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European Journal of Medical Genetics 57 (2014) 44e46
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European Journal of Medical Genetics
journal homepage: http : / /www.elsevier .com/locate/ejmg
Short clinical reports
RORB gene and 9q21.13 microdeletion: Report on a patient withepilepsy and mild intellectual disability
Maria Giuseppina Baglietto a, Gianluca Caridi b, Giorgio Gimelli c, Margherita Mancardi a,Giulia Prato a, Patrizia Ronchetto c, Cristina Cuoco c, Elisa Tassano c,*
aCentro Epilessie, UO Neuropsichiatria Infantile, Istituto Giannina Gaslini, Genova, Italyb Laboratorio di Fisiopatologia dell’Uremia, Istituto Giannina Gaslini, Genova, Italyc Laboratorio di Citogenetica, Istituto Giannina Gaslini, Largo G. Gaslini 5, I-16148 Genova, Italy
a r t i c l e i n f o
Article history:Received 7 August 2013Accepted 6 December 2013Available online 17 December 2013
Keywords:RORB gene9q21.13 MicrodeletionEpilepsyMild intellectual disabilityArray-CGHCopy number variants
* Corresponding author. Tel.: þ39 (0) 10 5636922.E-mail addresses: [email protected], elisat
(E. Tassano).
1769-7212/$ e see front matter � 2013 Elsevier Masshttp://dx.doi.org/10.1016/j.ejmg.2013.12.001
a b s t r a c t
Copy number variants represent an important cause of neurodevelopmental disorders including epi-lepsy, which is genetically determined in 40% of cases. Epilepsy is caused by chromosomal imbalances ormutations in genes encoding subunits of neuronal voltage- or ligand-gated ion channels or proteinsrelated to neuronal maturation and migration during embryonic development. Here, we report on a girlwith mild intellectual disability and idiopathic partial epilepsy. Array-CGH analysis showed a 1.040 Mb denovo interstitial deletion at 9q21.13 band encompassing only four genes, namely RORB, TRPM6, NMRK1,OSTF1, two open reading frames (C9orf40, C9orf41), and a microRNA (MIR548H3). RORB encodes a nu-clear receptor highly expressed in the retina, cortex, and thalamus. We hypothesize its role in producingthe phenotype of our patient and compare this case with other ones previously reported in the literatureto better identify a genotypeephenotype correlation.
� 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Copy number variants (CNVs) collectively represent an impor-tant cause of neurodevelopmental disorders such as developmentaldelay (DD)/intellectual disability (ID), autism, and epilepsy.
Epilepsy is one of the most common neurological disordersaffecting up to 1% of the general population. It has been estimatedthat up to 40% of epilepsies are genetically determined [Gardiner,2000; Hauser et al., 1996; Jozwiak et al., 2005; Pal et al., 2010].
Different types of epilepsy have been reported in patients withchromosomal imbalances and numerous genemutations have beenfound to cause epilepsy, particularly in genes encoding subunits ofneuronal voltage- or ligand-gated ion channels or proteins relatedto neuronal maturation and migration during embryonicdevelopment.
Recently, nine patients with a novel microdeletion syndrome at9q21.13 presenting mental retardation, speech delay, epilepsy, andcharacteristic facial features have been reported [Boudry-Labiset al., 2013]. The deleted segments spanned from 2.2 to 12.6 meg-abases and included a variable number of genes. The smallest
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region of deletion overlap extending 750 kb included four genesamong which the authors proposed RORB as a strong candidate fora neurological phenotype [Boudry-Labis et al., 2013].
RORB (RAR-related orphan receptor b) is an orphan nuclear re-ceptor, forming a subfamily with the closely related nuclear re-ceptors ROR-alpha (RORA) and ROR-gamma (RORC) (OMIM601972). Jabaudon et al. [2012] demonstrated that RORB expressionlevels control the cytoarchitectural patterning of neocortical neu-rons during development. Moreover, it has been reported thatRORB is specifically expressed in the temporal cortex, as demon-strated on cortical samples from patients with temporal lobe epi-lepsy [Rossini et al., 2011].
Here we report on a girl with mild intellectual disability, idio-pathic partial epilepsy, and a 9q21.13 microdeletion of about 1 Mbin size, encompassing only four genes including RORB.
2. Materials and methods
Cytogenetic analysis was performed on cultured lymphocytesby G-banding according to standard procedures. Array-CGH wasperformed on the patient and her parents using the Agilent kit2x400K (Human Genome CGH Microarray, Agilent Technologies,Santa Clara, CA, USA) according to the manufacturer’s protocol.Quality of experiments was assessed using Feature Extraction QCMetric v10.1.1 (Agilent). The derivative log ratio spread (DLR) value
M.G. Baglietto et al. / European Journal of Medical Genetics 57 (2014) 44e46 45
was calculated using the Agilent Genomics Workbench software.CNVs reported in the Database of Genomic Variants http://projects.tcag.ca/variation/and in in-house databases of benign CNVs wereexcluded from further analysis. Genomic positions refer to theHuman Genome February 2009 assembly (GRCh37/hg19).
