ARTICLE OPEN ACCESS

Novel Variations in the KDM5C Gene CausingX-Linked Intellectual DisabilityPo-Ming Wu MD Wen-Hao Yu MD Chi-Wu Chiang PhD Chen-Yu Wu MD Jia-Shing Chen PhD and

Yi-Fang Tu MD PhD

Neurol Genet 20228e646 doi101212NXG0000000000000646

Correspondence

Dr Tu

nckutugmailcom

or Dr Chen

cjshing77gmailcom

AbstractBackground and ObjectivesTo investigate the pathogenicity of 2 novel KDM5C variations report the clinical and neuro-imaging findings and review the available literature

MethodsPhysical examinations structural neuroimaging studies and exome sequence analysis wereperformed KDM5C constructs were used to study the effect of the variations in transfectedcells

ResultsWe identified 2 novel variations c2233CgtG and c3392_3393delAG in the KDM5C geneharboring from 2 Chinese families with X-linked intellectual disability (ID) The affected malepatients exhibited severe ID short stature and facial dysmorphism The 1 with c3392_3393delAG additionally had epilepsy and autistic spectrum disorder (ASD) Transientlytransfected mutant KDM5C constructs both reduced protein expression and stability anddecreased histone demethylase activities in cells Reviewing the available literature we foundthat the associated ASD tended to occur in patients with variations near the C-terminus ofKDM5C

DiscussionWe report the clinical molecular genetic and pathologic features in patients with novel vari-ations of KDM5C The variability of the clinical phenotype in addition to an ID may associatewith altered particular parts of KDM5C

These authors are co-first authors

From the Department of Pediatrics (P-MW W-HY C-YW Y-FT) National Cheng Kung University Hospital College of Medicine National Cheng Kung University Tainan School ofMedicine for International Students (J-SC) I-Shou University Kaohsiung Institute of Clinical Medicine (W-HY Y-FT) College of Medicine National Cheng Kung University TainanInstitute of Molecular Medicine (C-WC) College of Medicine National Cheng Kung University Tainan Taiwan

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Intellectual disability (ID) is a disability of intellectual andadaptive functions including reasoning problem solvingplanning abstract thinking judgment academic learning andpersonal independencesocial responsibility developing12 Itis an important medical issue and affects 15ndash3 of thepopulation worldwide13 Underlying causes of ID were verydiverse including brain malformation metabolic disordersbrain traumatic injury vascular disorders nerve system in-fection genetic abnormalities or even environmentalfactors24 As the quality of clinical care is improved the ge-netic contribution to ID becomes even more significant5

Studies of numerous large cohorts of patients with ID showeda significant excess of males with male to female ratio about14 for moderate to severe ID (IQ lt 50) and 19 for mild form(IQ 50ndash70)6 These indicate the male predominance in pa-tients with ID and that X-linked gene defects are consideredto be the informant causes of ID

It had been estimated that 285 of patients with moderate tosevere ID and 50 of patients with mild ID are X-linked ID(XLID)6 Clinical observations and linkage studies in familiesrevealed that XLID is a highly heterogeneous condition and issubdivided into syndromic and nonsyndromic forms Themost common form of syndromic XLID is the fragile X syn-drome which is associated with a cytogenetic marker in thedistal region of the long arm of the X chromosome involvingthe FMR1 gene6 In addition to fragile X syndrome there aremore than 140 syndromic forms of XLID described and inalmost half of them causative genetic defects have beenidentified7

Among these causative genes of XLID KDM5C is one of themost frequently mutated genes in XLID and estimated to beassociated with approximately 07ndash28 of all XLID cases89

The KDM5C (MIM 300534) gene is located at Xp1122 andencodes a ubiquitous 1560-aa protein that catalyzes the re-moval of methyl groups from dimethylated and trimethylatedhistone H3 lysine 4 (H3K4)10 H3K4me23 the substrate ofKDM5C is generally associated with promoters of tran-scriptionally active or poised genes and plays important rolesin gene transcription11 Previous reports suggest a centraltranscriptional repressive role for KDM5C in chromatin dy-namics and epigenetic regulation during cell growth differ-entiation and development and alterations in chromatinlandscape may contribute to disease1213

