RESEARCH ARTICLE

3p25.3 Microdeletion of GABA Transporters SLC6A1 and SLC6A11 Results in Intellectual Disability, Epilepsy and Stereotypic Behavior Nicola Dikow,1* Bianca Maas,1 Stephanie Karch,2 Martin Granzow,1 Johannes W.G. Janssen,1 Anna Jauch,1 Katrin Hinderhofer,1 Christian Sutter,1 Susanne Schubert-Bast,2 Britt Marie Anderlid,3 Bruno Dallapiccola,4 Nathalie Van der Aa,5 and Ute Moog1 1

Institute of Human Genetics, Heidelberg University, Heidelberg, Germany

2

Center for Child and Adolescent Medicine Pediatric Neurology, Heidelberg University Hospital, Heidelberg, Germany

3

Institution of Molecular Medicine and Surgery, CMM, Karolinska Institutet and Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden 4

Ospedale Pediatrico Bambino Gesu—IRCCS Roma, Italy

5

Department of Medical Genetics, Antwerp University Hospital and University of Antwerp, Antwerp, Belgium

Manuscript Received: 2 May 2014; Manuscript Accepted: 10 August 2014

Small interstitial deletions affecting chromosome region 3p25.3 have been reported in only five patients so far, four of them with overlapping telomeric microdeletions 3p25.3 and variable features of 3p- syndrome, and one patient with a small proximal microdeletion and a distinct phenotype with intellectual disability (ID) and multiple congenital anomalies. Here we report on three novel patients with overlapping proximal microdeletions 3p25.3 of 1.1–1.5 Mb in size showing a consistent non-3pphenotype with ID, epilepsy/EEG abnormalities, poor speech, ataxia and stereotypic hand movements. The smallest region of overlap contains two genes encoding sodium- and chloridedependent GABA transporters which have not been associated with this disease phenotype in humans so far. The protein function, the phenotype in transporter deficient animal models and the effects of specific pharmacological transporter inhibition in mice and humans provide evidence that these GABA transporters are plausible candidates for seizures/EEG abnormalities, ataxia and ID in this novel group of patients. A fourth novel patient deleted for a 3.16 Mb region, both telomeric and centromeric to 3p25.3, confirms that the telomeric segment is critical for the 3p- syndrome phenotype. Finally, a region of 643 kb is suggested to harbor one or more genes causative for polydactyly which is part of the 3p- syndrome. Ó 2014 Wiley Periodicals, Inc.

Key words: Ataxia, epilepsy; GABA transporter; microdeletion 3p25.3; intellectual disability

INTRODUCTION 3p- syndrome, resulting from terminal 3p deletions is characterized by a distinct association of intellectual disability (ID), short stature, microcephaly, hypotonia and recognizable facial features, including

Ó 2014 Wiley Periodicals, Inc.

How to Cite this Article: Dikow N, Maas B, Karch S, Granzow M, Janssen JWG, Jauch A, Hinderhofer K, Sutter C, Schubert-Bast S, Anderlid BM, Dallapiccola B, Van der Aa N, Moog U. 2014. 3p25.3 microdeletion of GABA transporters SLC6A1 and SLC6A11 results in intellectual disability, epilepsy and stereotypic behavior. Am J Med Genet Part A 164A:3061–3068.

triangular face, ptosis, hypertelorism, short nose with broad nasal tip, long philtrum, and micrognathia. Additional malformations such as congenital heart defect (CHD), cleft palate, and postaxial polydactyly

DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources. Firth, H.V. et al. (2009). Am. J. Hum. Genet. 84, 524–533 (DOI: dx.doi.org/10/1016/j.ajhg.2009.03.010). This study makes use of data generated by the DECIPHER Consortium. A full list of centres which contributed to the generation of the data is available from http://decipher.sanger.ac.uk and via email from [email protected]. Conflict of interest: none. Grant sponsor: Wellcome Trust.
Correspondence to: Dr. Nicola Dikow, Institute of Human Genetics INF 366, 69120 Heidelberg, Germany. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 24 September 2014 DOI 10.1002/ajmg.a.36761

