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RORB gene and 9q21.13 microdeletion: Report on a patient with epilepsy and mild intellectual disability
European Journal of Medical Genetics 57 (2014) 44e46
Contents lists available at ScienceDirect
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 with epilepsy 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, * a b c
Centro Epilessie, UO Neuropsichiatria Infantile, Istituto Giannina Gaslini, Genova, Italy Laboratorio di Fisiopatologia dell’Uremia, Istituto Giannina Gaslini, Genova, Italy 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
a b s t r a c t
Article history: Received 7 August 2013 Accepted 6 December 2013 Available online 17 December 2013
Copy number variants represent an important cause of neurodevelopmental disorders including epilepsy, which is genetically determined in 40% of cases. Epilepsy is caused by chromosomal imbalances or mutations in genes encoding subunits of neuronal voltage- or ligand-gated ion channels or proteins related to neuronal maturation and migration during embryonic development. Here, we report on a girl with mild intellectual disability and idiopathic partial epilepsy. Array-CGH analysis showed a 1.040 Mb de novo 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 nuclear receptor highly expressed in the retina, cortex, and thalamus. We hypothesize its role in producing the phenotype of our patient and compare this case with other ones previously reported in the literature to better identify a genotypeephenotype correlation. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: RORB gene 9q21.13 Microdeletion Epilepsy Mild intellectual disability Array-CGH Copy number variants
1. Introduction Copy number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. Epilepsy is one of the most common neurological disorders affecting up to 1% of the general population. It has been estimated that 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 with chromosomal imbalances and numerous gene mutations have been found to cause epilepsy, particularly in genes encoding subunits of neuronal voltage- or ligand-gated ion channels or proteins related to neuronal maturation and migration during embryonic development. Recently, nine patients with a novel microdeletion syndrome at 9q21.13 presenting mental retardation, speech delay, epilepsy, and characteristic facial features have been reported [Boudry-Labis et al., 2013]. The deleted segments spanned from 2.2 to 12.6 megabases and included a variable number of genes. The smallest
* Corresponding author. Tel.: þ39 (0) 10 5636922. E-mail addresses: [email protected], [email protected] (E. Tassano). 1769-7212/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmg.2013.12.001
region of deletion overlap extending 750 kb included four genes among which the authors proposed RORB as a strong candidate for a neurological phenotype [Boudry-Labis et al., 2013]. RORB (RAR-related orphan receptor b) is an orphan nuclear receptor, forming a subfamily with the closely related nuclear receptors ROR-alpha (RORA) and ROR-gamma (RORC) (OMIM 601972). Jabaudon et al. [2012] demonstrated that RORB expression levels control the cytoarchitectural patterning of neocortical neurons during development. Moreover, it has been reported that RORB is specifically expressed in the temporal cortex, as demonstrated on cortical samples from patients with temporal lobe epilepsy [Rossini et al., 2011]. Here we report on a girl with mild intellectual disability, idiopathic partial epilepsy, and a 9q21.13 microdeletion of about 1 Mb in size, encompassing only four genes including RORB. 2. Materials and methods Cytogenetic analysis was performed on cultured lymphocytes by G-banding according to standard procedures. Array-CGH was performed on the patient and her parents using the Agilent kit 2x400K (Human Genome CGH Microarray, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. Quality of experiments was assessed using Feature Extraction QC Metric v10.1.1 (Agilent). The derivative log ratio spread (DLR) value
M.G. Baglietto et al. / European Journal of Medical Genetics 57 (2014) 44e46