KDM5C-associated XLID is also called Claes-Jensen syn-drome The clinical manifestations included severe ID

epileptic seizure slowly progressive spastic paraplegia shortstature microcephaly maxillary hypoplasia and smallfeet1415 The truncation or other types of variations ofKDM5C have been found in patients with Claes-Jensensyndrome The majority of missense variations functionallytested to date result in reduced demethylation activity ofKDM5C1216 Here we present 2 unrelated families with pa-tients with XLID harboring 2 novel KDMC variationspQ745E and pE1131Afs17 Phenotypes clinical manifesta-tions and neurologic developmental courses of them wereprovided In addition the pathogenicity of these 2 novelvariations was confirmed by in vitro functional assays

MethodsPatientsFrom our previously reported cohort of 61 patients with ID weidentified 2 unrelated families with patients with XLID har-boring 2 novel KDMC variations pQ745E and pE1131Afs17

The previous cohort included patients with ID with unexplainedetiology ID was defined by a performance score at least 2 SDsbelow the mean for an appropriate test including the BayleyScales of Infant Development (BSID-III) orWechsler Preschooland Primary Scale of Intelligence (WPPSI-IV)17 All medicalrecords and laboratory results especially MR neuroimages andmetabolic disorder surveys were reviewed17 Patients with IDwith any possible known etiology were excluded17

Standard Protocol Approvals Registrationsand Patient ConsentsWritten informed consent was obtained from the parents ofeach patient Experiments were conducted after obtainingapproval from the ethics committee at National Cheng KungUniversity Hospital

Targeted Panel GenemdashWhole-Exome SequencingGenomic DNA isolated from blood was qualified by the QubitFluorometer (Thermo Fisher) The SeqCap EZ MedExomeTarget Enrichment Kit (Roche Sequencing) was used for thegDNA library preparation and exome enrichment followingthe manufacturerrsquos protocol We subjected the enrichedfragmental DNAs to sequencing by using the nextseq500high-output sequencing system (Illumina) to produce 2 times 150bp paired-end sequencing raw data17 Paired sequence readswere aligned to the human reference genome (hg 19) usingBurrows-Wheeler Aligner (BWA version 078) and thenvariants were called using SAMtools which were provided by

GlossaryASD = autistic spectrum disorder BSID = Bayley Scales of Infant Development BWA = Burrows-Wheeler Aligner FBS = fetalbovine serumH3K4 =H3 lysine 4 ID = intellectual disabilityHEK = human embryonic kidney PHD = plant homeodomainUTR = untranslated regionWPPSI =Wechsler Preschool and Primary Scale of IntelligenceWT = wild type XLID = X-linkedintellectual disability

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Partek Flow (Partek Inc) Rare variants were filtered accordingto the related records from genomic sequences databases in-cluding 1000 Genomes Project ExAC dbSNP and gnomADand the allele frequency of rare variant also was checked withthe information from Taiwan Biobank to pick up meaningfulvariant To isolate the variant that may directly affect thefunction of a particular protein stepwise filtering was used toremove common single nucleotide variations (formerly singlenucleotide polymorphisms) 39 and 59 untranslated region(UTR) variants nonndashsplice-related intronic variants and syn-onymous variants located at the nonconserved position or hadless likely splicing impact The functional variant was furthersubjected for genotype to phenotype analysis and manuallyreviewed by checking withClinVar andOMIMdatabase17 Thepotential pathogenic variants that passed the above filteringsteps were subsequently validated by Sanger sequencing toconfirm the calling without bias17

Validation of Variations by Sanger SequencingCandidate pathogenic variants were validated by Sanger se-quencing For PCR amplification of KDM5C the primerswere listed in eTable 1 (linkslwwcomNXGA499) PCRwas performed in a total volume of 20 μL containing 10 μLAmpliTaq Gold Fast PCR Master Mix (Applied BiosystemsFoster City CA) 02 μL of each primer with a concentrationof 10 pmolμL and 4 μL genomic DNA in a concentration of10 ngμL18 The PCR amplicons were sequenced using theBigDye Terminator v11 Cycle Sequencing Kit (AppliedBiosystems by Life Technologies Corporation) Sequencingwas performed using the 3500 Genetic Analyzer (AppliedBiosystems by Life Technologies Corporation)18