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3062 can be associated [Cargile et al., 2002; Drumheller et al., 1996]. Interstitial 3p25.3 deletions in the range of 0.64–6.3 Mb, variably overlapping with the terminal deletions, have been reported in at least seven patients [Cargile et al., 2002; Czeschik et al., 2014; Gunnarsson and Foyn Bruun, 2010; Kellogg et al., 2013; Peltekova et al., 2012; Riess et al., 2012; Shuib et al., 2009]. They display variable features of 3psyndrome. Deleted genes possibly responsible for CHD, ID and dysmorphic features have been suggested, although their large number in the critical region has prevented the identification of convincing candidates for different features. A proximal 3p25.3 microdeletion has been described in one patient so far [Czeschik et al., 2014]. Here we report on three unrelated patients with a non-3p- phenotype including developmental delay (DD)/ID, epilepsy or EEG abnormalities, poor speech and stereotypic hand movements who are heterozygous for proximal 3p25.3 deletions, ranging in size from 1.13–1.5 Mb. We also report on a fourth patient with an overlapping although more distal microdeletion, presenting with features of 3p- syndrome. We re-assess the critical region for the 3p- phenotype and discuss the genes candidate to the neurodevelopmental disorder of patients with proximal 3p25.3 deletions.

MATERIALS AND METHODS Patients Four unrelated patients were assessed by experienced clinical geneticists [UM (patient 1), NA (patient 2), BD (patient 3), BA (patient 4)]. Clinical data, photographs and results of laboratory testing were obtained with informed consent, including the consent to use the photographs in this report, which has been documented in protocols approved by institutional review boards at all participating institutions.

Molecular karyotyping High resolution molecular karyotyping was performed on a clinical basis for evaluation of ID, seizures, and/or dysmorphic features in the 4 index patients using different array platforms. Blood samples of the parents were analyzed in order to discern familial from de novo cases.

Fluorescence in situ hybridization (FISH) Deletion 3p25.3 was confirmed in patient 1 by two-color FISH analysis using BAC clone RP11-94A14 (red, [hg19] 19.638.350– 10.813.431 bp) together with a reference probe for the short arm of chromosome 3 (pcp3p, green). Microscopic evaluation of 10 metaphase spreads was performed using a DM RXA fluorescence microscope (Leica, Wetzlar, Germany) equipped with a Sensys CCD camera (Photometrics, Tucson, AZ) and microscope was controlled by the Leica Q-FISH software (Leica Microsystems Imaging solutions, Cambridge, United Kingdom).

RESULTS Clinical reports Clinical data of patients 1–4, all born to healthy non-consanguineous parents, are summarized in Table I; the face of patient 1 is shown in Fig. 1.