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 were excluded from further analysis. Genomic positions refer to the Human Genome February 2009 assembly (GRCh37/hg19). Mutational analysis of RORB was performed by direct sequencing of the 10 exons and flanking introns. Primer design was done by ExonPrimer (UCSC Genome Browser, hg19). 3. Clinical report The proposita is a 12-year-old girl. She was admitted to our observation for mild intellectual disability and idiopathic partial epilepsy. She showed mild facial dysmorphism with mild hypertelorism, low-set hair, slight divergent strabismus and upslanting palpebral fissures, thin upper lip, pavilions anteroversi bilaterally and, long nose filter. She was born full-term to non consanguineous healthy parents after an uneventful pregnancy. At birth, weight, length, and head circumference were normal. Vesicoureteral reflux was present as an abnormal widening of the ureter before birth. When medical management failed to prevent recurrent urinary tract infections, surgery was performed (at the age of 10 months). Her early developmental milestones were normal although she did not socialize normally with other children as a preschool child. At 4 years 6 months of age, she developed epilepsy with daily secondarily generalized seizures (SGE) with absences and apparently generalized tonic-clonic seizures. EEG showed left centrotemporal spikes and generalized spike-wave discharges. Seizure
45
control was achieved after administration of sodium valproate. At 7 years 3 months, neuropsychological assessment for school problems showed mental age of 5 years 7 months, evaluated with Griffths Mental Developmental Scale (QS 77). She remained seizure-free until the age of 8 years when she had two SGE episodes, during the adjustment of the dose of sodium valproate, while falling asleep. EEG was abnormal with frequent left frontal and centrotemporal spike and spike-wave discharges. Partial epilepsy was easy to control with an adequate dosage of antiepileptic drug. At the neurological examination, fine motor skills resulted inadequate for age, but no focal neurologic signs were detected. Cranial CT and MRI were normal. Neuropsychological assessment, evaluated with WISC-III at ten years of age, revealed mild intellectual disability. The neuropsychological profile was disharmonic with lower values at the verbal test: QIT 69, QIV 65, QIP 80. This result can be correlated with the left focal anomalies in her intercritical EEG. She was educated in a mainstream school with additional support. Laboratory investigations revealed normal values of the electrolytes Na, Ca, Mg, P, Cl. Today, at 13 years of age, she is seizurefree 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 9q21.13 band. The deleted region spans w1.040 Mb from probe A_18_P16874789 (77,283,017 bp) to probe A_16_P38754346
Fig. 1. Results of array-CGH analysis: AeB) Array-CGH analysis showed a de novo interstitial deletion at 9q21.13 band. The deleted region spans 1.040 Mb from probe A_18_P16874789 (77,283,017 bp) to probe A_16_P38754346 (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 UCSC Genome Browser (http://genome.ucsc.edu/; GRCh37/hg19, February 2009) B) Extract from the UCSC genome browser (http://genome.ucsc.edu/) GRCh37/hg19 shows the deleted region of our patient (arrowheads) and its gene content, including the RORB, TRPM6, NMRK1, OSTF1 loci, two open reading frames (C9orf40, C9orf41) and a microRNA (MIR548H3), compared to the deletion identified by Boudry-Labis et al. [2013].
46
M.G. Baglietto et al. / European Journal of Medical Genetics 57 (2014) 44e46
(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 UCSC Genome Browser (http://genome.ucsc.edu/GRCh37/hg19February 2009) (Fig. 1, AeB). The deleted segment contains only four genes, namely RORB (RAR-related orphan receptor B), TRPM6 (transient receptor potential cation channel, subfamily M, member 6), NMRK1 (nicotinamide riboside kinase 1), OSTF1 (osteoclast stimulating factor 1), two open reading frames (C9orf40, C9orf41), and a microRNA (MIR548H3) (Fig. 1, C). The proximal breakpoint of the deletion disrupts RORB between exons 5 and 8 while distal breakpoint disrupts the microRNA (MIR548H3) (Fig. 1, C). Mutation analysis of RORB gene was negative on the homologous allele. Surely, the deletion breaks the last three exons and, in particular at protein level, it disrupts a number of alpha-elices forming the ligand binding domain (LBD) and the activation function 2 (AF2) in H12 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 at 9q21.3 presenting epilepsy and mild intellectual disability. The deleted region encompasses only four genes including RORB. RORB, a nuclear orphan receptor, regulates neuronal patterning during cortical development [Jabaudon et al., 2012]. Neuropathologic