Computational ModelingThe deduced full-length and mutant proteins were subjected for3D structure prediction by using the SWISS-MODEL server(swissmodelexpasyorg) according to the suggested proceduresThe predicted model of protein structure was visualized and thesubstituted residue was labeled by YASARA (yasaraorg) Theprotein contained domains were analyzed by the NCBI Con-served Domains Database (ncbinlmnihgovcdd)

Functional Assays

KDM5C cDNA ConstructsThe wild-type (WT) human KDM5C cDNA in pcDNA31+C-(K)DYK vector was purchased from GenScript Technol-ogies Inc (NJ) To generate the Q745E and pE1131Afsmutants we used a mutagenesis kit (QuikChange II Site-Directed Mutagenesis Kit Stratagene La Jolla CA) followingthe manufacturerrsquos protocol Plasmids were purified with aplasmid DNA kit (Qiagen Plasmid Mini Kit Qiagen VenloLimburg and PureLink HiPure Plasmid Midiprep Kit LifeTechnologies Corp Carlsbad CA)19 All plasmid constructsequences were confirmed by direct sequencing

Cell CultureHuman embryonic kidney cells (HEK293) were obtained fromATCC Cells were maintained in Dulbeccorsquos Modified Eagle

Medium (DMEM Invitrogen Carlsbad CA) with 10 fetalbovine serum (FBS) and 100 unitsmL of penicillin-streptomycin(Invitrogen) in a humidified 5 (volvol) CO2 atmosphere

Transient TransfectionThe targeting construct was transiently transfected into cellsusing Lipofectamine 2000 reagent (Invitrogen) according to themanufacturerrsquos instructions In brief cells (3 x 106) were seededovernight The mixture of plasmid DNA (1 μg) and 2 μg ofLipofectamine 2000 diluted in Opti-MEM was added to cellsand cells were cultured for 48 hours before experiments

Western BlottingBriefly the protein samples (50 μglane) are separated by so-dium dodecyl sulfatendashpolyacrylamide gel electrophoresis andthen subjected to electrotransfer onto the polyvinylidenedifluoride membrane The membrane was subsequently in-cubated with primary antibodies (anti-KDM5C [Proteintech14426-1-AP] and anti-H3K4me3 [Cell Signaling 9727]) at 4degCfor overnight Thereafter the membranes were incubated withan HRP-conjugated secondary immunoglobulin G antibody for2 hours at room temperature Protein bands of interest in themembrane were visualized using the Super Signal Western Durasubstrate Following stripping membranes were incubated withanti-actin antibody (Sigma-Aldrich A5441) or anti-GAPDHantibody (Abcam ab181602) for protein semiquantification

Protein StabilityHEK293 cells transfected with WT or mutant clones ofKDM5C were incubated with cycloheximide (Sigma 20 mgmL) for 0 (DMSO vehicle only) 1 2 4 8 and 24 hours lysedand subjected to Western blotting11

Quantitative RT-PCRTotal RNA was isolated using the FavorPrep Tissue Total RNAMini Kit (Favorgen) according to the manufacturerrsquos instruc-tions Polyadenylated RNA was purified from at least 50 μg oftotal RNA using PolyA + Track mRNA Isolation System IIIfrom Promega (Z5300) according to the manufacturerrsquos in-structions First-strand cDNA was synthesized from total RNAusing a GoScript Reverse Transcription System (Promega) andT100 Thermal Cycler (Bio-Rad) according to the manufac-turerrsquos manual Transcript abundance was determined byquantitative PCR using a StepOnePlus Real-Time PCR (Ap-plied Biosystems) and primers specific for each transcript(eTable 1 linkslwwcomNXGA499) Expression levels werenormalized to the GAPDH housekeeping gene

Immunofluorescence for In SituDemethylation AssayCells transfected with WT and mutant clones of KDM5Cgrown on glass coverslips were fixed with 2 formaldehyde for10 minutes and then with 100 ice-cold methanol for 10 mi-nutes Cells were permeabilized with 01 NP-40 in PBSblocked with 20 FBS in PBS for 1 hour and then incubatedwith the appropriate primary antibodies including anti-H3K4me3 (Genetex GTX50897) anti-KDM5C (Proteintech