AMERICAN JOURNAL OF MEDICAL GENETICS PART A Patient 1 was born after an uneventful pregnancy with normal birth measurements. Motor delay was first noticed at eight months, when she stopped social smiling and laughing. She developed stereotypic hand movements and purposeful hand function was impaired at the age of one year. The ability to sit was achieved at two years. Epileptic seizures with atypical absences were diagnosed at the age of four. EEG showed generalized high amplitude background slowing and multifocal epileptic discharges, mainly over the posterior regions, similar to patients with Angelman syndrome. Under anticonvulsant treatment with valproate acid and sultiam she showed better motor development, however active speech had at the time of evaluation not yet developed. On clinical examination at 4 years and five months she was friendly, able to smile, although no eye contact could be established. She showed a normal growth with a head circumference within the low-normal range, mild hypotonia and truncal ataxia. Permanent stereotypic hand movements were noticed. She showed brachycephaly, without obvious facial dysmorphisms or malformations (Fig. 1). Brain MRI at four years showed unspecific white matter deficiency and periventricular gliosis. Hearing tests yielded normal results. Molecular genetic testing by methylation-specific MLPA for Angelman syndrome and molecular analyses of MECP2, FOXG1 and CDKL5 gave normal results. Patient 2 was born after an uneventful pregnancy with normal birth measurements. He was referred at the age of 5 years because of severe ID, epilepsy, impaired social interaction, and stereotypic behavior with handwringing. After an uneventful neonatal period, motor development was delayed. He started crawling at 18 months and was able to walk at four years of age, when he still had no speech. At age five years, he had a vocabulary of about ten words. Seizures first appeared at the age of 12 months and under treatment with valproate he had short episodes of weekly recurring myoclonic seizures. He had a happy demeanor with frequent laughter, constant handwringing and poor purposeful hand use. His gait was broadbased and ataxic. He was brachycephalic and had body measurements according to the 25th centile. Neither malformations nor dysmorphic features were recorded. MRI of the brain, audiometry, ophthalmic examination and an extensive metabolic workup showed normal results. Genetic testing for Fragile X syndrome, Angelman syndrome (methylation test as well as UBE3A mutation analysis) and mutation analysis of MECP2 revealed normal results. Patient 3 was born at term with normal birth measurements. At age of 8 months he showed EEG abnormalities with changes in the NREM pattern, which were confirmed in subsequent recordings. However, no seizure was observed. An onset of stereotypic behavior, mainly hand washing, was reported at the end of his second year of life. At three years he was evaluated for DD. Clinical examination showed a prominent forehead with bitemporal narrowing, depressed nasal bridge, and high arched palate. Long tapering fingers and flat feet were also noted. Patient 4 was born after 35 þ 5 weeks of gestation with hypotrophy (weight 2 SD; length 3.3 SD), microcephaly (2.9 SD) and bilateral postaxial polydactyly of hands and feet. Prenatally, an inoperable CHD with AVSD (atrioventricular septal defect) with an insufficient valve, small right ventricle, large left ventricle, and hypoplastic aortic arch were detected by ultrasound examination. She had a round face, downslanting palpebral fissures, a long philtrum, and micrognatia. The baby died on her first day of life.

 þ – – –

–

þ 

– –

Postaxial polydactyly Brachycephaly Downslanting palpebral fissures Epicanthus Depressed or flat nasal bridge



þ myoclonic (1y) þ na Stereotypic hand movements

10 words 5 y þ normal (5y)

–

þ þ Stereotypic hand movements

þ absence (4y)

þ reduced white matter volume (4y 8m)

5y normal

3y 0.02/0.70/0.75 þþþ no speech 4 y þþþ no speech 4 y

normal normal

1.5 10.241.22311.740.887 de novo m Belgian

Pat. 2 3p25.3

40 0.27/0.14/-0.31

1.125 10.391.28411.516.688 de novo f German

Pat. 1 3p25.3

CHD

Seizures/EEG (onset) Ataxia Hypotonia Behavior

Motor delay cMRI (age)

Birth at gw. Birth weight/length/ OFC (SD) Age at investigation Weight/length/OFC (SD) ID Speech

Size (Mb) Position Chr 3 (Hg19) Inheritance Sex Ethnicity

Patient/ Reference Deletion 3p

– þ

– –

–

 – Stereotypic hand movements, attention deficit –

EEG abnomalies

– unremarkable (3y)

40 0.65/-0.77/0.69 3y 0.87/-1.49/0.34 þþþ delay

1.33 10.975.73212.306.975 de novo f Italian

Pat. 3 3p25.3-p25.2

na na

– þ

þ 4x

þ

na

35 þ 5 1.73/-3.03/2.68 1 d; † 1 d

3.16 8.744.12711.903.456 de novo f Swedish

Pat. 4 3p25.3-p25.2

– –

na þ

þ PDA, PS, ASD, –

þ grand mal (newborn) na na na

þ na

39 0.77/-0.95/ -1.23 29y 1.50/-3.14/ -2.17 þþ(þ) delay

0.225 11.393.8911.618.468 de novo f German

Czeschik et al., 2014 3p25.3

na – þ Chinese þ

þA þ

–

–

na þ þ Autism spectrum disorder

–

þ normal (2 y)

term 0.30/0.73/ na 11y 1.55/-0.61/ -1.63 þ borderline na

0.684 9.005.0989.689.733 de novo f Chinese

Kellog et al., 2013 3p25.3

– –

–

þ ASD, VSD

þ transient (4 y) na þ na

þ asymmetry of thalamus (4 y)

þþþ no speech 4 y

37 þ 5 1.59/-2.21/ -1.65 4y n a/norm/n a

Gunnarsson and Foyn Bruun, 2010 3p26.1p25.3 1.6 8.330.4269.910.334 de novo f Caucasian