investigations frequently reveal the presence of architectural cortical dysplasia in patients with temporal lobe epilepsy (TLE), sometimes as an isolated finding but more commonly associated with 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 hippocampal abnormalities are the consequence of repeated seizures but also that genetic mechanisms could play a significant role in the development of hippocampal damage [Kobayashi et al., 2001]. Recently, in an array-CGH study on 102 patients with epilepsy and additional neurodevelopmental disorders, the authors reported on a 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 dysmorphisms have been reported [Boudry-Labis et al., 2013]. These patients carried 2.2 Mbe12.6 Mb interstitial deletions of chromosome 9q21.11eq21.32 [Boudry-Labis et al., 2013]. Our study describes a patient sharing similar features with the cases reported by BoudryLabis et al. [2013] and Bartnik et al. [2012], such as epilepsy and mild developmental delay. Mild dysmorphic features were present in our patient and in case 5 reported by Boudry-Labis et al. [2013]. Interestingly, our patient presented a small deletion at 9q21.3 overlapping the 750 Kb of the small overlapping region (SRO) reported by Boudry-Labis et al. [2013], who suggested a significant role of RORB in the development of the mild intellectual disability and epilepsy phenotype. In all patients reported by Boudry-Labis et al. [2013], RORB was completely deleted while in our patient RORB was partially deleted, although the phenotype of our patient was similar to those reported. The deletion breaks exons 8, 9, and 10 of RORB gene in the ligand binding domain (LBD). The LBDs of nuclear receptors are multifunctional, playing a role in ligand binding, nuclear localization, receptor dimerization, and provide an interface for the interaction with coactivators and corepressors. In addition to the typical 12 canonical a-helices (H1-12), the LBD of RORB contains two additional helices, H20 and H110. The activation function 2 (AF2) in H12 consists of PLYKELF, which is 100% conserved among RORs. Deletion of H12 or point mutations within H12 cause loss of ROR transactivation activity and result in a dominant-negative ROR [Kurebayashi et al., 2004; Lau et al., 1999].
RORB, TRPM6, NMRK, and OSTF1 are the genes included in the deleted region. NMRK1 and OSTF1 are not expressed in brain and do not seem to be relevant candidates for neurodevelopmental disorders. TRPM6 belongs to the transient receptor potential (TRP) superfamily [Schlingmann & Gudermann, 2005; Schmitz et al., 2005]. Mutations of the gene have been associated with hypomagnesaemia with secondary hypocalcemia (HSH). Though TRPM6 sequencing to exclude a mutation of the remaining allele has not been performed, magnesium and calcium levels in the intercritical period were normal in our patient. Interestingly, urinary tract disorders (enuresis and pyelonephritis) have been reported in four patients (patients 1, 3, 6, and 7) described by Boudry-Labis et al. [2013] and also our patient showed a vesico-ureteral reflux of unknown origin. Therefore, in agreement with Boudry-Labis et al. [2013], we could speculate that, among the genes of the interstitial deletion at 9q21.13, 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 interests related to this manuscript. Acknowledgements We thank the patient’s parents for their kind participation and support. We are grateful to Marco Bertorello and Corrado Torello for their technical assistance. This work was supported by “ Cinque per mille dell’IRPEF- Finanziamento della ricerca sanitaria” and “Finanziamento Ricerca Corrente, Ministero Salute” (contributo per la ricerca intramurale). References ska K, Wisniowiecka-Kowalnik B, Gambin T. Bartnik M, Szczepanik E, Derwin Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders. Am J Med Genet B Neuropsychiatr Genet 2012;159B:760e71. Boudry-Labis E, Demeer B, Le Caignec C, Isidor B, Mathieu-Dramard M, et al. A novel microdeletion syndrome at 9q21.13 characterised by mental retardation, speech delay, epilepsy and characteristic facial features. Eur J Med Genet 2013;56:163e70. Gardiner RM. Impact of our understanding of the genetic aetiology of epilepsy. J Neurol 2000;247:327e34. Hauser WA, Annegers JF, Rocca WA, et al. Descriptive epidemiology of epilepsy: contributions of population-based studies from Rochester, Minnesota. Mayo Clin Proc 1996;71:576e86. Jabaudon D, Shnider SJ, Tischfield DJ, Galazo