Mutational analysis of RORB was performed by directsequencing of the 10 exons and flanking introns. Primer designwasdone by ExonPrimer (UCSC Genome Browser, hg19).
3. Clinical report
The proposita is a 12-year-old girl. She was admitted to ourobservation for mild intellectual disability and idiopathic partialepilepsy. She showed mild facial dysmorphism with mild hyper-telorism, low-set hair, slight divergent strabismus and upslantingpalpebral fissures, thin upper lip, pavilions anteroversi bilaterallyand, long nose filter.
She was born full-term to non consanguineous healthy parentsafter an uneventful pregnancy. At birth, weight, length, and headcircumference were normal. Vesicoureteral reflux was present asan abnormal widening of the ureter before birth. When medicalmanagement failed to prevent recurrent urinary tract infections,surgery was performed (at the age of 10 months).
Her early developmental milestones were normal although shedid not socialize normally with other children as a preschool child.
At 4 years 6 months of age, she developed epilepsy with dailysecondarily generalized seizures (SGE) with absences and appar-ently generalized tonic-clonic seizures. EEG showed left cen-trotemporal spikes and generalized spike-wave discharges. Seizure
Fig. 1. Results of array-CGH analysis: AeB) Array-CGH analysis showed a de novo inteA_18_P16874789 (77,283,017 bp) to probe A_16_P38754346 (78,323,705 bp) flanked by procording to UCSC Genome Browser (http://genome.ucsc.edu/; GRCh37/hg19, February 2009shows the deleted region of our patient (arrowheads) and its gene content, including themicroRNA (MIR548H3), compared to the deletion identified by Boudry-Labis et al. [2013].
control was achieved after administration of sodium valproate. At 7years 3 months, neuropsychological assessment for school prob-lems showed mental age of 5 years 7 months, evaluated withGriffths Mental Developmental Scale (QS 77).
She remained seizure-free until the age of 8 years when she hadtwo SGE episodes, during the adjustment of the dose of sodiumvalproate, while falling asleep. EEGwas abnormal with frequent leftfrontal and centrotemporal spike and spike-wave discharges. Par-tial epilepsy was easy to control with an adequate dosage of anti-epileptic drug.
At the neurological examination, fine motor skills resultedinadequate for age, but no focal neurologic signs were detected.Cranial CT and MRI were normal.
Neuropsychological assessment, evaluated with WISC-III at tenyears of age, revealed mild intellectual disability. The neuropsy-chological profile was disharmonic with lower values at the verbaltest: QIT 69, QIV 65, QIP 80. This result can be correlated with theleft focal anomalies in her intercritical EEG.
She was educated in a mainstream school with additional sup-port. Laboratory investigations revealed normal values of theelectrolytes Na, Ca, Mg, P, Cl. Today, at 13 years of age, she is seizure-free and she is reducing the dosage of sodium valproate.
4. Results
Karyotypes of the girl and her parents were found to be normal.Array-CGH analysis showed a de novo interstitial deletion at 9q21.13band. The deleted region spans w1.040 Mb from probeA_18_P16874789 (77,283,017 bp) to probe A_16_P38754346
rstitial deletion at 9q21.13 band. The deleted region spans 1.040 Mb from probebe A_16_P38752003 (77,275,561 bp) and probe A_18_P16872507 (78,328,090 bp) ac-) B) Extract from the UCSC genome browser (http://genome.ucsc.edu/) GRCh37/hg19RORB, TRPM6, NMRK1, OSTF1 loci, two open reading frames (C9orf40, C9orf41) and a
M.G. Baglietto et al. / European Journal of Medical Genetics 57 (2014) 44e4646
(78,323,705 bp) flanked by probe A_16_P38752003 (77,275,561 bp)and probe A_18_P16872507 (78,328,090 bp) according to UCSCGenome Browser (http://genome.ucsc.edu/GRCh37/hg19February2009) (Fig. 1, AeB). The deleted segment contains only fourgenes, namely RORB (RAR-related orphan receptor B), TRPM6(transient receptor potential cation channel, subfamily M, member6), NMRK1 (nicotinamide riboside kinase 1), OSTF1 (osteoclaststimulating factor 1), two open reading frames (C9orf40, C9orf41),and a microRNA (MIR548H3) (Fig. 1, C). The proximal breakpoint ofthe deletion disrupts RORB between exons 5 and 8 while distalbreakpoint disrupts the microRNA (MIR548H3) (Fig. 1, C). Mutationanalysis of RORB gene was negative on the homologous allele.
