Mutations of protocadherin 19 in female epilepsy (PCDH19-FE) lead to allopregnanolone deficiency

OR I G INA L ART I C L E

Mutations of protocadherin 19 in female epilepsy(PCDH19-FE) lead to allopregnanolone deficiencyChuan Tan1, Chloe Shard2, Enzo Ranieri4, Kim Hynes1, Duyen H. Pham1,3,Damian Leach5, Grant Buchanan5, Mark Corbett1, Cheryl Shoubridge1,3,Raman Kumar1,3, Evelyn Douglas4, Lam S. Nguyen1,6, Jacinta Mcmahon7,Lynette Sadleir8, Nicola Specchio9, Carla Marini10, Renzo Guerrini10,Rikke S. Moller11,12, Christel Depienne13,14, Eric Haan1,3,15, Paul Q. Thomas2,Samuel F. Berkovic7,16,17, Ingrid E. Scheffer7,16,17 and Jozef Gecz1,2,4,3,*1School of Paediatrics and Reproductive Health, 2School of Molecular and Biomedical Sciences, 3RobinsonResearch Institute, The University of Adelaide, Adelaide, SA, Australia, 4SA Pathology, Adelaide, Australia,5Basil Hetzel Institute for Translational Health Research, The Queen Elizabeth Hospital, Adelaide, SA, Australia,6INSERMUMR 1163, Laboratory of Molecular and Pathophysiological Bases of Cognitive Disorders, Paris Descartes—Sorbonne Paris Cité University, Imagine Institute, Necker-Enfants Malades Hospital, Paris 75015, France,7Epilepsy Research Centre, The University of Melbourne, Melbourne, VIC, Australia, 8Department of Paediatricsand Child Health, School of Medicine and Health Sciences, University of Otago, Wellington, New Zealand,9Division of Neurology, Department of Neuroscience, Bambino Gesù Children’s Hospital IRCCS, P.za S. OnofrioRome 400165, Italy, 10Neuroscience Department, Children’s Hospital A. Meyer, University of Florence, Firenze,Italy, 11Danish Epilepsy Centre, Dianalund, Denmark, 12Institute of Regional Health Services Research, Universityof Southern Denmark, Odense, Denmark, 13Inserm, CNRS, UM 75, U 1127, UMR 7225, ICM, Sorbonne Universités,UPMCUniv Paris 06, Paris F-75013, France, 14Département de Génétique et de Cytogénétique, AP-HP, Hôpital de laPitié-Salpêtrière, Paris F-75013, France, 15South Australian Clinical Genetics Service, SA Pathology, NorthAdelaide, Australia, 16Epilepsy Research Centre, The University of Melbourne, Melbourne, Australia and17Florey Institute of Neuroscience and Mental Health, Melbourne, Australia

*To whom correspondence should be addressed at: School of Paediatrics and Reproductive Health, University of Adelaide at Women’s and Children’sHospital, 72 King William Road, North Adelaide, SA 5006, Australia. Tel: + 61 881616339; Fax: + 61 881617342; Email: [email protected]

AbstractProtocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited tofemales, EFMR; MIM300088) is an infantile onset epilepsy syndromewith or without intellectual disability (ID) and autism. Weinvestigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolizeneurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E−47, two-tailedFisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymeswhose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly

Received: May 1, 2015. Revised: June 18, 2015. Accepted: June 22, 2015

© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Human Molecular Genetics, 2015, 1–10

doi: 10.1093/hmg/ddv245Advance Access Publication Date: 29 June 2015Original Article

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decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced.In conclusion,we show that thedeficiencyof neurosteroid allopregnanolone, oneof themost potentGABA receptormodulators,may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism andcreate realistic opportunities for targeted therapeutic interventions.

IntroductionProtocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE;also known as epilepsy andmental retardation limited to females,EFMR; MIM300088) is an infantile onset epileptic encephalopathy(EIEE), with or without intellectual disability (ID) and autism(1,2). PCDH19-FE eluded geneticmapping due to its female-limitedexpression until 1997 when Ryan et al. (3) mapped the responsiblegene, perhaps surprisingly, to the X-chromosome. It was another10yearsbefore systematic sequencingofX-chromosomeexons re-vealed mutations of the protocadherin 19 (PCDH19) gene as thecause of the disorder (4). In general, the EFMR phenotype was re-stricted to females while males transmitting the mutations wereapparently unaffected (1). However, serendipitous identificationof amalewith somatic mosaicism for PCDH19 deletion and a seiz-ure disorder resembling Dravet syndrome led Depienne and col-leagues (5) to identify PCDH19 mutations in small families andsingletons with SCN1A negative Dravet-like infantile epileptic en-cephalopathy. Many reports of PCDH19-positive cases followed[see example inRef. (6) for review] and the PCDH19-FE-related clin-ical spectrum broadened (6,7). Most typical characteristics areearly-onset seizures (6–36 months) (1) followed by clusters of re-current seizures throughout childhood. The severity and fre-quency of seizures, as well as the presence of other clinicalfeatures such as autistic and obsessive compulsive behavior, de-pression and schizophrenia, vary among the affected females(8). No obvious correlation between the type of PCDH19 mutationand the phenotype has been ascertained. The majority of muta-tions cluster in the extracellular domain of PCDH19 protein. Onlyframeshift mutations, but no missense mutations, have been de-scribed in the intracellular domain (6). Variable clinical expressiv-ity was noted originally when comparing large EFMR pedigreeswithPCDH19-FE-reproducing females (1), andwhencomparing re-producing females with severely affected singletons (5), twins (9),sibs (10) and mother–daughter pairs (11). The broad clinical vari-ability of PCDH19 mutation expressivity, including non-pene-trance, suggests the presence of environmental, genetic orstochastic (e.g. X-chromosome inactivation) modifiers. We postu-late that interindividual variability in steroid and neurosteroidmetabolism together with variation in X-chromosome inactiva-tion may explain the variable penetrance of PCDH19-FE. Here,we provide evidence in support of a role for neurosteroids, allo-pregnanolone, in particular in the pathophysiology of PCDH19-FE.

ResultsExpression profiling

Using genome-wide gene expression (Affymetrix Human Exon 1.0ST arrays) analysis, we investigated two cohorts of PCDH19-FE pa-tients. Cohort 1 (AF1-O) comprisedolder PCDH19-FE females,withameanageof 25 years, themajorityofwhomno longer suffered fromseizures (n = 6). Cohort 2 (AF2-Y) comprised young PCDH19-FE fe-males, with a mean age of 8.8 years, the majority of whom werestill experiencing seizures (n = 6). These femaleswere fromdifferentfamilies and had different PCDH19 mutations (see Table 1 for fur-ther detail and references). We also investigated transmittingmales (TM; n = 3) skin cell lines from age and passage-matched,

control males (n = 3) and females (n = 3). Comparison of gene ex-pression between PCDH19-FE females and female controls (FC)identified 192 (AF1-O) and 140 (AF2-Y) significantly dysregulatedgenes (P < 0.05, fold change greater than ±2, one-way ANOVA;Fig. 1A), respectively. Therewere 94 dysregulated genes in commonbetweenAF1-O and AF2-Y, of which 73were annotated (Fig. 1B, seefull list of dysregulated gene in Supplementary Material, Table S2).Of the 73 genes, 92% are known to be expressed in the brain (Uni-gene). Comparison between the expression profiles of TM andmale controls (MC) identified 136 significantly dysregulated genes(P < 0.05, fold change greater than ±2, one-way ANOVA; Fig. 1A), ofwhich four genes (IL1R1, STEAP1, OSR1 and ASPA) were also identi-fied in the 94 shared dysregulated genes fromAF1-O/AF2-Y versusFC comparison.

The unusual, gender reversed X-chromosome inheritance ofPCDH19-FE ledus to speculate that geneswithdifferent expressionbetween the two sexes, that is gender-biased genes, may play arole. To investigate this, we first generated a list of likely gender-biased genes by comparing genome-wide expression between FCandMCwith 223 genes identified (Fig. 1A and Supplementary Ma-terial, Fig. S1). Subsequently,we compared this set of genes againstthe 94 significantly dysregulated genes from the affected femaleversus control female comparisons (see above). Interestingly, 43of these 94 genes (68%) had gender-biased expression (Fig. 1B).This enrichment of gender-biased genes was statistically highlysignificant compared with the number of gender-biased genes incontrol skin fibroblast cells (P = 2.51 × 10−47, two-tailed Fisher’sexact test; Fig. 1C). To further test this observation, we took advan-tage of publicly available (Array Express < EMBL—EBI—EuropeanBioinformatics Institute) expression array data sets with largernumbers of control skin fibroblasts (25 females and 23 males)and generated refined gender-biased gene lists (SupplementaryMaterial, Fig. S1). Despite the reduction of overall number (Supple-mentary Material, Fig. S1B), the enrichment of gender-biasedgenes in PCDH19-FE remained highly statistically significant (P =1.58 × 10−18 or P = 2.91 × 10−15 when looking at two different datasets, respectively; Supplementary Material, Fig. S1B). Additionally,we performed an in silico permutation assay with randomly gener-ated lists of 100 genes and assessed the enrichment of gender-biased genes. After 100 simulations, we found on average onlyone (minimum=0,maximum= 5) gender-biased gene in each ran-domly generated gene list. The difference between the observedand the simulated enrichment of gender-biased genes was alsostatistically significant (observed = 43/94 versus simulated =5/100, P = 7.8 × 10−8, two-tailed Fisher’s exact test).

