ARHGEF9 Mutations in Epileptic Encephalopathy/Intellectual Disability: Toward Understanding the Mechanism Underlying Phenotypic Variation

Neurogenetics (2018) 19:9–16 https://doi.org/10.1007/s10048-017-0528-2

ORIGINAL ARTICLE

ARHGEF9 mutations in epileptic encephalopathy/intellectual disability: toward understanding the mechanism underlying phenotypic variation

Jing-Yang Wang1,2 & Peng Zhou1,2 & Jie Wang1,2 & Bin Tang1,2 & Tao Su 1,2 & Xiao-Rong Liu1,2 & Bing-Mei Li1,2 & Heng Meng3,4 & Yi-Wu Shi 1,2 & Yong-Hong Yi 1,2 & Na He1,2 & Wei-Ping Liao1,2

Received: 7 September 2017 /Accepted: 25 October 2017 /Published online: 13 November 2017 # Springer-Verlag GmbH Germany 2017

Abstract ARHGEF9 resides on Xq11.1 and encodes Missense mutations with severe molecular alteration in the collybistin, which is crucial in gephyrin clustering and DH domain, or located in the DH-gephyrin binding region,

GABAA receptor localization. ARHGEF9 mutations have or adjacent to the SH3-NL2 binding site were associated with been identified in patients with heterogeneous phenotypes, severe epilepsy, implying that the clinical severity was poten- including epilepsy of variable severity and intellectual disabil- tially determined by alteration of molecular structure and lo- ity. However, the mechanism underlying phenotype variation cation of mutations. Male patients with ARHGEF9 mutations is unknown. Using next-generation sequencing, we identified presented more severe phenotypes than female patients, which a novel mutation, c.868C > T/p.R290C, which co-segregated suggests a gene-dose effect and supports the pathogenic role with epileptic encephalopathy, and validated its association of ARHGEF9 mutations. This study highlights the role of with epileptic encephalopathy. Further analysis revealed that molecular alteration in phenotype expression and facilitates all ARHGEF9 mutations were associated with intellectual dis- evaluation of the pathogenicity of ARHGEF9 mutations in ability, suggesting its critical role in psychomotor develop- clinical practice. ment. Three missense mutations in the PH domain were not associated with epilepsy, suggesting that the co-occurrence of epilepsy depends on the affected functional domains. Keywords ARHGEF9 . Collybistin . Epileptic encephalopathy . Epilepsy . Intellectual disability . Whole Jing-yang Wang and Peng Zhou are co-first authors due to their equal exome sequencing contribution to this work.

* Na He [email protected] Introduction * Wei-Ping Liao [email protected] The rho guanine nucleotide exchange factor 9 gene (ARHGEF9, MIM# 300429) encodes collybistin, a member of 1 Institute of Neuroscience and Department of Neurology of the the family of Dbl-like GDP/GTP exchange factors, and is Second Affiliated Hospital of Guangzhou Medical University Please expressed predominantly in the brain. Collybistin plays a critical check if the affiliations are presented correctly.The affiliations are presented correctly., Chang-Gang-Dong Road 250, role in the clustering of gephyrin-dependent glycine receptors γ Guangzhou 510260, China and -aminobutyric acid type A (GABAA) receptors at inhibi- 2 Key Laboratory of Neurogenetics and Channelopathies of tory postsynaptic sites [1]. Deficiency of collybistin due to Guangdong Province and the Ministry of Education of China, knockout of ARHGEF9 in mice induces a region-specific loss Guangzhou 510260, China of gephyrin and GABAA receptors at inhibitory synapses, lead- 3 Department of Neurology, The First Affiliated Hospital of Jinan ing to a significant change in hippocampal synaptic plasticity. University, Guangdong 510630, China Consequently, ARHGEF9-knockout mice exhibited decreased 4 Clinical Neuroscience Institute of Jinan University, exploration in a new environment, increased anxiety-related Guangdong 510630, China response, cognitive aptitude defects, and convulsions [2–4]. 10 Neurogenetics (2018) 19:9–16