NeurologyorgNG Neurology Genetics | Volume 8 Number 1 | February 2022 3

14426-1-AP) and anti-Flag (Sigma-Aldrich F3165) overnightat 4degC11 After washing cells were incubated with Alexa 594ndashconjugated donkey anti-rabbit or Alexa 488-conjugated goatanti-mouse secondary antibodies (Molecular Probes) for 30minutes at room temperature11 Coverslips were thenmountedwith Vectashield containing DAPI (Vector Laboratories) fornuclear localization Images were acquired by laser confocalmicroscopy (Olympus)

StatisticsA commercial program (SPSS version 200 SPSS InstituteChicago IL) was used for the statistical analysis Data werepresented as mean plusmn SD (SD) In case of multiple comparisonsthe 1-way ANOVA plus Scheffe post hoc test was applied

Data AvailabilityThe data that support the findings of this study are alwaysavailable from the corresponding author

ResultsClinical Features of the PatientsThese 2 Taiwanese families both have 2 affected male siblingsrespectively The proband in the 1st family a 12-year-old boyis the first child born to healthy nonconsanguineous parentsHis perinatal history was unremarkable The patient startedwalking at age 23 months and riding a tricycle at age 45 yearsHe started babbling at age 12ndash14 months and spoke his firstwords after age 3 years yet he could not reliably use sen-tences The formal intelligence test revealed severe ID The

brain MRI at age 11 years was normal (Figure 1) His siblingwas diagnosed with severe ID later These boys had somedysmorphic features including short stature (less than 3rdpercentile) microcephaly (48 cm at age 5 years 3rd percen-tile) facial dysmorphism (prognathism of the mandible slightmaxillary hypoplasia flat philtrum strabismus and diastema)(Figure 1) Besides being severely ID these 2 patients did nothave seizures

In the 2nd family there were also 2 affected boys of healthynonconsanguineous parents The perinatal histories of themwere also unremarkable Both had a similar clinical coursepresenting with severe developmental delay precocious pu-berty and epilepsy They both started walking at around age 24months and still had poor daily coping skills like using spoonsThey spoke a few meaningful words after age 3 years yet theycould not reliably use sentences The formal intelligence testrevealed severe ID in both patients Focal seizures and gener-alized tonic-clonic seizures occurred in both patients since earlychildhood The EEG revealed slow background and activeepileptiform discharges at each side of frontotemporal areasOver the years a variety of anticonvulsants were tried to controlseizures including valproic acid oxcarbazepine lamotriginelevetiracetam and topiramate but epilepsy became intractableto anticonvulsants thereafter The proband is the youngersibling who was also diagnosed with autistic spectrum disorderat age 3 years and his MRI at age 14 years showed smallnodular T2 hyperintensity lesions over bilateral periventricularwhite matter (Figure 1) Besides they both had short statureand facial dysmorphism (prognathism of the mandible flat

Figure 1 Clinical Features and Neuroimages

Frontal and lateral views of the older sibling at age 9 years (A andB) and the younger sibling at age 6 years and 4months (C andD) in the 1st family BrainMRI ofthe older sibling of the 1st family at age 10 years (E and F T2-weighted images) and of the younger sibling of the 2nd family at age 13 years (G T1-weightedimage H FLAIR image) There were small white matter lesions (arrow) in the patient with pE1131Afs variation of the 2nd family

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philtrum and diastema) as well These 4 affected boys now areat the Special Education School in their hometown

Identification of Novel Variants in KDM5C byWhole-Exome SequencingWe identified sequence variants in KDM5C by whole-exomesequencing in 3 patients of these 2 families Overall we detected2 unique variants including c2233CgtG (pQ745E) in 2 siblingsof the 1st family and c3392_3393delAG (pE1131Afs) in 1sibling of the 2nd family (Figure 2A B) The pQ745E occurs athighly conserved residues and was predicted to be deleteriousand was absent in population‐based databases (gnomAD and

Taiwan Biobank) (Figure 2C) The other variant (pE1131Afs)had a small indel resulting in a truncated protein (Figure 2C) andis predicted to be deleterious as well It has not been documentedin gnomAD and Taiwan Biobank either In accordance with theACMG criteria these variants were all classified as pathogenic