Pathway analysis and data validation

To investigate molecular pathways affected in PCDH19-FE skinfibroblasts, we assessed the significant annotated gene list(73 genes) using Database for Annotation, Visualization and Inte-grated Discovery (DAVID) pathway and Ingenuity Pathway Ana-lysis (IPA). Highlighted by DAVID analysis were pathways thatare highly relevant to PCDH19 protein function, including cell ad-hesion and integrin-related signaling (Supplementary Material,Table S3). IPA analysis showed similar pathway enrichment,with cell-to-cell signaling and interaction ranking the highest

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Table 1. Summary of clinical and genetic data of 12 PCDH19-FE patients studied in expression array

AF1-O (Cohort 1) AF2-Y (Cohort 2)

1 2 3 4 5 6 7 8 9 10 11 12

Age at the

time of the

study

51 years 22 years 24 years 20 years 21 years 12 years 9 years 8 years 12 years 11 months 10 years 10 years

Age of

seizures

onset

8 months 24 months 18 months 9 months 16 months 7 months 8 months 6 months 7 months 7 months 14 months 24 months

Seizure type

at onset

Focal seizure Right

hemiclonic

seizures

Simple febrile

generalized

tonic clonic

seizures

Febrile

generalized

tonic clinic

seizure

Afebrile

generalized

tonic clonic

seizures

Febrile

generalized

tonic clonic

seizures

Focal febrile

seizures

Focal Focal Focal Febrile seizure

and cluster of

focal seizures

Cluster of febrile

seizures

Seizure

frequency

Monthly cluster

of seizures

Monthly cluster

of seizures

Monthly

cluster of

seizures

Cluster of

convulsions

every couple

of years,

regular

myoclonic

seizures

Yearly cluster

of seizures/

status

epilepticus

Monthly

cluster of

seizures

Clusters of seizures

every 2–3

months, yearly

status

epilepticus

(↑fever)

Monthly to

yearly

cluster of

seizures

and status

epilepticus

Single seizure

every 2–3

months,

clusters every

2 years

(↑fever)

3 clusters of

seizures

Monthly to

yearly clusters

No seizures

Age of

seizures

offset

12 years 16 years 12.5 years 10 years 10 years − − − − − − Currently seizure

free (no

medication)

Family history + + + + + + − − − + + +

Development Delayed

development

(no regression)

Delayed

development

(no

regression)

− Regression

at 9 months

Regression at

16 months

Regression at

7 months

− − − − − −

Intellect Normal Borderline Normal (no

regression)

Mild mental

retardation

Mild mental

retardation

Mild mental

retardation

Severe mental

retardation

Moderate

mental

retardation

Mild mental

retardation

Borderline Severe

mental

retardation

Normal

Psychiatric

features

Depression Attention

deficit

hyperactivity

disorder

Asperger

syndrome,

obsessional

features

Autistic

features

None Autistic

features

Autistic features Autistic

features

Autistic features Hyperactivity Autistic features normal

Initial

assessment

(epilepsy

syndrome)

EFMR EFMR ERMR EFMR EFMR EFMR Epileptic

encephalopathy

with prolonged

febrile and

afebrile seizures

Focal epilepsy Dravet

syndrome

Focal

epilepsy

Epilepsy with

focal and

generalized

seizures

Focal epilepsy

(rare seizures)

PCDH19

mutation

c.1322T>A

p.V441E

c.1671C>G

p.N557K

c.1671C>G

p.N557K

c.2012C>G

p.S671X

c.2012C>G

p.S671X

c.253C>T

p.Q85X

c.119G>C

p.Asp377His

(de novo)

c.2675–6A>G

p.? (de novo)

c.[608A>C;617 T]

p.[H203P;

F206C]

(de novo)

c.2697dupA

p.E900Rfs*8

(de novo)

c.1300_1301delCA

p.Q434EfsX11

(de novo)

c.1300_1301delCA

p.Q434EfsX11

(de novo)

Publication

reference

Ref. (1) and (4) Ref. (4) Ref (4). Ref. (1) and (4) Ref. (1) and (4) Ref. (1) and (4) Ref. (12) Ref. (12) Ref. (12) Ref. (12) Ref. (13) Ref. (13)