MJ, Macklis JD. RORb induces barrellike neuronal clusters in the developing neocortex. Cereb Cortex 2012;22: 996e1006. Jozwiak S, Lason W, Bijak M, Katulska K. Research advances in molecular genetics of epilepsies. Neurol Neurochir Pol 2005;39:497e508. Kobayashi E, Lopes-Cendes I, Guerreiro CAM, Sousa SC, Guerreiro MM, et al. Seizure outcome and hippocampal atrophy in familial mesial temporal lobe epilepsy. Neurology 2001;56:166e72. Kurebayashi S, Nakajima T, Kim SC, Chang CY, McDonnell DP, Renaud JP, Jetten AM. Selective LXXLL peptides antagonize transcriptional activation by the retinoidrelated orphan receptor RORgamma. Biochem Biophys Res Commun 2004;315: 919e27. Lau P, Bailey P, Dowhan DH, Muscat GE. Exogenous expression of a dominant negative RORalpha1 vector in muscle cells impairs differentiation: RORalpha1 directly interacts with p300 and myoD. Nucleic Acids Res 1999;27: 411e20. Pal DK, Pong AW, Chung WK. Genetic evaluation and counseling for epilepsy. Nat Rev Neurol 2010;6:445e53. Rossini L, Moroni RF, Tassi L, Watakabe A, Yamamori T, et al. Altered layer-specific gene expression in cortical samples from patients with temporal lobe epilepsy. Epilepsia 2011;52:1928e37. Schlingmann KP, Gudermann T. A critical role of TRPM channel-kinase for human magnesium transport. J Physiol 2005;15:301e8. Schmitz C, Dorovkov MV, Zhao X, Davenport BJ, Ryazanov AG, et al. The channel kinases TRPM6 and TRPM7 are functionally nonredundant. J Biol Chem 2005;280:37763e71.
Contents lists available at ScienceDirect
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 with epilepsy 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, * a b c
Centro Epilessie, UO Neuropsichiatria Infantile, Istituto Giannina Gaslini, Genova, Italy Laboratorio di Fisiopatologia dell’Uremia, Istituto Giannina Gaslini, Genova, Italy 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
a b s t r a c t
Article history: Received 7 August 2013 Accepted 6 December 2013 Available online 17 December 2013
Copy number variants represent an important cause of neurodevelopmental disorders including epilepsy, which is genetically determined in 40% of cases. Epilepsy is caused by chromosomal imbalances or mutations in genes encoding subunits of neuronal voltage- or ligand-gated ion channels or proteins related to neuronal maturation and migration during embryonic development. Here, we report on a girl with mild intellectual disability and idiopathic partial epilepsy. Array-CGH analysis showed a 1.040 Mb de novo 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 nuclear receptor highly expressed in the retina, cortex, and thalamus. We hypothesize its role in producing the phenotype of our patient and compare this case with other ones previously reported in the literature to better identify a genotypeephenotype correlation. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: RORB gene 9q21.13 Microdeletion Epilepsy Mild intellectual disability Array-CGH Copy number variants
1. Introduction Copy number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. Epilepsy is one of the most common neurological disorders affecting up to 1% of the general population. It has been estimated that 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 with chromosomal imbalances and numerous gene mutations have been found to cause epilepsy, particularly in genes encoding subunits of neuronal voltage- or ligand-gated ion channels or proteins related to neuronal maturation and migration during embryonic development. Recently, nine patients with a novel microdeletion syndrome at 9q21.13 presenting mental retardation, speech delay, epilepsy, and characteristic facial features have been reported [Boudry-Labis et al., 2013]. The deleted segments spanned from 2.2 to 12.6 megabases and included a variable number of genes. The smallest
* Corresponding author. Tel.: þ39 (0) 10 5636922. E-mail addresses: [email protected], [email protected] (E. Tassano). 1769-7212/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmg.2013.12.001