Further segregation analysis by Sanger sequencing was con-ducted in the 1st family and showed that the pQ745E wasinherited from their mother Because both mothers have anormal intellectual phenotype and social adjustment abilitythe inheritance pattern of pQ745E should be an X-linkedrecessive type For some family issues the parents in the 2nd

Figure 2 Variations

The pedigree of the 1st family (A) and 2nd family (B) and sequence chromatograms of parts of the KDM5C gene The c2233CgtG nucleotide change of the 1stfamily is indicated with a circle (pQ745E) and the c3392_3393delAG nucleotide change of the 2nd family is indicated with an arrow (pE1131Afs) (C)Schematic of functional domains on humanKDM5Cproteins encodedby our 2 reported variations (D)Molecularmodeling of the variantModel generation isbased on template structure PDB 5fwj1A by using SWISS-MODEL server pQ745E found in affected individuals shown in red Zinc finger domain is in blueand zinc ions are shown as a yellow ball The surfacemodel of the zinc finger domain is presented as light gray Because of the lack of a suitable 3D structurethemodeling of pE1131Afs is not available now JmN = jumonji-N domain ARID = AT-rich interacting domain PHD = plant homeodomain box domain JmjC =jumonji-C catalytic domain ZF = zinc finger domain PLU-1 = PLU-1ndashlike domain

NeurologyorgNG Neurology Genetics | Volume 8 Number 1 | February 2022 5

family refused the segregation analysis to validate the in-heritance pattern

Functional Complementation

RNA Expression Protein Expression and ProteinStabilityThe effects of the genetic variations on the RNA expression levelof KDM5C in HEK293 cells transfected with WT and mutantclones were detected using qRT-PCR which showed that

KDM5C mRNA levels did not reduce in pQ745E andpE1131Afsmutants of KDM5C comparedwith that in KDM5CWT (Figure 3A) However the protein levels of KDM5CpQ745E and pE1131Afs were significantly reduced comparedwith those of KDM5C WT (Figure 3B) The reduction ofKDM5C (a demethylase enzyme) protein levels correlated withthe increases of its substrate H3K4me3 (Figure 3B) To in-vestigate whether the stability of KDM5C protein was affectedby pQ745E and pE1131Afs we treated cells with

Figure 3 Impacts of the pQ745E and pE1131Afs Variations on Expression of KDM5C

(A) Representative gel image of semiquantitative (above) and quantitative RT-PCR analysis (below) of KDM5C mRNA expression in HEK293 cells transfectedwith control plasmid WT KDM5C and mutants of KDM5C The expression levels were normalized with that of housekeeping gene GAPDH of the control(HEK293) cells (N = 3) (B) Whole cell lysates fromHEK293 cells transfectedwith control plasmid WT KDM5C andmutants were analyzed for the expression ofKDM5C and H3K4me3 protein (N = 4) (C) Cycloheximide (CHX) chase analysis was performed Immunoblotting was in the left panel and graphs in the rightpanel represented the percentage of remaining KDM5C The level of WT KDM5C decreased to 78 whereas the level of pQ745E and pE1131Afs KDM5Cdecreased to 47 and 11 at 24 hours after the CHX treatment The half-life of WT KDM5C was more than 24 hours and the half-life of pQ745E andpE1131Afs KDM5C was near 21 hours and 18 hours respectively Dotted line indicates 50 of initial normalized KDM5C protein level (N = 3) Data areexpressed as mean plusmn SD p lt 005 p lt 001 CHX = cycloheximide H3K4 = H3 lysine 4 WT = wild type

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cycloheximide an inhibitor of protein synthesis for 0ndash24 hoursAs shown in Figure 3C the increased degradation of pQ745Eand pE1131Afs KDM5C was reflected by a reduction of theirhalf-life from more than 24 hours (WT) to near 21 hours(pQ745E) and 18 hours (pE1131Afs) The data suggested thatprotein stability is largely affected by both variations