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Figure 1. PCDH19-FE Affymetrix Human Exon 1.0 ST arrays analysis. (A) Comparison of number of dysregulated gene reported in each group. Fibroblast samples were

grouped into affected female (Cohort 1; AF1-O; O = old) (n = 6), affected female (Cohort 2; AF2-Y; Y = young) (n = 6), female controls (n = 3; FC), male controls (n = 3; MC)

and transmitting male (n = 3; TM). All expression-profiled samples were imported and analyzed simultaneously in Partek Genomic suite V6.6. One-way ANOVA tests

were performed to detect differentially expressed genes between selected status groups (P-value < 0.05 and fold change greater than or equal to ±2). (B) Venn diagram

illustrating the overlap of three genes lists. AF1-O and AF2-Y shared 94 differentially expressed genes compared with FC, in which 43 overlapped with the gender-

biased gene list generated from comparing FC with MC. (C) Graph illustrating the percentage enrichment of gender-biased genes in each gene list generated from one-

way ANOVA analysis of the microarray results. P-values were derived from Fisher’s exact t-test with two-tailed analysis for each ratio of gender-biased genes in the gene

lists comparedwith the ratio of gender-biased genes present in the fibroblast pool. These analyses showed significant enrichment of gender-biased genes (P-value < 0.05).

(D) Hierarchical clustering analysis using 43 significantly dysregulated genes originally identified by comparison of affected PCDH19-FE females against FC. Note that

PCDH19-FE female’s cluster with TM and MC instead of FC.


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(Supplementary Material, Table S3). None of the highlightedpathways showed enrichment of the dysregulated gender-biasedgenes. However, when we examined the upstream regulatory re-gions of the 73 annotated PCDH19-FE dysregulated genes (part ofthe IPA analysis), we found that 22% (16/73) of these genes areregulated by chorionic gonadotropin (Cg), progesterone and es-trogen through their respective receptors, that is progesteronereceptor (PGR) and estrogen receptor alpha (ESR1) (Fig. 2A).Cross-examination of these 16 genes with IPA pathway analysis,in particular molecular and cellular functions, showed that 56%of these were involved in cell-to-cell signaling and interactionand that all 16 genes are also bona fide targets of nuclear steroidhormone receptors (Supplementary Material, Table S3). We se-lected 5 (WISP2, OXTR, AKR1C3, APOD andGRIA1) for reverse tran-scriptase-quantitative polymerase chain reaction (RT-qPCR)validation and showed that all five genes had the same trend ofdysregulation as identified by microarray expression analysis(Fig. 2B). To ascertain the biological relevance of this finding, add-itional controls (3 males and 3 females) were included. We vali-dated 4/5 genes (WISP2, AKR1C3, APOD and GRIA1)(Supplementary Material, Fig. S2A). OXTR, while it did not valid-ate across all PCDH19-FE individuals, showed statistically

significant difference (P < 0.01) for the AF-O cohort (Supplemen-tary Material, Fig. S2A). Intriguingly, when examining these andseveral other genes that we tested (data not shown), PCDH19-FEfemales showed expression levels more similar to control malesthan to control females. This observation was reinforced by su-pervised hierarchical clustering analysis (see Fig. 1D). This‘male-like’ expression was further validated on skin fibroblastcell lines from additional six PCDH19-FE females (data notshown). When we performed RT-qPCR analysis of three genes(OXTR, AKR1C3 and GRIA1) in primary skin fibroblast cell linefrom the single reported affected mosaic male (5), we foundthat all three genes were dysregulated and were expressed atthe levels more similar to FC than to MC (Fig. 2B). In addition togenes linked to hormone regulation, we selected six non-hormo-nal regulated genes with potential functional relevance toPCDH19-FE for RT-qPCR validation. In total, we validated >60%(technical) and 45% (biological) of these genes (Fig. 2B).

Neurosteroid enzyme dysregulation and measurements

Two genes, AKR1C2 and AKR1C3, were of particular interestamong the significantly dysregulated genes. AKR1C2 and

Figure 2. IPA and RT-qPCR validation of dysregulated genes selected from the microarray analysis. (A) In PCDH19-FE, dysregulated genes are involved in sex hormone

regulation. Seventy-three annotated genes from the shared 94 dysregulated genes in PCDH19-FE were analyzed by IPA. Figure illustrates the direct and indirect

relationship of 16/73 genes that are regulated by chorionic gonadotropin (Cg), progesterone and estrogen through their respective receptors, that is PGR and ESR1. (B)Bar graphs illustrating RT-qPCR validation of selected targeted genes. Mean expression level of the targeted genes, after normalization against a control gene is

shown. Value of each status group (except FC) was relative to FC. Error bar indicates the standard deviation of each status group. Top panel, RT-qPCR validation of