TABLE I. Clinical data of patients with interstitial deletion 3p25-p26.

na –

– þ

þ 4x

þ

þ parenchymal volume loss/ atrophy of all structures (20 y) þ tonic clonic (1y) na na na

0.643 9.392.27410.035.209 not maternal f þA Caucasian 37 0.86/-1.88/ -0.64 22y 2.68/-6.27/ -2.54 þþþ non verbal adult

Peltekova et al., 2012 3p25.3

(Continued)

na þ

na A



na

na þ na

–

þ na

þ delay

39 0.84/-0.95/ 0.31 3y n a/-1.22/0.08

1.24 8.275.5419.516.586 de novo f Caucasian

Riess et al., 2012 3p26.1-p25.3

DIKOW ET AL. 3063

þ right ear pit, proximally placed thumbs þ downturned corners of mouth, overlapping toes na bypertrophic pyloric stenosis, CP, clavicula pseudoarthrosis, sensorineural hearing loss, broad thumbs and first toes – sacral dimple – bitemporal narrowing, long tapering fingers, high arched palate –  Ear anomalies Others

þ: present; –: absent; † deceased; A: deduced from the patient picture in the paper; ASD: atrial septum defect; cMRI: cranial MRI; CP: cleft palate; f: female; m.: male; gw.: weeks of gestation; ID: intellectual disability; LD: learning diability; m: months; n a: not available; OFC: occipitofrontal circumference; Pat.: Patient; PDA: persisting ductus arteriosus; PS: pulmonary stenosis; US: ultrasound; VSD: ventricular septum defect; y: years.

na thin upper lip vermillion

A

 prognathism þ knee contractures, muscle atrophy, small hands and feet, microphthalmia, blepharophimosis, syndactyly 2/3, CP, scoliosis A þ – –

–

þ

A

– þ  short þ – –

–

Pat. 4 na Pat. 2 – Pat. 1 –

Pat. 3 –

Czeschik et al., 2014 þ

Patient/ Reference Broad/prominent nose Long/prominent philtrum Micrognathia

TABLE I. (Continued )

Gunnarsson and Foyn Bruun, 2010 þ

Kellog et al., 2013 anteverted nares þ

Peltekova et al., 2012 na

þ

AMERICAN JOURNAL OF MEDICAL GENETICS PART A

Riess et al., 2012 þA

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FIG. 1. Patient 1 at 3 years of age. Note facial hypotonia without obvious facial dysmorphisms. (Photographs submitted with written consent from the patients’ legal guardians for publication in print and online). [Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley. com/journal/10.1002/(ISSN)1552-4833.]

Array analyses and confirmation investigations Molecular data of array analyses are depicted in Fig. 2. In patient 1, copy number-SNP-array analysis using an Affymetrix1 Cyto Scan HD array revealed a 1.1 Mb copy number loss in 3p25.3 (arr[hg19] 3p25.3(10,391,284–11,516,688)1). The interstitial deletion of 3p25.3 was confirmed by metaphase FISH analysis and shown to be de novo as both parents showed normal diploid FISH results. In patient 2, array analysis was performed using a HumanCytoSNP-12v2.0 beadchip (Illumina). The analysis showed two aberrations: a de novo 1.5 Mb deletion of 3p25.3 (arr[hg19] 3p25.3 (10,241,223–11,740,887)1 dn) and a small deletion in 7q11.21 encompassing 3 genes of unknown phenotypic effect (ZNF107; ZNF138; ZNF273) which was inherited from the healthy mother (arr[hg19] 7q11.21(64,150,849–64,419,612)1 mat). In patient 3, array analysis of the index and the parents using an Agilent Technologies 44 K chip disclosed a de novo 1.33 Mb deletion (arr[hg19] 3p25.3p25.2(10,975,732–12,306,975)1). In patient 4, analysis with Agilent oligoarray 244 k using samples of the index and the parents showed a de novo 3.16 Mb deletion (arr[hg19] 3p25.3p25.2(8,744,127–11,903,456)1 dn).