Enzymatic ActivityA majority of disease-associated KDM5C variations exhibit adecrease in histone demethylase activity suggesting a loss-of-function pathogenic mechanism1720 The demethylation ac-tivities were assessed ex vivo in cells (Figure 4) A significant

decrease ofH3K4me3 signals was observed in cells with ectopicexpression of KDM5C WT In contrast signals of H3K4me3were preserved in cells with ectopic expression of pQ745E orpE1131Afs KDM5C indicating that the demethylation activityof KDM5C was nearly abrogated by these 2 variations

DiscussionKDM5C encodes a ubiquitous protein which contains severalhighly conserved domains including the catalytic JmjC do-main the ARID DNA binding domain the zinc finger do-main and the JmjN domain for protein stability and 2 planthomeodomains (PHDs) for chromatin association21

KDM5C medicates the demethylation of trimethylated anddimethylated lysines on H3K4 (H3K4me3 and H3K4me2respectively) and possesses functions as a transcriptional co-repressor and an enhancer modulator121622 This chromatin-modifying enzyme is mainly expressed in brain and exertstight dose- and time-dependent control over neuronal de-velopment through silencing germline genes fine-tune theneuronal epigenome and preclude spurious transcription23

Thus KDM5C dysfunction plays a role in the etiology of IDand other neurologic disorders It was reported that germinalloss of KDM5C in mice causes dendritic and spine anomaliesand recapitulates cognitive adaptive and social abnormalitiesseen in patients with KDM5C variations including increasedaggression decreased anxiety and social behavior and defectsin learning and memory24 Furthermore RNA interferencendashmediated silencing of the KDM5C gene resulted in the de-repression of genes including SCN2A encoding sodiumchannel CACNA1B and CACNA1H encoding calciumchannels and SCL4A3 SLC18A1 and SLC6A12 encoding

Figure 4 In Situ Assay on Demethylation Activity of KDM5C

Representative immunofluorescence images of KDM5C and H3K4me3 incells transfected with WT and variations of KDM5C (N = 4) Cells transfectedwith WT KDM5C showed reduced H3K4me3 signals implicating demethy-lase activity of KDM5C (arrow) Cells transfectedwith pQ745E or pE1131Afsvariation retained high H3K4me3 signals implicating the reduction ofdemethylase activity of KDM5C (open arrowhead) Nuclei were stained withDAPI Scale bar = 50 μm H3K4 = H3 lysine 4

Figure 5 Distribution of Variations and Associated Phenotypes

Distribution of variations was shown on the KDM5Cprotein Each column in the heatmap represents a patient ranked by the position of the variant carried bythe patient and each row represents a clinical phenotype from patient surveys in the literature (references 9141526ndash36)

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monoamine transporters1225 All these KDM5C target geneshave been previously implicated in neurologic disorders suchas epilepsy autism or schizophrenia12

In this studywe present 2Taiwanese familieswithXLIDcaused bynovel variations c2233CgtG (pQ745E) and c3392_3393delAG(pE1131Afs) in theKDM5C gene and bothwere confirmed to bepathogenic The first variation (c2233CgtG pQ745E) is a mis-sense variation located in the coding region of the zinc fingerdomain This highly conserved domain contains potential zincligand-binding residues and may have a DNA-binding function12

Previous studied showed that variations in the zinc finger causeddifferent degrees of decreased histone demethylase activities andthe associated neurodevelopmental disability relied on the residualenzyme activities1926 The pQ745E variation of KDM5C identi-fied in our study decreased KDM5C protein levels and reducedprotein stabilities compared with cell expression with that of theWT KDM5C The in situ demethylation assay showed thatKDM5C with this pQ745E variation cannot efficiently reducelevels of its substrate H3K4me3 suggesting loss of its histonedemethylase activities The second variation (c3392_3393delAGpE1131Afs) is a frameshift variation located in the C-terminus ofthe KDM5C Although most of the functional domains werepreserved in this truncated KDM5C caused by pE1131Afs thelevel and stability of the truncated KDM5C were tremendouslyattenuated compared with WT KDM5C and KDM5C withpQ745E variation

To date 57 pathogenic variations on the KDM5C gene havebeen described in the English literature914151825-36 We sum-marized their loci and clinical manifestations in Figure 5 Ofinterest the missense and splice site variations commonly occurin functional domains and the nonsense and frameshift varia-tions are frequently in nonfunctional regions The phenotypevaries within and between families it is probably due to thefunctional severity of the variations the existence of possiblecompensatory mechanisms by alternative histone demethylasesand other epigenetic and environmental modifying factors25