RNA samples (different passages) from the microarray analysis showing a trend of dysregulation of gene expression in PCDH19-FLE, where AF gene expressions were

similar of MC than FC. Bottom panel, RT-qPCR analysis of OXTR, AKR1C3 and GRIA1 expression on PCDH19-FE mosaic male and control groups (FC and MC). PCDH19-

FE mosaic male showing significant dysregulation of OXTR and AKR1C3 expression compared with control group (FC or MC), with a P-value of 0.06 compared with MC

in GRIA1. Statistical analysis was performed using Student’s t-test (two-tailed, unequal variance). *P < 0.05, **P < 0.01.

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AKR1C3 aremembers of the aldo-keto reductase 1C (AKR1C) fam-ily that consists of four members, AKR1C1-4, of which onlyAKR1C1-3 are expressed in brain. In mammalian brain, these en-zymes are responsible for reducing oroxidizing steroid hormonesinto their downstream metabolites, namely neurosteroids.AKR1C3 has several enzymatic activities, including 20α-hydro-xysteroid dehydrogenase (20α-HSD), 3α-HSD, 17β-HSD and 11-ketoprostaglandin reductase activity, indicating that it cancoordinate ligand access to progesterone, androgen, estrogenand peroxisome proliferator-activated nuclear steroid receptorslocalized in tissues in which it is present (14). Together withAKR1C1 and AKR1C2, AK1RC3 is able to metabolize 5α-hydroxy-progesterone (5αDHP; immediate downstream product of proges-terone) into allopregnanolone (3α-hydroxy-5α-pregnan-20-one).Neurosteroids exert multiple biological activities, conventionalgenomic as well as various membrane receptor facilitated. Neu-rosteroids act not only on GABAA, but also on NMDA, kainate andAMPA glutamate receptors or glycine and serotonin receptors(15). Allopregnanolone acts as a positive allosteric modulator ofGABAA inhibitory neurotransmitter receptors, causing prolongedhyperpolarization of neurons andhence inhibiting the over excit-ability of neurons associated with an epileptic seizure (16). How-ever, allopregnanolone can also modulate glutamate release viapresynaptic GABAA receptors (17). We confirmed significant

downregulation of AKR1C3 mRNA in PCDH19-FE females by RT-qPCR (Figs 2B and 3A). AKR1C3 dysregulation was also confirmedat the protein level (Fig. 3B). AKR1C3 was confirmed to be ex-pressed in a gender-biased manner (Fig. 3B). TM investigatedshowed slight upregulation of AKR1C3 (data not shown); theimportance of this remains to be determined (e.g.whether poten-tially protective).AKR1C2mRNAwas significantly downregulatedprimarily in old (AF1-O) PCDH19-FE females (data not shown).Wewere unable to determine the protein level of AKR1C2 due to thelack of AKR1C2-specific antibody.

Given that AKR1C1-3 enzymes are crucial for allopregnano-lone production, we speculated that PCDH19-FE females mightbe allopregnanolone deficient. We therefore recruited further 9PCDH19-FE females, different to those 12, in whom skin fibro-blasts were analyzed by microarrays. We measured their bloodallopregnanolone using two alternative assays. Additionalseven young PCDH19-FE and seven age-matched control females’blood allopregnanolone levels were measured by a third, goldstandard test, radioimmunoassay (RIA). Altogether we tested 18different PCDH19-FE females for AKR1C3 mRNA and protein le-vels in their skin fibroblasts and 16 PCDH19-FE females forblood allopregnanolone levels. While allopregnanolone levelsvary with age (Supplementary Material, Fig. S3), our data usingRIA, ELISA and high-pressure liquid chromatography–mass

Figure 3. Downregulation of AKR1C3 mRNA and protein level in AF and involvement of AKR1C family in progesterone metabolism. (A) Biological validation of AKR1C3

expression. We detected lower mRNA level in AF (cohort AF1-O and AF2-Y) compared with FC (n = 6). In addition to the control samples used in the microarray, three

other female and four MC were included. Error bar indicates the standard deviation. Student’s t-tests (two-tailed, unequal variance) showed significant differences

between AF1-O and FC, while AF2-Y and FC yielded a P-value of 0.06. (B) Western blot. The results illustrate overall lower AKR1C3 protein level in AF (cohort 1 and 2)

compared with FC. Samples were probed with the AKR1C3 antibody. β-Tubulin antibody (TUBB) (Abcam, ab6046) was used for loading control. (C) Plasma

allopregnanolone levels. Allopregnanolone was measured by either ELISA or HPLC/MS assays showing lower metabolite level in AF. Plasma was isolated from blood

collected from age-matched patients (n = 5) and controls (n = 9). Each sample was measured twice by both ELISA and HPLC/MS assays. ELISA measurement is plotted

against the left Y-axis (Concentration ng/ml); HPLC/MS measurement is plotted against the right Y-axis (area ratio relative to D6–5αDHP). Error bars indicate the

standard deviation of biological repeat. *P < 0.05 and **P < 0.01.