region of deletion overlap extending 750 kb included four genes among which the authors proposed RORB as a strong candidate for a neurological phenotype [Boudry-Labis et al., 2013]. RORB (RAR-related orphan receptor b) is an orphan nuclear receptor, forming a subfamily with the closely related nuclear receptors ROR-alpha (RORA) and ROR-gamma (RORC) (OMIM 601972). Jabaudon et al. [2012] demonstrated that RORB expression levels control the cytoarchitectural patterning of neocortical neurons during development. Moreover, it has been reported that RORB is specifically expressed in the temporal cortex, as demonstrated on cortical samples from patients with temporal lobe epilepsy [Rossini et al., 2011]. Here we report on a girl with mild intellectual disability, idiopathic partial epilepsy, and a 9q21.13 microdeletion of about 1 Mb in size, encompassing only four genes including RORB. 2. Materials and methods Cytogenetic analysis was performed on cultured lymphocytes by G-banding according to standard procedures. Array-CGH was performed on the patient and her parents using the Agilent kit 2x400K (Human Genome CGH Microarray, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. Quality of experiments was assessed using Feature Extraction QC Metric v10.1.1 (Agilent). The derivative log ratio spread (DLR) value
M.G. Baglietto et al. / European Journal of Medical Genetics 57 (2014) 44e46
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 were excluded from further analysis. Genomic positions refer to the Human Genome February 2009 assembly (GRCh37/hg19). Mutational analysis of RORB was performed by direct sequencing of the 10 exons and flanking introns. Primer design was done by ExonPrimer (UCSC Genome Browser, hg19). 3. Clinical report The proposita is a 12-year-old girl. She was admitted to our observation for mild intellectual disability and idiopathic partial epilepsy. She showed mild facial dysmorphism with mild hypertelorism, low-set hair, slight divergent strabismus and upslanting palpebral fissures, thin upper lip, pavilions anteroversi bilaterally and, long nose filter. She was born full-term to non consanguineous healthy parents after an uneventful pregnancy. At birth, weight, length, and head circumference were normal. Vesicoureteral reflux was present as an abnormal widening of the ureter before birth. When medical management failed to prevent recurrent urinary tract infections, surgery was performed (at the age of 10 months). Her early developmental milestones were normal although she did not socialize normally with other children as a preschool child. At 4 years 6 months of age, she developed epilepsy with daily secondarily generalized seizures (SGE) with absences and apparently generalized tonic-clonic seizures. EEG showed left centrotemporal spikes and generalized spike-wave discharges. Seizure
45
control was achieved after administration of sodium valproate. At 7 years 3 months, neuropsychological assessment for school problems showed mental age of 5 years 7 months, evaluated with Griffths Mental Developmental Scale (QS 77). She remained seizure-free until the age of 8 years when she had two SGE episodes, during the adjustment of the dose of sodium valproate, while falling asleep. EEG was abnormal with frequent left frontal and centrotemporal spike and spike-wave discharges. Partial epilepsy was easy to control with an adequate dosage of antiepileptic drug. At the neurological examination, fine motor skills resulted inadequate for age, but no focal neurologic signs were detected. Cranial CT and MRI were normal. Neuropsychological assessment, evaluated with WISC-III at ten years of age, revealed mild intellectual disability. The neuropsychological profile was disharmonic with lower values at the verbal test: QIT 69, QIV 65, QIP 80. This result can be correlated with the left focal anomalies in her intercritical EEG. She was educated in a mainstream school with additional support. Laboratory investigations revealed normal values of the electrolytes Na, Ca, Mg, P, Cl. Today, at 13 years of age, she is seizurefree 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 9q21.13 band. The deleted region spans w1.040 Mb from probe A_18_P16874789 (77,283,017 bp) to probe A_16_P38754346
Fig. 1. Results of array-CGH analysis: AeB) Array-CGH analysis showed a de novo interstitial deletion at 9q21.13 band. The deleted region spans 1.040 Mb from probe A_18_P16874789 (77,283,017 bp) to probe A_16_P38754346 (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 UCSC Genome Browser (http://genome.ucsc.edu/; GRCh37/hg19, February 2009) B) Extract from the UCSC genome browser (http://genome.ucsc.edu/) GRCh37/hg19 shows the deleted region of our patient (arrowheads) and its gene content, including the RORB, TRPM6, NMRK1, OSTF1 loci, two open reading frames (C9orf40, C9orf41) and a microRNA (MIR548H3), compared to the deletion identified by Boudry-Labis et al. [2013].