In humans, patients with ARHGEF9 defects have been re- used for linkage and segregation analysis. Whole exome ported to present with extensive anomalies, including epilep- sequencing was performed on an Illumina HiSeq 2000 sy, hyperekplexia, intellectual disability, dysmorphism, and sequencer. Paired-end reads with a length of 90 bp were anxiety [5–7]. Mutation of ARHGEF9 was initially discovered generatedbymassiveparallelsequencingwithmorethan in a patient with epileptic encephalopathy (EE) [5]. However, 125 times average depth and more than 98% coverage of later studies identified mutations in patients with other milder the target region, which fulfills the quality criteria. types of epilepsy, and those patients were found to have The raw data were aligned to the reference human genome favorable responses to drug therapies [6, 8, 9]. Several (GRCh37) using Burrows−Wheeler Alignment (BWA). mutations have also been detected in patients with Stepwise filtering was applied to derive potential pathogenic dysmorphism or hyperekplexia without epilepsy [10–12]. variants. First, population-based filtration removed common Recent studies have further identified mutations in patients variants presenting a minor allele frequency (MAF) ≥ 0.005 in with distinct phenotypes of epilepsy or in patients without the 1000 Genomes Project, Exome Aggregation Consortium epilepsy [13, 14]. Therefore, the role of ARHGEF9 in epilep- (ExAC), or ExAC-East Asian Population (ExAC-EAS), ex- sy, particularly in EE, remains elusive. It is unknown whether cept for those previously reported to be associated with dis- phenotype variation correlates with ARHGEF9 mutation ease in the Human Gene Mutation Database (HGMD) and/or genotypes. Online Mendelian Inheritance in Man (OMIM) database. In this study, we performed whole exome sequencing in a Second, functional impact-based filtration prioritized func- family with four male siblings with EE and intellectual tional variants (protein-altering) that are missense, nonsense, disability. A novel mutation in ARHGEF9 was identified indel, frameshift, and splicing variants. Potential pathogenic and co-segregated with EE in this family. We further sys- variants were flagged if predicted as damaging by SIFT tematically reviewed all ARHGEF9 mutations and ana- (http://sift.jcvi.org/), PolyPhen2 (http://genetics.bwh.harvard. lyzed their molecular heterogeneity, aiming to define the edu/pph2/), and Mutation Taster (http://www.mutationtaster. genotype−phenotype correlation and the role of ARHGEF9 org/). Third, inheritance-based filtration filtered variants based mutations in epilepsy and intellectual disability. on family history and possible inheritance models. Sanger sequencing was used to validate the identified potential disease-causing variant. A total of 296 healthy Materials and methods volunteers were recruited as normal controls.

Patients Mutation analysis

A two-generation family with four affected male siblings To evaluate the pathogenicity of candidate variants, protein was recruited at the Epilepsy Centre of the Second modeling was performed using Iterative Threading Affiliated Hospital of Guangzhou Medical University. ASSEmbly Refinement (I-TASSER) software [18]basedon Clinical data were collected, including age at seizure onset, data from the Protein Data Bank (http://www.rcsb.org). The seizure types and frequency, response to antiepileptic drugs three-dimensional structures were shown using PyMOL 1.7. (AEDs), general and neurological examination results, and To explore the genotype−phenotype association, mutations detailed family history. Brain magnetic resonance imaging of the candidate gene and related phenotypes were systemat- (MRI) was performed to exclude symptomatic epilepsy. ically retrieved on the PubMed database. Video electroencephalography (EEG) was performed, and the results were reviewed by two qualified electroencephalogra- phers. Epileptic seizures and epilepsy were diagnosed and clas- Results sified according to the criteria of the Commission on Classification and Terminology of the International League Mutation screening and clinical features Against Epilepsy (ILAE) [15–17]. This study was approved by the ethics committee of the In the family in this study, four male siblings were affected, hospital, and written informed consent was obtained from the whereas four female siblings and their parents were asymp- patients and their parents. tomatic (Fig.1a). All affected patients showed phenotypic ho- mogeneity in EE and intellectual disability. We performed Whole exome sequencing whole exome sequencing in individual II-7 (proband), II-6, and their father. Sanger sequencing was used to validate the Genomic DNA was extracted from the peripheral blood using positive findings and the variants in II-1, II-8, and their moth- the Qiagen Flexi Gene DNA Kit (Qiagen, Germany). Blood er. We detected three variants that co-segregated with the phe- samples of available parents and siblings were collected and notype in this family. These variants were located in three Neurogenetics (2018) 19:9–16 11

