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Clin Genet 2010: 78: 471–477 2010 John Wiley & Sons A/S Printed in Singapore. All rights reserved Short Report
Refining the phenotype associated withMEF2C haploinsufficiency Novara F, Beri S, Giorda R, Ortibus E, Nageshappa S, Darra F, F Novaraa, S Berib, R Giordab,
dalla Bernardina B, Zuffardi O, Van Esch H. Refining the phenotype E Ortibusc, S Nageshappad,
associated with MEF2C haploinsufficiency.
F Darrae, B dalla Bernardinae,
Clin Genet 2010: 78: 471–477. John Wiley & Sons A/S, 2010 O Zuffardia,f and H Van Eschd
Recently, submicroscopic deletions of the 5q14.3 region have been aGenetica Medica, Universit a di Pavia, described in patients with severe mental retardation (MR), stereotypic Pavia, 27100 PV, Italy, bBiologiaMolecolare, IRCCS ‘‘E. Medea'', Bosisio movements, epilepsy and cerebral malformations. Further delineation of a Parini, Lecco, Italy, cDepartment of critical region of overlap in these patients pointed to MEF2C as the Pediatrics and dCenter for Human responsible gene. This finding was further reinforced by the identification Genetics, University Hospitals Leuven, of a nonsense mutation in a patient with a similar phenotype. In brain, Leuven, Belgium, eNeuropsichiatria MEF2C is essential for early neurogenesis, neuronal migration and Infantile, Policlinico GB Rossi, Verona, differentiation. Here we present two additional patients with severe MR, Italy, and fIRCCS Fondazione C.
autism spectrum disorder and epilepsy, carrying a very small deletion Mondino, Pavia, Italy encompassing the MEF2C gene. This finding strengthens the role of this Key words: aCGH – chromosome gene in severe MR, and enables further delineation of the clinical 5q14.3 – epilepsy – haploinsufficiency –MEF2C – microdeletion – severe mental Corresponding author: Hilde Van Esch,MD, PhD, Centre for Human Genetics,University Hospitals Leuven, Herestraat49, 3000 Leuven, Belgium.
Tel.: +32 16 345903;fax: +32 16 346051;e-mail: Hilde.Vanesch@med.
kuleuven.be Received 6 November 2009, revisedand accepted for publication 22February 2010 Recently, submicroscopic deletions of the 5q14.3 factors are expressed in overlapping but distinct region have been described in patients featuring regions of the central nervous system (CNS) that severe mental retardation (MR), stereotypic move- correlate with the withdrawal of neurons from ments, epilepsy and cerebral malformations (1–3).
the cell cycle and acquisition of a differentiated Further delineation of a critical region of overlap phenotype (4). In mouse, Mef2c is the first of in these patients pointed to the MEF2C gene as four Mef2 genes to be expressed in the CNS. In the responsible gene. This finding was further rein- the adult brain, Mef2c is highly expressed in the forced by the identification of a nonsense mutation frontal cortex, entorhinal cortex, dentate gyrus, and in a patient with a similar phenotype. MEF2C, amygdala. Recently it was shown that the dele- encoding transcription factor myocyte enhancer tion of Mef2c transcription factor in the CNS of factor 2C, plays a crucial role during several mice impairs hippocampal-dependent learning and embryological processes, including hematopoiesis, memory by negative regulation of synapse num- cardiogenesis and neurogenesis. In brain, members bers and function (5, 6).
of the MEF2 family of MADS (MCM1, agamous, Here we present two additional patients with deficiens, serum response factor) box transcription severe MR, autism spectrum disorder and epilepsy, Novara et al.
carrying a very small deletion encompassing the Milan, Italy) with the following protocol: 30 s at MEF2C gene. This finding strengthens the role 96◦C, 35 cycles of 15 s at 94◦C/20 s at 58◦C/10 of this gene in severe MR, and enables further min at 68◦C, 10 min final elongation time. Primers delineation of the clinical phenotype.
were: for Patient 1, Del5-11F (5-CATCATTGCCCCACATCACA-3) and Del5-13R (5-TGAAGGAGAGCTGGCTGTGA-3); for Patient 2, Del5-10F (5-TGTGGCTGAGCTGCTTCTAACA-3) and Del The protocol was approved by the appropri- 5-11R (5-TTCCTGCCCTACCTTCATGTG-3). All ate Institutional Review Boards involved in the sequencing reactions were performed with a Big research (Universities of Leuven, Belgium and Dye Terminator Cycle Sequencing kit 3.1 (Applied Pavia, Italy). Informed consent was obtained from Biosystems) and run on an ABI Prism 3130xl the parents of the affected patients.