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spectrometry (HPLC/MS) showed that all PCDH19-FE femalestested so far had lower blood allopregnanolone levels than theirage-matched controls (Fig. 3C).

PCDH19-FE seizure onset and offset

PCDH19-FE females suffer fromavariety of seizure types (6,7,9,10).The initial seizures typically start during a young age (6–18months) (1–3,5), and interestingly, many PCDH19-FE females areseizure free by the time they reach puberty. In view of our findingsof the involvement of neurosteroid-metabolizing enzymes inPCDH19-FE, we summarized available seizure onset and offsetages of >150 published PCDH19-FE females. The period betweenonset of seizures at median age of 8 months and offset at medianage of 12 years (Fig. 4) delineates a well-documented period (18)during which steroid hormone levels are low between so-called‘mini-puberty’ (0–6 months) and puberty ∼12 years of age (Fig. 4).

DiscussionMutations in PCDH19 are an important cause of epilepsy in fe-males. The unusual X-chromosome inheritance of PCDH19-FE,withmale sparing of the phenotype, variable seizure type and se-verity, the possibility of non-penetrance, and the frequent pres-ence of neurological and neuropsychiatric comorbidities, haspuzzled the scientist and clinician for years. Using primary skinfibroblasts from affected females, TM and the only known af-fected mosaic male (5), we have shown that steroid metabolism,and neurosteroid metabolism in particular, is associated withPCDH19-FE. We identified that skin fibroblasts of PCDH19-FE fe-males have dramatically altered gene expression, favoring, atleast for a specific set of genes, a male-like expression pattern.Recent advances in genome-wide systematic expression profilingusing arrays and RNA-Seq technologies reveal considerable dif-ferences in gene expression between males and females [for

review, see Ref. (17)]. These gender-biased genes are under evolu-tionary selection and may explain differences in fitness betweenmales and females (19,20). Of the 73 significantly dysregulatedannotated genes in PCDH19-FE females, 43 showedmale–femalebiased expression. We took specific interest in the AKR1C2 andAKR1C3 genes, which code for crucial neurosteroid-metabolizingenzymes. These genes are among a family of four paralogousgenes, AKR1C1-4, that cluster on chromosome 10p14-p15 andall four share high similarity in amino acid sequence (21).A role for these genes (and their encoded enzymes) in regulatinggene expression in a sex-specific manner has recently been rein-forced by the identification of mutations in AKR1C2 and AKR1C4in 46, XY male patients with disordered sexual development(DSD) (22). Minimum activity of AKR1C2 has been detected inall patients, suggesting that low enzymatic activities of AKR1C2together with alternative splicing of AKR1C4 result in a dose-de-pendent 46, XY DSD with ambiguous genitalia (22). We observedthat fibroblasts from PCDH19-FE females have lower expressionlevel of AKR1C3, at both mRNA and protein, and considerabledownregulation of AKR1C2. Although we are yet to determinethe underlying mechanism(s) of AKR1C gene dysregulation inPCDH19-FE, we show that AKR1C gene downregulation leads toallopregnanolone deficiency in the blood of all PCDH19-FE fe-males tested so far. This would be expected, as one of the mainenzymatic activities of AKR1C3/AKR1C2 is conversion of 5α-DHP to allopregnanolone. While we cannot comment on AKR1Cenzyme activities and their dysregulation in the regions of thebrain involved in PCDH19-FE seizures or related cognitive per-formance, there is evidence that skin has the neurosteroidogenicenzymatic apparatus (15,23), which underscores the relevance ofour findings for PCDH19-FE.

Fluctuations in sex steroid hormone levels have been linkedto epilepsy in the past. Neurosteroids have anticonvulsant andproconvulsant properties mediated by their effect as allostericmodulators of neurotransmitter ion channel receptors (23). A

Figure 4. Seizure onset and offset in PCDH19-FE females coincides with period of low sex hormones during humanmaturation. This figure shows the age of seizure onset

and offset of PCDH19-FE females, plotted on a diagram of hormonal fluctuation during humanmaturation reproduced fromOber et al. (18). Data fromover 150 PCDH19-FE

patientswere collected frompublicly available resources (see references in SupplementaryMaterial, File). Thediagramshows that seizure onset occurs aftermini-puberty,

which seems to start after the fall in in utero sex steroid levels. Themedian age of onsetwas 8months old, while themedian age of offset was 12 years old, when sex steroid

hormones are elevated in association with puberty.