Fig. 1 ARHGEF9 mutation and electroencephalogram (EEG) changes in the proband. a Pedigree of the family with epileptic encephalopathy. Clear symbols represent normal individuals and filled squares represent the affected men. Individuals tested for nucleotide substitution are indicated with either a T (mutant allele) or a C (normal allele). b Sequencing chromatogram for c.868C > T. c The amino acid sequence alignment of collybistin showed that the residue R290 was highly conserved in various species. d Interictal EEG changes in the proband demonstrated generalized and multifocal discharges, including generalized polyspikes (left), focal spike −slow waves in the bilateral temporal lobes (medium), and focal spike−slow waves in the right anterior frontal lobe (right panel)

genes, including ARHGEF9, CACNA1F,andMAP3K15. All MAP3K15, c.1872-8delT, was identified in the present family; these candidate genes were X-linked, consistent with the clin- this variation was present at an allele frequency of 0.004 in the ical pattern of X-linked recessive inheritance. 1000 Genomes Project. It was predicted that it most probably A novel mutation in ARHGEF9, c.868C > T/p.R290C affects splicing. However, as MAP3K15 is not expressed in (based on the longest transcript NM_01518.2), was identified the central nervous system, we considered that this variation (Fig. 1b). This mutation was not present in the general was not likely to play a pathogenic role in epilepsy. population of the 1000 Genomes Project, ExAC, and Clinical manifestations of the four male siblings are listed ExAC-EAS. It was also not in our 296 normal controls. It in Table 1. The proband (II-7) was an 8.5-year-old boy who was predicted to be Bprobably damaging^ by Polyphen had his first febrile seizure at 2 years of age. It was a (score = 1) and Bdeleterious^ by SIFT (score = 0). In a generalized tonic−clonic seizure (GTCS), which occurred cross-species comparison, the amino acid sequence alignment 5−6 times per month. Subsequently, he suffered from daily of ARHGEF9 showed that R290 was highly conserved in myoclonic seizures and complex partial seizures that occurred various species (Fig. 1c). ARHGEF9 has previously been re- once or several times per month. Seizures were refractory to ported to be an epilepsy-related gene and associated with EE any monotherapy or combination of carbamazepine, (MIM#300607), or other types of epilepsy, or developmental valproate, and topiramate. He had been born by normal delay, and intellectual disability [5, 9, 19], which is consistent delivery. His brain MRI findings was normal. Video EEG with the phenotypic characteristics of the affected individuals showed frequent interictal generalized polyspikes and fo- in the present study. cal spike−slow waves in the bilateral temporal and right CACNA1F has been associated with X-linked eye disorders anterior frontal lobes (Fig.1d). He was observed to have a [20]. The variation c.1373G > A/p.R458H was observed psychomotor developmental delay after birth. At the time with an allele frequency of 0.013 in ExAC-EAS and was of the study, he showed poor comprehension of verbal considered as a single nucleotide polymorphism (SNP). instructions, could not utter meaningful words, and was MAP3K15 is predominantly expressed in steroidogenic completely disabled in terms of the activities of daily living. tissue and has been associated with osmotic stress and blood A diagnosis of EE was considered. The other three male pressure regulation [21, 22]. An intronic variation in siblings shared a similar presentation and disease evolution. 12 Neurogenetics (2018) 19:9–16