Genetic Analyzer.
The UCSC Genome Browser (May 2004 assem- bly; http://genome.ucsc.edu/) maps and sequence Cytogenetic analysis were used as references. The primer sequences are available on request.
according to routine protocol. Arrays were per-formed using the Agilent array 105 K according to the manufacturer's protocol.
Patient 1 is the first child of healthy non-relatedparents, born at term with normal birth parame- ters after an uneventful pregnancy. Two younger Genotyping of polymorphic loci in patient 2 and siblings are normal. A brother and a cousin of his parents was performed by amplification with the mother have a benign form of epilepsy, well primers labeled with fluorescent probes (ABI responding to therapy and not interfering with 6-Fam and 8-Hex), followed by analysis on ABI daily functioning or cognition. The boy came to 3100 Genetic Analyzer (Applied Biosystems, Fos- medical attention at the age of 3 months because ter City, CA). Primers were designed using the of absent eye contact and social smile, hypoto- database tool Tandem Repeats Finder (http:// nia and irritable behavior. MRI scan at that age showed a cystic lesion and leucoencephalopathyin the left frontal region, probably due to a peri-natal hemorrhage. Metabolic investigations includ- Real-time quantitative PCR ing coagulation were all normal. Subsequently, his Specific target sequences were selected for Real- psychomotor development was severely delayed time quantitative PCR (qPCR) using Primer with sitting with little support at the age of 2 years.
Express software (Applied Biosystems). A control An MRI scan of the brain at that age showed signs amplicon was selected with the same parameters of periventricular leucomalacia and atrophy of the in the MAPK1 gene on 22q11.2; size (approxi- frontal cortex at the left side. He never reached mately 60 bp) and Tm (59◦C) were the same for all independent ambulation because of persistent amplicons. Amplification and detection were per- severe axial hypotonia. Epilepsy started in the first formed on an ABI PRISM 7700 Sequence Detec- year of life, initially as isolated myoclonic jerks, tion System (Applied Biosystems) using SYBR later evolving toward infantile spasms with con- Green PCR Master Mix (Applied Biosystems); tinuous epileptic activity bi-posterior with no basic thermal cycling conditions were 50◦C for 2 min rhythm on EEG. Because of further deterioration and 95◦C for 10 min, followed by 40 cycles of of the drug-resistant epilepsy, he was admitted at 95◦C for 15 s and 60◦C for 1 min; all samples a special epilepsy unit for intensive care at the age were amplified in duplicate. Relative quantifica- of 2 years. After this period there was a regres- tion of the amount of DNA was obtained using sion of his motor functions, necessitating a wheel the comparative CT method (described in Applied chair and orthopedic ortheses. Speech was absent.
Biosystems User Bulletin #2, December 11, 1997: Now at the age of 14 years, his growth parameters ABI PRISM 7700 Sequence Detection System).
are within the normal range (OFC at 50th centile).
He has a cerebral palsy with severe axial hypoto-nia and compensatory peripheral hypertonia. His eye contact is very poor with external strabismus Long-range PCRs were performed with Jump- of the right eye. He does not exhibit stereotypic Start Red ACCUTaq LA DNA polymerase (Sigma, movements. He has only mild dysmorphisms with

Deletion of MEF2C causes severe MR
Fig. 1. Clinical picture of patient 1 at the age of 14 years (a) and patient 2 at the age of 3 years (b). Note the facial hypotonia,
strabismus, prominent philtrum with cupid's bow in both boys. Patient 1 has in addition macrodontia. (c) EEG record of patient 2 at
the age of 13 months showing slow background activity with theta waves degraded over the central regions of the two hemispheres
and degraded diffuse discharges, together with rhythmic sharp wave activity; EEG velocity: 15 mm/s; amplitude: 50 μV. (d,e) MRI
images of patient 2: axial section showing dilatation of the lateral ventricles (d) and sagittal section showing hypoplasia of the
distal part of the corpus callosum (e).
prominent ear lobes, short prominent philtrum with His mother suffered from hypothyroidism and was a cupid's bow and macrodontia (Fig. 1a).
treated with levothyroxin during pregnancy. At Patient 2 was born at term with birth weight of 4 months of age, lack of reactivity was observed 3600 g and length of 51 cm (both 75th centile).
in addition to severe hypotonia, dystonic motor Novara et al.