46
M.G. Baglietto et al. / European Journal of Medical Genetics 57 (2014) 44e46
(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 UCSC Genome Browser (http://genome.ucsc.edu/GRCh37/hg19February 2009) (Fig. 1, AeB). The deleted segment contains only four genes, namely RORB (RAR-related orphan receptor B), TRPM6 (transient receptor potential cation channel, subfamily M, member 6), NMRK1 (nicotinamide riboside kinase 1), OSTF1 (osteoclast stimulating factor 1), two open reading frames (C9orf40, C9orf41), and a microRNA (MIR548H3) (Fig. 1, C). The proximal breakpoint of the deletion disrupts RORB between exons 5 and 8 while distal breakpoint disrupts the microRNA (MIR548H3) (Fig. 1, C). Mutation analysis of RORB gene was negative on the homologous allele. Surely, the deletion breaks the last three exons and, in particular at protein level, it disrupts a number of alpha-elices forming the ligand binding domain (LBD) and the activation function 2 (AF2) in H12 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 at 9q21.3 presenting epilepsy and mild intellectual disability. The deleted region encompasses only four genes including RORB. RORB, a nuclear orphan receptor, regulates neuronal patterning during cortical development [Jabaudon et al., 2012]. Neuropathologic investigations frequently reveal the presence of architectural cortical dysplasia in patients with temporal lobe epilepsy (TLE), sometimes as an isolated finding but more commonly associated with 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 hippocampal abnormalities are the consequence of repeated seizures but also that genetic mechanisms could play a significant role in the development of hippocampal damage [Kobayashi et al., 2001]. Recently, in an array-CGH study on 102 patients with epilepsy and additional neurodevelopmental disorders, the authors reported on a 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 dysmorphisms have been reported [Boudry-Labis et al., 2013]. These patients carried 2.2 Mbe12.6 Mb interstitial deletions of chromosome 9q21.11eq21.32 [Boudry-Labis et al., 2013]. Our study describes a patient sharing similar features with the cases reported by BoudryLabis et al. [2013] and Bartnik et al. [2012], such as epilepsy and mild developmental delay. Mild dysmorphic features were present in our patient and in case 5 reported by Boudry-Labis et al. [2013]. Interestingly, our patient presented a small deletion at 9q21.3 overlapping the 750 Kb of the small overlapping region (SRO) reported by Boudry-Labis et al. [2013], who suggested a significant role of RORB in the development of the mild intellectual disability and epilepsy phenotype. In all patients reported by Boudry-Labis et al. [2013], RORB was completely deleted while in our patient RORB was partially deleted, although the phenotype of our patient was similar to those reported. The deletion breaks exons 8, 9, and 10 of RORB gene in the ligand binding domain (LBD). The LBDs of nuclear receptors are multifunctional, playing a role in ligand binding, nuclear localization, receptor dimerization, and provide an interface for the interaction with coactivators and corepressors. In addition to the typical 12 canonical a-helices (H1-12), the LBD of RORB contains two additional helices, H20 and H110. The activation function 2 (AF2) in H12 consists of PLYKELF, which is 100% conserved among RORs. Deletion of H12 or point mutations within H12 cause loss of ROR transactivation activity and result in a dominant-negative ROR [Kurebayashi et al., 2004; Lau et al., 1999].