DISCUSSION Few patients with small interstitial deletions in 3p have been reported so far. In four of them the telomeric part and in one the proximal part of 3p25.3 had been affected (Fig. 2) [Czeschik

DIKOW ET AL.

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FIG. 2. Chromosome 3. A: Array profile of Patient 1 showing deleted region 3p25.3. B: Representation of the distal short arm of chromosome 3 including deleted regions of the index patients 1–4, and previously published cases with smallest region of overlap (SRO, dotted box) and localization of genes in this region. Positions of deletions according to Genome Reference Consortium Human Build 37 (GRCh37/hg19). [Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1552-4833.]

et al., 2014; Gunnarsson and Foyn Bruun, 2010; Kellogg et al., 2013; Peltekova et al., 2012; Riess et al., 2012]. Here we discuss overlapping microdeletions of proximal 3p25.3, ranging in size from 1.13–1.33 Mb. They were detected by molecular karyotyping in three unrelated index patients (patients 1–3), all evaluated for ID. The larger deletion of a fourth patient (patient 4) overlaps with that of the other three patients, as well as with previously reported 3p25.3 deletions (Fig. 2). Patients 1–3 presenting with overlapping deletions in the proximal 3p25.3 sub-band show a consistent phenotype comprising DD/ ID, absent or poor speech, epilepsy or EEG abnormalities, and conspicuous stereotypic hand movements, without distinct physical anomalies (Table I). The association of ID with severe speech

impairment, seizures and ataxia in patients 1 and 2 led to the differential diagnostic consideration of Angelman syndrome. ID and the development of stereotypic hand movements and seizures in patients 1 and 2 and loss of eye-to-eye contact in patient 1 were suggestive for Rett syndrome; both patients were tested negative for MECP2 mutations. Comparison of clinical and molecular data of patients 1–3 allowed the mapping of the smallest region of overlap (SRO) in 3p25.3, which spans a segment of approximately 540 kb. The SRO comprises four genes including SLC6A1 (OMIM 137165) and the coding 3’ end of SLC6A11 (OMIM 607952), coding for two sodiumand chloride-dependent GABA (Gamma-aminobutyric acid) transporters. GABA acts as a major inhibitory neurotransmitter

3066 in the brain, mediated by GABA receptors. Different types of GABAA receptor activation (GABAA receptor: ionotropic type) have been described [Farrant and Nusser, 2005]. Transient (or phasic) activation of synaptic GABAA receptors generates local, short inhibitory postsynaptic currents, whereas tonic GABAA receptor activation by low perisynaptic GABA concentration leads to persistent random receptor activation, generating a tonic inhibitory current. Tonic activation of GABAA receptors is evident in embryonic neurons before synapse formation has taken place [Farrant and Nusser, 2005]. GABAergic neurotransmission via GABAA receptors is terminated by the uptake of GABA into the presynaptic terminal and the surrounding cells by sodiumdependent GABA transporters such as SLC6A1 and SLC6A11, the two major GABA transporters in brain [Borden et al., 1994; Hirunsatit et al., 2009; Zhou and Danbolt, 2013]. No disease-causing mutations in SLC6A11 and SLC6A1 have been published so far with the exception of two de novo SLC6A1 sequence variants listed among “probable disease causing variants” in two patients with ID and autism respectively [Rauch et al., 2012; Sanders et al., 2012]. SLC6A1 encodes the most abundantly expressed GABA transporter in the brain, which removes GABA from the synaptic cleft. The human protein is highly homologous to that from the rat brain (GAT1) [Hirunsatit et al., 2009; Lam et al., 1993]. The GABAtransporter protein encoded by SLC6A11 (rat homolog GAT3) also terminates the GABAergic inhibition by the uptake of GABA into the presynaptic terminal and surrounding astroglial cells [Borden et al., 1994]. In GAT1 deficient mice, uptake of GABA is reduced and overactivation of peri- or extrasynaptic GABA receptors by ambient GABA increases the magnitude of tonic current. Enhanced tonic inhibition was shown to be sufficient for inducing seizures in rats, while GAT1-knockout mice developed spontaneous absence seizures with characteristic spike-wave discharges [Farrant and Nusser, 2005], [Cope et al., 2009]. GABA uptake assays on cerebellar synaptosomes confirmed reduced (