activity, absent head control and poor visual track- (patient 2) mechanisms. (Table S1, Supporting ing. At the age of 5 months, psychomotor delay information). Parental origin was not investigated and myoclonus were observed. EEG showed slow in patient 1. In patient 2, the chromosomal imbal- background activity with theta waves degraded ance originated at the paternal meiosis. Three out over the central regions of the two hemispheres of four microsatellites analyzed in the deleted and degraded diffuse discharges, sometimes with region showed the presence of the maternal allele episodes of rhythmic sharp wave activity, asso- only (data not shown).
ciated with revulsion of eyes and myoclonus ofthe limbs (Fig. 1c). Clinical evaluation showed occipital plagiocephaly, hypertelorism, flattenednasal bridge, small and hooked nose, ogival Recently, MEF2C haploinsufficiency has been palate, and low-set and dysmorphic ears (Fig. 1b).
described in five patients presenting a severe and Marked myopia with alternating esotropia was also syndromic form of MR and carrying a submicro- observed. Several MRI scans of the brain were scopic deletion on chromosome 5q14.3 of variable performed at 5, 8 and 19 months of age, respec- size ranging from 216 kb to 8.8 Mb (1). Moreover, tively; they showed moderate dilatation of lateral the same authors detected a nonsense mutation ventricles and hypoplasia of the corpus callosum in MEF2C in an additional patient exhibiting a with abnormal aspect of the splenius (Fig. 1d,e).
similar phenotype and thus underscoring the clin- The development quotient was calculated using the ical relevance of this new autosomal dominant Griffith mental development scales at 13 and 19 MR gene. MEF2C encodes myocyte enhancer months of age (35 and 30, respectively). At the factor 2 that functions as a transcription factor, last evaluation at 3 years and 10 months of age, with MEF2C as the predominant isoform in the he presented a severe cognitive deficit with numer- brain. The role of MEF2C during brain develop- ous behavioral stereotypes. Head circumference ment and functioning, including neurogenesis, neu- was at the 50th centile. Neurological examination ronal migration and synaptic plasticity, has been showed severe axial hypotonia with partial control well established in murine models and in vitro of the head. In the lower limbs an increased mus- functional studies (4–7). Its role in learning and cular tonus was noticed with dystonic–dyskinetic memory as well as maintaining the critical bal- movements. He was able to fix and follow objects ance between inhibitory and excitatory synapses and wore glasses for myopia. Language was is consistent with the human haploinsufficiency absent. He still suffered from myoclonic and phenotype we and others have observed. Both myoclonic–atonic seizures with falling of the patients we describe here carry a very small dele- head, not responding to any anti-epileptic ther- tion, involving only the MEF2C in patient 1. The apy (Valproic Acid, Nitrazepam, Levetiracetam, deletion in patient 2 also affects the TMEM161B Hydrocortison, Clobazam, and Ethosuximide).
gene, a gene of unknown function that is predictedto encode a transmembrane protein. We can notexclude an involvement of haploinsufficiency of this gene in the clinical phenotype of patient 2.
In the absence of an etiological diagnosis in both When we compare the clinical phenotype in patients, array CGH analysis using the 105 K our patients with the reported patients, the pheno- array (Agilent) was performed. In both patients, type remains very consistent (Table 1). All patients a small deletion in 5q14.3 was detected (Fig. 2).
present with severe primary developmental delay All breakpoint locations were refined by qPCR, reflected by early hypotonia, delayed motor devel- and then both junctions were amplified by long- opment and no speech development. Thus far, our range PCR and sequenced. Patient 1 has a deletion patients and previously reported patients who are of 318357 bp (87,978,527–88,296,884) harboring hemizygous for the gene did not reach indepen- only MEF2C, while patient 2 has a slightly larger dent walking and remain very hypotonic, while deletion of 1140131 bp (87,234,127–88,374,258).
the patient with a reported point mutation started Besides MEF2C, the latter deletion also includes to walk at 3 years of age without hypotonia (1) the TMEM161B gene. The mother of patient 1 (Table 1). This might point toward a more severe does not carry the deletion, while his father could effect of the deletion compared to the intragenic not be tested. In patient 2 the deletion occurred mutation, but more cases are needed to make de novo. Breakpoint junctions are outside low any genotype–phenotype correlation. Stereotypic copy repeats and have no particular identity.
movements and poor eye contact are present in They are consistent with microhomology-mediated many patients, suggesting the diagnosis of autism (patient 1) or non-homologous end-joining (NHEJ) spectrum disorder (ASD). Interestingly, a role for

Deletion of MEF2C causes severe MR
Fig. 2. Detailed view of the chromosome 5q14.3 aCGH profile showing the deletion in patient 1 (right) and patient 2 (left).