Surely, the deletion breaks the last three exons and, in particularat protein level, it disrupts a number of alpha-elices forming theligand binding domain (LBD) and the activation function 2 (AF2) inH12 consisting of PLYKELF, which is 100% conserved among RORs.
5. Discussion
Here we report on a girl with a w1.040 Mb de novo deletion at9q21.3 presenting epilepsy and mild intellectual disability. Thedeleted region encompasses only four genes including RORB.
RORB, a nuclear orphan receptor, regulates neuronal patterningduring cortical development [Jabaudon et al., 2012]. Neuropatho-logic investigations frequently reveal the presence of architecturalcortical dysplasia in patients with temporal lobe epilepsy (TLE),sometimes as an isolated finding but more commonly associatedwith hippocampal sclerosis (HS) and white matter abnormalities[Rossini et al., 2011]. The same authors showed that, in all cases,RORB mRNA was intensely expressed in layer IV.
MRI studies of TLE families showed not only that hippocampalabnormalities are the consequence of repeated seizures but alsothat genetic mechanisms could play a significant role in thedevelopment of hippocampal damage [Kobayashi et al., 2001].Recently, in an array-CGH study on 102 patients with epilepsy andadditional neurodevelopmental disorders, the authors reported ona patient (patient 12) with a 2.5 Mb deletion at 9q21.13 including,among others, the RORB and TRPM6 genes [Bartnik et al., 2012].
More recently, 13 patients with developmental delay, epilepsy,neuro-behavioural disorders, and moderate facial dysmorphismshave been reported [Boudry-Labis et al., 2013]. These patientscarried 2.2 Mbe12.6 Mb interstitial deletions of chromosome9q21.11eq21.32 [Boudry-Labis et al., 2013]. Our study describes apatient sharing similar features with the cases reported by Boudry-Labis et al. [2013] and Bartnik et al. [2012], such as epilepsy andmild developmental delay. Mild dysmorphic features were presentin our patient and in case 5 reported by Boudry-Labis et al. [2013].Interestingly, our patient presented a small deletion at 9q21.3overlapping the 750 Kb of the small overlapping region (SRO) re-ported by Boudry-Labis et al. [2013], who suggested a significantrole of RORB in the development of the mild intellectual disabilityand epilepsy phenotype. In all patients reported by Boudry-Labiset al. [2013], RORB was completely deleted while in our patientRORB was partially deleted, although the phenotype of our patientwas similar to those reported. The deletion breaks exons 8, 9, and10 of RORB gene in the ligand binding domain (LBD). The LBDs ofnuclear receptors are multifunctional, playing a role in ligandbinding, nuclear localization, receptor dimerization, and provide aninterface for the interaction with coactivators and corepressors.
In addition to the typical 12 canonical a-helices (H1-12), the LBDof RORB contains two additional helices, H20 and H110. The activa-tion function 2 (AF2) in H12 consists of PLYKELF, which is 100%conserved among RORs. Deletion of H12 or point mutations withinH12 cause loss of ROR transactivation activity and result in adominant-negative ROR [Kurebayashi et al., 2004; Lau et al., 1999].
RORB, TRPM6, NMRK, and OSTF1 are the genes included in thedeleted region. NMRK1 and OSTF1 are not expressed in brain and donot seem to be relevant candidates for neurodevelopmental dis-orders. TRPM6 belongs to the transient receptor potential (TRP)superfamily [Schlingmann & Gudermann, 2005; Schmitz et al.,2005]. Mutations of the gene have been associated with hypo-magnesaemia with secondary hypocalcemia (HSH). Though TRPM6sequencing to exclude a mutation of the remaining allele has notbeen performed, magnesium and calcium levels in the intercriticalperiod were normal in our patient.
Interestingly, urinary tract disorders (enuresis and pyelone-phritis) have been reported in four patients (patients 1, 3, 6, and 7)described by Boudry-Labis et al. [2013] and also our patient showeda vesico-ureteral reflux of unknown origin.
Therefore, in agreement with Boudry-Labis et al. [2013], wecould speculate that, among the genes of the interstitial deletion at9q21.13, RORB could best explain the phenotype of our patient,characterized by mild intellectual disability and epilepsy.
Competing interests
The authors declare that they have no competing interestsrelated to this manuscript.
Acknowledgements
We thank the patient’s parents for their kind participation andsupport.We are grateful to Marco Bertorello and CorradoTorello fortheir technical assistance. This work was supported by “ Cinque permille dell’IRPEF- Finanziamento della ricerca sanitaria” and“Finanziamento Ricerca Corrente, Ministero Salute” (contributo perla ricerca intramurale).
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