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good example is catamenial epilepsy (24), which is character-ized by increased seizure sensitivity in women at differenttimes of the menstrual cycle due to hormone fluctuations. Inthe perimenstrual form (most common), the seizures oftenoccur at the time of menstruation when circulating estradiol:progesterone ratio is high (24,25). This has parallels with the ob-servation that the seizure-active phase in PCDH19-FE femalesfalls between post mini-puberty and pre-puberty, a develop-mental window during which sex hormone levels are low (18).This suggests that for a period of time, levels of particular ef-fector steroids in PCDH19-FE females are low, confirmed byour detection of reduced allopregnanolone levels in blood ofseizure-active PCDH19-FE girls. Neurosteroids, and allopregna-nolone in particular, are known to have anticonvulsive effects(23). Supplementation with ganaxolone, a synthetic methylanalog of allopregnanolone, was able to rescue audiogenicseizures in the fragile X syndrome (Fmr1) knockout mousemodel (25). Ganaxolone also had promising outcomes for agroup of epilepsy patients, who were likely to have had hetero-geneous etiologies, as genetic and non-genetic cases were notdistinguished (26–28). More recently, allopregnanolone wasused successfully to treat status epilepticus in both adultsand children (29–31). These antiepileptic properties of allopreg-nanolone and its analog ganaxolone underline the importanceof investigating involvement of AKR1C2 and AKR1C3 in PCDH19-FE epileptogenesis.

We are yet to address the question of how mutations of thePCDH19 gene converge on the regulation of steroid-metabolizingenzymes andwhether PCDH19 gene itself is regulated by steroids.The role of cell adhesion molecules in sexual differentiation ofthe brain, and their regulation by sex steroids, has been observedbefore. Two members of the focal adhesion complex, FAK andpaxillin, have been implicated in feminization of the brain.These genes are highly expressed in the hypothalamus of fe-males compared with males, and in a study using rats treatedwith estradiol, an effector of masculinization in the brain,their levels were decreased to that of their male counterparts(32). Similarly the expression of N-cadherin, a known interactorof PCDH19 (33), has been shown to be upregulated by androgens,testosterone and dihydroprogesterone in motor neurons ofrats (34).

Our data do not resolve the proposed mechanism of cellularinterference of the PCDH19-FE (4,5). However, it is tempting tospeculate that the two different PCDH19-positive and PCDH19-deficient cell populations, as a consequence of randomX-chromosome inactivation in the affected females (4) or somaticmosaicism in the affected males (5), present with two differentexpression profiles. Such expression differences, for example ingenes like AKR1C genes, GRIA1 or OXTR, might then drive devel-opmental, structural andmost importantly functional, includingnetwork, differences in the brain, which likely underpin the clin-ical presentations of PCDH19-FE.

Taken together, our findings on AKR1C gene dysregulationand subsequent allopregnanolone deficiency suggest that ster-oids and in particular neurosteroids (e.g. allopregnanolone)play an important role in PCDH19-FE and represent a realistictherapeutic target.

Materials and MethodsPatients

Clinical presentations of patients investigated in this study havebeen reported previously (1,12,13).

Human materials

Primary skin fibroblast cell linesSkin biopsies were collected and primary skin fibroblast culturesestablished and cultured as previously described (4). Collectionsof skin biopsies were coordinated by I.E.S., S.F.B., R.S.M., C.D. andC.M. The relevant institutional human research ethics commit-tee has approved the protocol for skin biopsy collection. Informedconsent has been obtained from all participants. Blood: Patientbloods were obtained under approved consent and coordinatedby J.M. and L.S. De-identified age-matched female control bloodswere kindly provided by Adelaide SA Pathology’s core laboratory.

Sample sizes and ages

Gene expression analysis: (i) Affected female 1-O (old), n = 6,mean age = 25 years; (ii) AF2-Y (young), n = 6, mean age = 8.8years; (iii) TM, n = 3, mean age = 51.3 years; (iv) FC, n = 3, age > 20years and (v) MC, n = 3, age > 20 years.

ELISA andHPLC/MS: (i) Young female patients (patient 13–17),n = 5, age as per indicated in the Supplementary Material, FigureS3 and (ii) Female control group (n = 3, 5-, 8- and 12-year-oldgroup). Five-year-old group, n = 6, mean age = 5 years; 8-year-oldgroup, n = 3, mean age = 8 years and 12-year-old group, n = 2,mean age = 12 years.