RORB, TRPM6, NMRK, and OSTF1 are the genes included in the deleted region. NMRK1 and OSTF1 are not expressed in brain and do not seem to be relevant candidates for neurodevelopmental disorders. TRPM6 belongs to the transient receptor potential (TRP) superfamily [Schlingmann & Gudermann, 2005; Schmitz et al., 2005]. Mutations of the gene have been associated with hypomagnesaemia with secondary hypocalcemia (HSH). Though TRPM6 sequencing to exclude a mutation of the remaining allele has not been performed, magnesium and calcium levels in the intercritical period were normal in our patient. Interestingly, urinary tract disorders (enuresis and pyelonephritis) have been reported in four patients (patients 1, 3, 6, and 7) described by Boudry-Labis et al. [2013] and also our patient showed a vesico-ureteral reflux of unknown origin. Therefore, in agreement with Boudry-Labis et al. [2013], we could speculate that, among the genes of the interstitial deletion at 9q21.13, 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 interests related to this manuscript. Acknowledgements We thank the patient’s parents for their kind participation and support. We are grateful to Marco Bertorello and Corrado Torello for their technical assistance. This work was supported by “ Cinque per mille dell’IRPEF- Finanziamento della ricerca sanitaria” and “Finanziamento Ricerca Corrente, Ministero Salute” (contributo per la ricerca intramurale). References ska K, Wisniowiecka-Kowalnik B, Gambin T. Bartnik M, Szczepanik E, Derwin Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders. Am J Med Genet B Neuropsychiatr Genet 2012;159B:760e71. Boudry-Labis E, Demeer B, Le Caignec C, Isidor B, Mathieu-Dramard M, et al. A novel microdeletion syndrome at 9q21.13 characterised by mental retardation, speech delay, epilepsy and characteristic facial features. Eur J Med Genet 2013;56:163e70. Gardiner RM. Impact of our understanding of the genetic aetiology of epilepsy. J Neurol 2000;247:327e34. Hauser WA, Annegers JF, Rocca WA, et al. Descriptive epidemiology of epilepsy: contributions of population-based studies from Rochester, Minnesota. Mayo Clin Proc 1996;71:576e86. Jabaudon D, Shnider SJ, Tischfield DJ, Galazo MJ, Macklis JD. RORb induces barrellike neuronal clusters in the developing neocortex. Cereb Cortex 2012;22: 996e1006. Jozwiak S, Lason W, Bijak M, Katulska K. Research advances in molecular genetics of epilepsies. Neurol Neurochir Pol 2005;39:497e508. Kobayashi E, Lopes-Cendes I, Guerreiro CAM, Sousa SC, Guerreiro MM, et al. Seizure outcome and hippocampal atrophy in familial mesial temporal lobe epilepsy. Neurology 2001;56:166e72. Kurebayashi S, Nakajima T, Kim SC, Chang CY, McDonnell DP, Renaud JP, Jetten AM. Selective LXXLL peptides antagonize transcriptional activation by the retinoidrelated orphan receptor RORgamma. Biochem Biophys Res Commun 2004;315: 919e27. Lau P, Bailey P, Dowhan DH, Muscat GE. Exogenous expression of a dominant negative RORalpha1 vector in muscle cells impairs differentiation: RORalpha1 directly interacts with p300 and myoD. Nucleic Acids Res 1999;27: 411e20. Pal DK, Pong AW, Chung WK. Genetic evaluation and counseling for epilepsy. Nat Rev Neurol 2010;6:445e53. Rossini L, Moroni RF, Tassi L, Watakabe A, Yamamori T, et al. Altered layer-specific gene expression in cortical samples from patients with temporal lobe epilepsy. Epilepsia 2011;52:1928e37. Schlingmann KP, Gudermann T. A critical role of TRPM channel-kinase for human magnesium transport. J Physiol 2005;15:301e8. Schmitz C, Dorovkov MV, Zhao X, Davenport BJ, Ryazanov AG, et al. The channel kinases TRPM6 and TRPM7 are functionally nonredundant. J Biol Chem 2005;280:37763e71.