Table 1 Clinical characteristics of the patients with ARHGEF mutation

Clinical features II-1 II-6 II-7 II-8

Current-age 21yrs 11yrs 9yrs 7yrs Gender Male Male Male Male FS onset age 4yrs 2yrs 2yrs 1yrs aFS onset age 5yrs 5yrs 3yrs 1.5yrs Initial seizure type FS (GTCS) FS (GTCS) FS (GTCS) FS (GTCS) Seizure types GTCS, CPS, MS GTCS, CPS, MS GTCS, CPS, MS GTCS, CPS, MS Seizure frequency GTCS: 3-4/month GTCS: 5-6/year GTCS: 5-6/month GTCS: 5-6/year MS: daily MS: daily MS: daily MS: daily Seizure outcome Refractory Refractory Refractory Refractory Intellectual disability Moderate Severe Severe Severe ADLs disability -+++ Speech disorder ++++ Dysmorphic features ---- Autistic features ----

CPS: complex partial seizures; GTCS: generalized tonic-clonic seizures; MS: myoclonic seizures; FS: febrile seizure; aFS: afebrile seizure; ADLs: activities of daily living

Collybistin deformation and epilepsy severity three main domains: SH3, DH, and PH (Fig.3a). Under normal conditions, the R290 residue (within the DH domain) An ARHGEF9 mutation in the R290 residue, R290H, was forms four hydrogen bonds with the E318, D430, N459, and previously identified in a patient diagnosed with X-linked R462 residues (Fig. 2a). E318 is located in the DH-PH linker, mental retardation [23]. The patients with R290H only had whereas the other three residues are located in the PH domain. simple GTCS. However, the present patients carrying These bonds are critical for the stable structure of collybistin. R290C showed obvious intractable seizures. The molecular When arginine is replaced by histidine at residue 290 (R290H, differences between R290H and R290C were further Fig. 2b), the original hydrogen bonds are all destroyed. analyzed. Instead, a new bond with S317 (within the DH-PH linker) is Mutation R290C was predicted to have more severe effects formed. These changes reduce the binding affinity for than R290H according to SIFT and Grantham scores (SIFT phosphatidylinositol-3-phosphate (PI3P) [24]. R290H also score 0 vs 0.07; Grantham score 180 vs. 29). Using I- leads to decreased positive charge and hydrophilicity. In con- TASSER, we further analyzed the effect of amino acid substi- trast, R290C destroys all hydrogen bonds, without forming tution on collybistin structure (Fig. 2). Collybistin consists of new bonds, and the region in the vicinity of this mutation

Fig. 2 Focal structure of collybistin in wild-type, R290H, and R290C. a formed a new bond between His-290 and Ser-317. c Substitution of Arginine 290 (blue) was located on α helix (yellow) within the DH arginine with cysteine at position 290 (R290C) destroyed all hydrogen domain. It interacted with Glu-318, Asp-430, Asn-459, and Arg-462 bonds without forming new bonds, which led to this area being neutral in through hydrogen bonds (green). b Substitution of arginine with histidine charge and losing hydrophilicity at position 290 (R290H) destroyed all original hydrogen bonds, but Neurogenetics (2018) 19:9–16 13