MEF2C in ASD was already shown in classical is very distinct in severity, we believe that another and conditional knock-out mouse models (4, 8).
genetic trait is reponsible for their epilepsia.
Moreover, Morrow et al. identified many MEF2 In most patients brain imaging is reported to target genes in their screen for autism genes by be abnormal, including anomalies of the cor- means of homozygosity mapping in pedigrees with pus callosum, enlarged ventricles, periventricular shared ancestry (9). The recent findings in humans white matter hyperintensities and cortical atrophy further reinforce the role of MEF2C during neu- (Table 1). None of these anomalies seems to be rogenesis and synaptogenesis. Epilepsy is another specific and some might be secondary to the severe frequent feature of MEF2C haploinsufficiency, epileptic activity. Interestingly, Cardoso et al.
although the type (myoclonic, tonic-clonic, infan- reported periventricular heterotopia in a patient tile spasms and febrile seizures) and age at onset with a larger deletion including MEF2C (2). For may vary considerably. In both patients the severe the moment it is unclear whether MEF2C haploin- drug-resistant infantile spasms even necessitated sufficiency is responsible for this variable spectrum admission at an epilepsy care center. Patient 1 has of features or whether other genes within larger two male relatives with a benign form of epilepsy, deletions exert an additional effect. The same holds not affecting cognition or daily functioning. As the true for the facial dysmorphisms that seem to deletion is de novo, and the phenotype in patient 1 be more pronounced in the patients with larger Novara et al.
Deletion of MEF2C causes severe MR
deletions (1). We did not observe any effect of MEF2C hemizygosity on growth and head circum- 1. Le Meur N, Holder-Espinasse M, Jaillard S et al. MEF2C hap- ference (Table 1).
loinsufficiency caused either by microdeletion of the 5q14.3 In summary, we present two new patients with region or mutation is responsible for severe mental retardation severe MR, epilepsy and ASD associated with with stereotypic movements, epilepsy and/or cerebral malfor- deletion of MEF2C.
mations. J Med Genet 2009: 47: 22 – 29.
2. Cardoso C, Boys A, Parrini E et al. Periventricular heterotopia, mental retardation, and epilepsy associated with 5q14.3 – q15deletion. Neurology 2009: 72: 784 – 792.
3. Engels H, Wohlleber E, Zink A et al. A novel microdeletion The following Supporting information is available for this article: syndrome involving 5q14.3 – q15: clinical and molecular cyto- Table S1. Cloning of the deletion breakpoints in patients 1 and 2.
genetic characterization of three patients. Eur J Human Genetics2009: 17: 1592 – 1599.
Additional Supporting information may be found in the online 4. Li H, Radford JC, Ragusa MJ et al. Transcription factor version of this article.
MEF2C influences neural stem/progenitor cell differentiation Please note: Wiley-Blackwell Publishing is not responsible for the and maturation in vivo. Proc Natl Acad Sci U S A 2008: 105: content or functionality of any supplementary materials supplied 9397 – 9402.
by the authors. Any queries (other than missing material) should 5. Barbosa AC, Kim MS, Ertunc M et al. MEF2C, a transcription be directed to the corresponding author for the article.
factor that facilitates learning and memory by negative regu-lation of synapse numbers and function. Proc Natl Acad SciU S A 2008: 105: 9391 – 9396.
6. Flavell SW, Cowan CW, Kim TK et al. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory We thank the families for their cooperation. H.V.E. is funded by synapse number. Science 2006: 311: 1008 – 1012.
the F.W.O. Vlaanderen. O.Z. is funded by Cassa di Risparmio 7. Shalizi A, Gaudilliere B, Yuan Z et al. A calcium-regulated delle Provincie Lombarde (CARIPLO: 2007.5197, bando 2007) and MEF2 sumoylation switch controls postsynaptic differentiation.
by Ministry of Health Grant (RF-AOM-2007-636538. ‘Genomic Science 2006: 311: 1012 – 1017.
structural variation studies in mentally retarded and normal 8. Lipton SA, Li H, Zaremba JD et al. Autistic phenotype from individuals in Italy').
MEF2C knockout cells. Science 2009: 323: 208.
9. Morrow EM, Yoo SY, Flavell SW et al. Identifying autism loci and genes by tracing recent shared ancestry. Science 2008: 321: Conflict of interest
218 – 223.
The authors do not have any affiliation with anygroup with a direct financial interest in the subjectmatter or materials discussed in the manuscript.
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