Patients with variations in the zinc finger domain including ourpatient with pQ745E variation usually have severe ID shortstature and some facial dysmorphism However none withvariations involving the zinc finger domain have the manifesta-tion of seizures when compared with variations involving inother domains (Figure 5) Moreover we found that autisticspectrum disorder is not a common manifestation in these pa-tients with KDM5C variations but it only occurs in patients withvariations near the C-terminus like our reported patient with thepE1131Afs variation This evidence indicates that involvementsof particular functional domains of KDM5C on the epigeneticregulations may have distinguished impacts on the CNSdevelopment

KDM5C is one of the most frequently mutated genes foundin XLID89 It should be taken into consideration whenpatients with ID in the families with at least 2 affected malesiblings and may accompany with short stature andorseizures Further studies identify that novel disease-causing

mechanisms associated with variations within a particulardomain of KDM5C will help understand the role ofKDM5C in the CNS development

AcknowledgmentThe authors express their gratitude to the patients theircaretakers and the clinical and laboratory staff from NationalCheng Kung University Hospital

Study FundingThis study was supported by grants from National ChengKung University Hospital (NCKUH-10802015) andMinistryof Science and Technology (MOST 106-2314-B-006-075 and108-2314-B-006 -066) of Taiwanfunding

DisclosureThe authors report no disclosures Go to NeurologyorgNNfor full disclosures

Publication HistoryReceived by Neurology Genetics April 27 2021 Accepted in final formOctober 13 2021

Appendix Authors

Name Location Contribution

Po-MingWu MD

Department of PediatricsNational Cheng Kung UniversityHospital College of MedicineNational Cheng Kung UniversityTainan Taiwan

Draftingrevision of themanuscript for contentincluding medical writingfor content and major rolein the acquisition of data

Wen-Hao YuMD

Department of PediatricsNational Cheng Kung UniversityHospital College of MedicineNational Cheng Kung UniversityInstitute of Clinical MedicineCollege of Medicine TainanTaiwan

Draftingrevision of themanuscript for contentincluding medical writingfor content major role inthe acquisition of data andanalysis or interpretation ofdata

Chi-WuChiangPhD

Institute of Molecular MedicineCollege of Medicine NationalCheng Kung University TainanTaiwan

Draftingrevision of themanuscript for contentincluding medical writingfor content and analysis orinterpretation of data

Chen-YuWu MD

Department of PediatricsNational Cheng Kung UniversityHospital College of MedicineNational Cheng Kung UniversityTainan Taiwan

Major role in the acquisitionof data and analysis orinterpretation of data

Jia-ShingChenPhD

School of Medicine forInternational Students I-ShouUniversity Kaohsiung Taiwan

Draftingrevision of themanuscript for contentincluding medical writingfor content study conceptor design and analysis orinterpretation of data

Yi-FangTu MDPhD

Department of PediatricsNational Cheng Kung UniversityHospital College of MedicineNational Cheng Kung UniversityInstitute of Clinical MedicineCollege of Medicine TainanTaiwan

Draftingrevision of themanuscript for contentincluding medical writingfor content major role inthe acquisition of datastudy concept or designand analysis orinterpretation of data

8 Neurology Genetics | Volume 8 Number 1 | February 2022 NeurologyorgNG

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nities in the new millenniumMent Retard Dev Disabil Res Rev 20028(3)117-1344 Kuo HT Muo CH Chang YT Lin CK Change in prevalence status for children with

developmental delay in Taiwan a nationwide population-based retrospective studyNeuropsychiatr Dis Treat 2015111541-1547 doi102147NDTS84088

5 Ropers HH Genetics of early onset cognitive impairment Annu Rev Genomics HumGenet 201011161ndash187

6 Ropers HH Hamel BC X-linked mental retardationNat Rev Genet 20056(1)46-577 Vissers LE Gilissen C Veltman JA Genetic studies in intellectual disability and

related disorders Nat Rev Genet 201617(1)9-188 de Brouwer AP Yntema HG Kleefstra T et al Mutation frequencies of X-linked

mental retardation genes in families from the EuroMRX consortium Hum Mutat200728(2)207-208