Fig. 3 Schematic illustration of mutation location and interaction of in red indicate mutant residues associated with epilepsy. Residues in blue collybistin in the postsynaptic membrane. a Schematic illustration of indicate mutant residues that are not associated with epilepsy and are the location of mutations in the three-dimensional structure of located in the PH domain. b Schematic illustration of interactions of collybistin that consists of SH3, DH, and PH domains. Green areas are collybistin with NL2, PI3P, gephyrin clusters, and GABAA receptor binding sites, which indicate NL2 binding in the SH3 domain, gephyrin located on the postsynaptic membrane. The PH domain and the PH- binding in the DH domain, and PI3P binding in the PH domain. Residues PI3P complex do not directly interact with GABAA receptors changes from positive to neutral charge and from hydrophilic mutations in the DH domain, L177P and R104Q, resulted to hydrophobic (Fig. 2c). in refractory epilepsy; R104Q was located in the gephyrin- binding region (L97−E110) (Fig. 3a). G55A in the SH3 Genotype−phenotype relationship in ARHGEF9 domain was identified in a severe case of EE. Several studies demonstrated that G55A, which is adjacent to the NL2- To explore the factors determining phenotypic variations, binding site (G56), resulted in the inability to activate we systematically reviewed ARHGEF9 mutations and an- collybistin [25, 26]. alyzed correlations between genotypes and phenotypes. To A nonsense mutation, Q2X, was associated with intractable date, 21 mutations have been identified: 10 missense, 1 epilepsy. Other mutations in males, including one splicing nonsense, 1 splicing mutation, 6 gross deletions, and 3 mutation and three gross deletions, led to mild epilepsy; the complex rearrangements (Table 2). All mutations were as- seizures in these individuals later improved or disappeared. sociated with intellectual disability, indicating that Six mutations were identified in female patients, including ARHGEF9 is a causative gene in intellectual disability. three gross deletions and three complex rearrangements. All affected individuals with point mutations were men These patients did not present with epilepsy or only had mild (Table 2). The location of the missense mutations in epilepsy, with good response to AEDs (Table 2). The female collybistin is illustrated in Fig. 3a.Threemissensemuta- carriers with point mutations (the mothers of the male tions (R338W, R356Q, E400K) in the PH domain were not patients) were all described as asymptomatic, but no data associated with epilepsy. The PH domain is responsible for on intelligence test were available.

PI3P binding involving R356−R357 (Fig. 3b). Thus, R356Q is located at the PH-PI3P-binding site. A previous study demonstrated that R338W resulted in decreased Discussion binding affinity of collybistin to PI3P[11]. In contrast, a mutation close to the DH-PH linker, G323R, resulted in In the present study, an ARHGEF9 mutation (c.868C > mild epilepsy with a favorable outcome. T/p.R290C) was identified in four male siblings affected by The other missense mutations outside the PH domain EE and intellectual disability. To our knowledge, this is the were all associated with epilepsy with varying severity. first report on ARHGEF9 mutation co-segregating with EE The mutation S317W in the DH-PH linker was identified in one family. This study highlights the pathogenic role of in two male siblings with mild seizures that partially ARHGEF9 in EE. Further analysis revealed a potential responded to AED therapy. R290H in the DH domain phenotype−genotype correlation in patients with was associated with mild epilepsy, whereas R290C, which ARHGEF9 mutations. was predicted to have a more detrimental effect on the ARHGEF9 is located on chromosome Xq11.1. This gene protein’s structure, was associated with the severe phenotype encodes collybistin, which is critical for gephyrin clustering of EE in the present study. The other two reported and GABAA receptor localization onto the postsynaptic 14 Neurogenetics (2018) 19:9–16

Table 2 Genetic and phenotypic features of patients with ARHGEF9 mutations

Mutation type Mutation (recurrence) Inheritance Location Exon GS Sex ID DD Seizures EE Prognosis of Ref. Ep

Missense c.164G>C/p.G55A NA SH3 2 60 M + + + + Intractable [5] c.311G>A/p.R104Q De novo DH 3 43 M + + + + Intractable [13] c.530T>C/p.L177P De novo DH 4 98 M + + + - Improved [13] c.869G>A/p.R290H NA DH 6 29 M + + + - NA [13] c.868C>T/p.R290C (4) Maternally DH 6 180 M + + + + Intractable This study c.950C>G/p.S317W (2) Maternally DH-PH 7 177 M + + + - Improved [13] linker c.967G>A/p.G323R (4) Maternally PH 7 54 M + + + - Seizure free [14] c.1012C>T/p.R338W (3) Maternally PH 7 101 M + + - - - [11] c.1067G>A/p.R356Q Maternally PH 8 43 M + - - - - [13] c.1198G>A/p.E400K Maternally PH 8 56 M + + - - - [10] Nonsense c.4C>T/p.Q2X Maternally - 1 - M + + + + Intractable [9] Splicing c.1300+2T>C Maternally - - - M + + + - NA [10] Gross deletion ChrX:61848414-63138698 De novo - - - M + + + - Well-controlled [6] ChrX:62321746-63058548 De novo - - - M + + + - Well-controlled [9] ChrX:62856174-63072861 De novo - - - M + + + - Well-controlled [8] ChrX:62970571-63052696 De novo - - - F + + - - - [12] ChrX:62838630-62865334 De novo - - - F + + - - - [13] ChrX:62854862-62862403 De novo - - - F + + + - Seizure free [13] Complex 46,X,inv(X)(q11.1;q27.3) De novo - - - F + + - - - [7] rearrangement 46,X,t(X;18)(q11.1;q11.21) De novo - - - F + + + - NA [30] 46,X,t(X;20)(q12;P13) De novo - - - F + + + - Improved [13]