RIA assay: (i) Young femalepatients (patient 18–24),n = 7,meanage = 7 years and (ii) Female control, n = 7, mean age = 8 years.

RNA extraction and cDNA synthesis

RNA extraction and cDNA synthesis were performed as previous-ly described (35).

Reverse transcriptase-quantitative polymerase chainreaction

RT-qPCR was performed as previously described (36). All primerswere designed to generate a specific 70–250 bp amplicon (seeprimer sequences in Supplementary Material, Table S1).

Western blotting

Protein extraction: Skin fibroblast cells were collected as men-tioned above. Cells were lysed with 1× lysis buffer [50 m Tris–HCl pH7.5; 250 m NaCl; 1 m EDTA; 50 m NaF; 1× CompleteProtease Inhibitor, EDTA-free (Roche, REF 11873580001); 1% Tri-ton-X-100 and 0.1 m Na3VO4], and proteins were extracted bytwo rounds of low-frequency sonication for 5 s, followed byquick centrifugation to remove cell debris.

Immunoblotting: Western blot was performed as previouslydescribed (36). Polyclonal anti-AKR1C3 (Abcam; ab27491) usedas primary and polyclonal Rabbit anti-goat conjugated to HRP(Dako; P044901-2) as secondary antibody. The blots were re-probed with rabbit polyclonal β-tubulin antibody (Abcam,ab6046) as primary and polyclonal goat anti-Rabbit conjugatedto HRP (Dako; P044701-2) as secondary antibody.

Expression arrays

Affymetrix Human Exon 1.0 ST array (Affymetrix, Santa Clara,CA, USA) has been used. The analysis was performed using Par-tek® software, version 6.3 Copyright© 2012 Partek Inc., St Louis,MO, USA. Data files were imported as .CEL files simultaneously,and quality control check was performed under default settings.Samples were grouped according to status, separated into


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affected female, transmitting male, control female or controlmale. Differential gene expression due to batch effect was dealtwith using ‘Remove Batch Effect’ tool. Principal component ana-lysis (PCA) graphs were generated from the QA/QC option inthe gene expression workflow menu to aid visualization ofsample clustering. A gene summary was then created to enableone-way ANOVA tests to detect for differentially expressedgenes between selected status groups. Gene lists were createdby setting a threshold P-value of <0.05 and fold change greaterthan ±2.

Allopregnanolone measurements

Steroid hormone extraction: Plasma was obtained by centrifuga-tion of blood samples at 3 000g at 4°C for 15 min. Plasma’s steroidhormones were extracted by subjecting them to vigorous mixingwith hexane:isopropanol (4:1, v/v) and subsequent centrifugationat 3000g for 15 min. Supernatant was air-dried (nitrogen gas andheat block) before resuspension in 100% methanol. D6-5αDHP(6 deuterium-labelled 5αDHP; Steraloids Inc.) was added to eachplasma sample prior to extraction and served as internalstandard.

HPLC/MS: Extracted samples were subjected to a C8 HPCL col-umn (Luna 3 micron, 50 × 3.00 mm) before analysis in API 5000LC/MSMS machine. The mobile phase was composed of 100%acetonitrile + 0.1% formic acid (v/v) (A) and 50% acetonitrile +0.1% formic acid in isopropanol (v/v) (B) using the following gra-dient program: 0–1 min, isocratic at 50% (A and B); 1.01–12 min,isocratic at 51% (A) to 49% (B); 12.01–15 min, isocratic at 100%(B); 15.01–20 min, isocratic at 50% (A and B). The flow rate was0.25 ml min−1. Each sample ratio (μ⍰ cps−1) was generated bycomparing the intensity (cps; count per second) of allopregnano-lone peak (m/z = 319.1/161.1) divided by intensity of an internalstandard (m/z = 323.1/95.1).

ELISA: ELISA measurement was performed according tomanufacturer’s instruction (USCN Life science Inc.) with somemodifications. Plasma volume used was 100 μl, and the loweststandard curve concentration was reduced to 0.41 ng ml−1 to in-crease the detection range of plasma allopregnanolone.

Supplementary MaterialSupplementary material is available at HMG online.

AcknowledgementsWe thank the patients and family members who contributedsamples for the purpose of this study.

Conflict of Interest statement. None declared.

FundingThis study was supported by Australian National Health andMedical Research Council (NHMRC) Program Grant (628952) andResearch Fellowship (1041920) to J.G. and Insieme per la RicercaPCDH19 – ONLUS.

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Mutations of protocadherin 19 in female epilepsy (PCDH19-FE) lead to allopregnanolone deficiency