9 Vallianatos CN Farrehi C Friez MJ Burmeister M Keegan CE Iwase S Alteredgene-regulatory function of KDM5C by a novel mutation associated with autism andintellectual disability Front Mol Neurosci 201811104

10 Merath K Ronchetti A Sidjanin DJ Functional analysis of HSF4 mutations found inpatients with autosomal recessive congenital cataracts Invest Ophthalmol Vis Sci 201354(10)6646-6654

11 Brookes E Laurent B Otildeunap K et al Mutations in the intellectual disability geneKDM5C reduce protein stability and demethylase activity Hum Mol Genet 201524(10)2861-2872

12 Tahiliani M Mei P Fang R et al The histone H3K4 demethylase SMCX links RESTtarget genes to X-linked mental retardation Nature 2007447(7144)601-605

13 Kouzarides T Chromatin modifications and their function Cell 2007128(4)693-705

14 Abidi FE Holloway L Moore CA et al Mutations in JARID1C are associated withX-linked mental retardation short stature and hyperreflexia J Med Genet 200845(12)787-793

15 Jensen LR Amende M Gurok U et al Mutations in the JARID1C gene which isinvolved in transcriptional regulation and chromatin remodeling cause X-linkedmental retardation Am J Hum Genet 200576(2)227-236

16 Iwase S Lan F Bayliss P et al The X-linked mental retardation gene SMCXJARID1C defines a family of histone H3 lysine 4 demethylases Cell 2007128(6)1077-1088

17 Chen JS Yu WH Tsai MC Hung PL Tu YF Comorbidities associated with geneticabnormalities in children with intellectual disability Sci Rep 2021116563

18 Laidoudi Y Davoust B Varloud M Niang EHA Fenollar F Mediannikov O De-velopment of a multiplex qPCR-based approach for the diagnosis of Dirofilariaimmitis D repens and Acanthocheilonema reconditum Parasit Vectors 202013(1)319

19 Merath K Ronchetti A Sidjanin DJ Functional analysis of HSF4 mutations found inpatients with autosomal recessive congenital cataracts Invest Ophthalmol Vis Sci 201354(10)6646-6654

20 Rujirabanjerd S Nelson J Tarpey PS et al Identification and characterization of twonovel JARID1C mutations suggestion of an emerging genotype-phenotype correla-tion Eur J Hum Genet 201018(3)330-335

21 Gonccedilalves TF Gonccedilalves AP Fintelman Rodrigues N dos Santos JM Pimentel MMSantos-Rebouccedilas CB KDM5C mutational screening among males with intellectualdisability suggestive of X-Linked inheritance and review of the literature Eur J MedGenet 201457(4)138-144

22 Collins BE Greer CB Coleman BC Sweatt JD Histone H3 lysine K4 methylationand its role in learning and memory Epigenetics Chromatin 201912(1)7

23 Scandaglia M Lopez-Atalaya JP Medrano-Fernandez A et al Loss of Kdm5c causesspurious transcription and prevents the fine-tuning of activity-regulated enhancers inneurons Cell Rep 201721(1)47-59

24 Iwase S Brookes E Agarwal S et al A mouse model of X-linked intellectual disabilityassociated with impaired removal of histone methylation Cel Rep 201614(5)1000-1009

25 Santos-Reboucas CB Fintelman-Rodrigues N Jensen LR et al A novel nonsensemutation in KDM5CJARID1C gene causing intellectual disability short stature andspeech delay Neurosci Lett 2011498(1)67-71

26 Ounap K Puusepp-Benazzouz H Peters M et al A novel c2T gt C mutation of theKDM5CJARID1C gene in one large family with X-linked intellectual disability Eur JMed Genet 201255(3)178-184

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NeurologyorgNG Neurology Genetics | Volume 8 Number 1 | February 2022 9

DOI 101212NXG000000000000064620228 Neurol Genet

Po-Ming Wu Wen-Hao Yu Chi-Wu Chiang et al Gene Causing X-Linked Intellectual DisabilityKDM5CNovel Variations in the

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