DD: developmental delay; EE: epileptic encephalopathy; Ep: epilepsy; F: female; GS: Grantham score; ID: intellectual disability; M: male; NA: not available membrane. To date, 21 mutations have been identified, includ- PH domain did not develop epilepsy [13]. The three non- ing 11 destructive mutations that cause gross protein malfor- epileptogenic mutations were located in exon 7 or exon 8 mation (nonsense, deletions, and complex rearrangements) (Table 2), implying that the occurrence of epilepsy potentially and ten missense mutations, of which three were shown to depends on the functional domains affected, but not on the impair the gephyrin clustering function of collybistin [5, 11, mutated exons per se. 24, 25]. ARHGEF9-knockout mice exhibited cognitive de- Missense mutations in the DH-PH linker and DH and SH3 fects, anxiety, and convulsions, and were associated with a domains were all associated with epilepsy of varying clinical selective loss of gephyrin-dependent GABAA receptor clus- severity, typically observed in mutations at residue R290 in ters in the amygdala and hippocampus [2–4]. These findings the DH domain. R290H caused mild GTCS, whereas R290C suggest that loss of function may act as a pathogenetic mech- resulted in severe EE. R290C was predicted to have more anism in ARHGEF9-associated diseases. All patients with detrimental structural effects than R290H (Fig. 2 and mutations had intellectual disability, indicating that Table 2), which may explain the severe phenotype in ARHGEF9 plays a critical role in psychomotor development. R290C. L177P in the DH domain also had a relatively higher However, not all patients with ARHGEF9 mutations pre- Grantham score and was associated with severe epilepsy. sented with epilepsy. Three missense mutations were not as- These findings suggest that clinical severity was correlated sociated with epilepsy and were all in the PH domain, includ- with the molecular effect. Another DH-located mutation, ing one located at the PH-PI3P binding site (R356Q). A PH R104Q, had a relatively low Grantham score, but was associ- domain-located mutation (G323R) was close to the DH-PH ated with severe epilepsy. However, R104Q is located in the linker and was associated with mild seizures. This evidence gephyrin-binding region, potentially explaining the severe suggested that dysfunction of the PH domain may not be phenotype. This suggests that the location of mutations is associated with epilepsy. A recent study has reported that pa- possibly one of the determinants of clinical severity. Severe tients with mutations in exon 9 or the last exon affecting the EE was also observed in the patient with a G55A mutation, Neurogenetics (2018) 19:9–16 15

