurobehavioral pheno- Plasmid preparation and siRNA information types, studies to date have failed to determine whether CIC GFP- CIC and HA- KPNA3 plasmids were provided to us by Dr mutations contribute to the aetiology of CFD. In fact, there are Carol MacKintosh at the University of Dundee. CIC variant no comprehensive trio- based genetic studies on patients with was introduced into the GFP- CIC plasmid using the GeneArt CFD. Since the aforementioned patient who carries the trun- Site- Directed Mutagenesis System (Cat# A14604, Thermofisher cated CIC mutation does not seem to carry any rare variant in Scientific). Ataxin-1 plasmids were provided by Dr Huda Zoghbi known CFD genes (FOLR1, PCFT and RFC1) in our whole at Baylor College of Medicine (Addgene Cat# 48189). exome sequencing (WES) dataset, we propose that the truncated Full-length human CIC and CIC variant were inserted via CIC protein may play a role in CFD progression. Thus, CFD EcoRI/SalI into the expression vector pLVX-IRES- puro (Takara may be caused from genetic factors other than variants in known Bio) containing an EGFP ORF. Vectors for production of lentiviral folate transport genes. In order to better understand the aeti- particles pMD2.G and psPAX2 were from Addgene (plasmids ology of CFD, a comprehensive genetic profiling of patients with Cat# 12259, Cat# 12260; deposited by Didier Trono).