which was adjacent to the SH3-NL2 binding site. G55A was gephyrin scaffold formation and subsequent GABAA receptor experimentally shown to abolish NL2 binding and to render localization [29]. However, previous studies have also dem- collybistin resistant to NL2 activation, which subsequently onstrated that deletion of the PH domain with abolished PI3P disrupted GABAA receptor and gephyrin clustering [25, 26]. binding does not affect the interaction of collybistin with All reported destructive mutations in men, except Q2X, gephyrin [4, 30]. As shown in Fig. 3b, the PH domain and were associated with mild epilepsy. Previous studies indicated the PH-PI3P complex do not directly interact with GABAA that nonsense mutations located in the first 100 coding nucle- receptors. This may explain why the dysfunction of the PH otides can evade nonsense-mediated mRNA decay (NMD) domain is not associated with epilepsy. On the other hand, a [27, 28], which exerts a protective effect by eliminating recent study indicated that collybistin interacts with the mam- mRNAs carrying premature stop codons. Translation of these malian target of rapamycin (mTOR) and affects mTOR com- aberrant mRNAs could, in some cases, lead to dominant- plex 1 (mTORC1) signaling activity, which might contrib- negative activity of the harmful truncated proteins. Q2X was ute to intellectual disability and behavioral abnormalities in located in the NMD-insensitive region and might escape patients with ARHGEF9 mutations [8]. NMD, explaining the severe EE observed. In conclusion, we identified a novel ARHGEF9 mutation The three male patients with ARHGEF9 deletions were that co-segregated with EE and intellectual disability in a associated with mild epilepsy with favorable outcomes family, confirming its pathogenic role in EE. A systematic (Table 2), whereas those with missense mutations presented analysis of ARHGEF9 mutations revealed that the co- epilepsy of varying severity or even without epilepsy, as occurrence of epilepsy is potentially related to the type of described above, indicating that the damaging effect of mutation and the affected functional domains; the epilepsy missense mutations differs. However, the pathogenicity severity was potentially determined by the severity of the of mutations is also affected by other modifying genetic structural effect on the molecule and the location of the or external factors, as manifested by phenotype variations missense mutations. This study highlights the role of the between family members who carry the same mutation. molecular effects of mutations in phenotype expression and is The current data of ARHGEF9 mutations are relatively expected to facilitate evaluation of the pathogenicity of limited. Further studies with high quality phenotyping are ARHGEF9 mutations in clinical or genetic counseling practice. required to confirm the genotype−phenotype correlation of ARHGEF9 mutations. In female patients, no epilepsy or only mild epilepsies were reported (Table 2). The female carriers with ARHGEF9 Acknowledgements We thank the family and physicians for participa- missense mutations were all asymptomatic, in contrast to tion in our study. We are grateful to the He Shanheng Charity Foundation their affected male children. This may be because females for contributing to the development of this institute. have two copies of the X chromosome. A pathogenic het- erozygous mutation in females would cause relatively milder genetic damage than that in males with hemizygous mutations. The differences in men and women suggested a Funding information This work was supported by the National gene-dose effect, which supported the pathogenic role of Natural Science Foundation of China (Grant Nos. 81571273, ARHGEF9 mutations. It was noted that the two missense 81501124, and 81571274), Omics-based Precision Medicine of Epilepsy being entrusted by Key Research Project of the Ministry of mutations associated with the severely affected boys had a Science and Technology of China (Grant No. 2016YFC0904400), and de novo or unknown origin (c.311 > A/p.R104Q, c.164G > Science and Technology Project of Guangzhou (Grant Nos. C/p.G55A). Thus, currently, it is difficult to define a genotype 201,508,020,011, 201,604,020,161, and 201,607,010,002). −phenotype correlation for missense mutations in females. Compliance with ethical standards Furthermore, the random deactivation of X-chromosome in − females may add complexities to the genotype phenotype Ethical approval All procedures performed in studies involving hu- correlation [7, 12, 13, 30]. man participants were in accordance with the ethical standards of the Reduced GABAergic transmission has been proposed as institutional and/or national research committee and with the 1964 an underlying pathophysiologic mechanism of ARHGEF9- Helsinki declaration and its later amendments or comparable ethical standards. associated disease, including epilepsy and intellectual disability [25]. It is not known why mutations in the PH domain, including the mutation at the PH-PI3Pbindingsite, Informed consent Informed consent was obtained from all individual are not associated with epilepsy. The PH domain is respon- participants included in the study. sible for binding of collybistin to PI3P on the membrane. The collybistin recruitment to cellular membranes together Conflict of interest The authors declare that they have no conflict of with gephyrin is believed to be essential for proper interest. 16 Neurogenetics (2018) 19:9–16

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