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Early Infantile Epileptic Encephalopathy with a de novo variant in ZEB2 identified by exome sequencing
European Journal of Medical Genetics 59 (2016) 70e74
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Early Infantile Epileptic Encephalopathy with a de novo variant in ZEB2 identified by exome sequencing Natalia Babkina a, b, *, Joshua L. Deignan c, Hane Lee c, Eric Vilain b, d, Raman Sankar e, Irina Giurgea f, David Mowat g, John M. Graham Jr. a, b, h a
Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA Department of Pediatrics, Division of Medical Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA d Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA e Department of Neurology, Pediatrics and Children's Discovery and Innovation Institute at Mattel Children's Hospital, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA f ^pital Henri Mondor, 94000 Cr Service de Biochimie G en etique, INSERM U955 Equipe 11, Ho eteil, France g Department of Medical Genetics, Sydney Children's Hospital, School of Women's and Children's Health, University of New South Wales, Australia h Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA 90502, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2015 Received in revised form 8 November 2015 Accepted 19 December 2015 Available online 22 December 2015
Early Infantile Epileptic Encephalopathy (EIEE) presents shortly after birth with frequent, severe seizures, a burst-suppression EEG pattern, and progressive disturbance of cerebral function. We present a case of EIEE associated with a de novo missense variant in ZEB2. Heterozygous truncating mutations or deletions in ZEB2 are known to cause Mowat-Wilson syndrome (MWS), which is characterized by seizures with onset in the second year of life, distinctive dysmorphic facial features and malformations that were absent in this patient. This unique case expands the range of phenotypes associated with variants in ZEB2 and indicates that this gene should be included in the molecular investigation of EIEE cases. © 2016 Published by Elsevier Masson SAS.
Keywords: Infantile Epileptic Encephalopathy (EIEE) ZEB2 MowateWilson syndrome (MWS) Seizures Infantile spasms Burst-suppression EEG pattern Developmental delay Cortical gray and white matter atrophy Clinical exome sequencing
1. Introduction Seizure disorder is a frequent finding in individuals seeking a medical genetics consultation. The etiology of seizures is very diverse and more than half of epilepsies reportedly have some genetic basis (Pal et al., 2010). EIEE is a group of disorders that present shortly after birth with frequent and severe seizures, a burst-suppression EEG pattern, and progressive disturbance of cerebral function. EIEE includes Dravet, Ohtahara, and West syndromes, but in many cases, the genetic cause remains unknown
* Corresponding author. Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. E-mail address: [email protected] (N. Babkina). http://dx.doi.org/10.1016/j.ejmg.2015.12.006 1769-7212/© 2016 Published by Elsevier Masson SAS.
(Noh et al., 2012). Identification of the genetic cause is valuable not only for diagnosis but also for guiding the therapy. Application of exome sequencing has enabled identification of genetic causes of many new syndromes and has expanded the phenotypes of known genetic disorders. We present a case of EIEE associated with a de novo heterozygous variant in ZEB2 that was identified by clinical exome sequencing. 2. Clinical description We describe a case of an eight-year-old Ashkenazi Jewish boy with severe EIEE and profound developmental delay. The patient was born at 39 weeks to a 42-year-old mother and a 36-year-old father. His growth and head circumference were appropriate for gestational age. The family history was negative for any known
N. Babkina et al. / European Journal of Medical Genetics 59 (2016) 70e74
genetic disorders, intellectual disability or seizures. The pregnancy was uncomplicated, prenatal serum screening was negative, chorionic villus sampling revealed normal male karyotype, and second trimester anatomy ultrasound was negative for structural abnormalities. Shortly after birth this neonate developed seizures and was diagnosed with EIEE at the age of 5 weeks, followed by infantile spasms with a burst-suppression EEG pattern. His physical examination was significant for postnatal-onset microcephaly (10th centile at birth and 4SD by age 4 months), short stature (6SD at 8.5 years), coarse facial features due to prolonged use of various antiepileptic drugs, and lack of distinctive dysmorphic features (Fig. 1). The patient had severe developmental delay, feeding problems and failure to thrive. Seizures were refractory to treatment and multiple antiepileptic medications were required. EEG was significant for background slowing with predominant delta frequencies and more significant involvement of the right hemisphere. MRI showed significant cortical gray and white matter atrophy and hypomyelination in the supratentorial regions, but no abnormalities of the corpus callosum. MR spectroscopy revealed increased glutamine/glutamate complex, decreased NAA/creatine ratio, decreased choline/creatine ratio consistent with hypomyelination, normal myoinositol peak, and no definite lactate peak. There was no evidence of focal cortical dysplasia. Metabolic evaluation included plasma amino acids, urine organic acids, plasma acylcarnitine profile, biotinidase, long chain fatty acids, CSF neurotransmitters, and enzyme essays for GM1 gangliosidosis, beta mannosidosis, Tay-Sachs, and Krabbe and these studies were all negative. Conjunctival biopsy was negative for storage. SNP microarray was performed using the Affymetrix Cytoscan HD using 743,000 SNP probes and 1,953,000 NPCN probes with a median spacing of 0.86 kb within genes. Microarray was negative for copy number variants or regions of homozygosity to suggest consanguinity. GLRA1 and MECP2 sequence analyses were normal.
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3. Methods Clinical Exome Sequencing (CES) was performed at the UCLA Clinical Genomics Center using clinically validated protocols from DNA extraction to variant analysis (Strom et al., 2014) and interpretation. The proband and both parents were sequenced simultaneously. Genomic DNA was extracted from blood specimen using standard methods. CES was performed using customized Agilent SureSelect Human All Exon 50 Mb kit for exome capture and an Illumina Hiseq2000 for sequencing as 50 bp paired end run. In total ~15 Gb of sequence was generated and uniquely aligned to the human reference genome, generating a mean coverage of 165 per base within Refseq protein coding bases of the human genome. All variants were annotated using annotation tool Variant Annotator X that retrieves information from databases such as OMIM, UniProt, NHLBI Exome Sequencing Project (ESP), SIFT and PolyPhen2 for each variant. All genes harboring de novo, homozygous or compound heterozygous variants with allele frequencies <1% in the general population [estimated using data from the 1000 Genomes Project (1 Kg), ESP, NIEHS project and HapMap] were evaluated by a Genomics Data Board consisting of physicians, pathologists, clinical geneticists, laboratory directors, genetic counselors, and informatics specialists. Clinically significant variants were confirmed using Sanger sequencing of the proband and both parents. 4. Results In total 22,024 DNA variants were identified across the exome, including 20,883 single nucleotide substitutions and 1141 small deletions/insertions (1e10 bp). The data were consistent with a high quality genomic sequence and fell within normal human genomic variation quality parameters. It was estimated that about 95% of the known disease-causing bases were reliably sequenced with at least 10 coverage. CES data summary is presented in
Fig. 1. Patient's facial characteristics from infancy till 8 years of age.
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Table 1 Next-generation sequencing summary for the trio.
RefSeq coding regions ± 2 bp average coverage RefSeq coding regions ± 2 bp with coverage 10 Total number of SNPs Total number of INDELs No. de novo variantsa No. homozygous variantsa,b No. genes with compound heterozygous variantsa No. hemizygous variantsa
Proband
Father
Mother
165 95% 20,883 1141 2 0 2 2
170 95% 20,861 1124 e e e e
153 95% 21,019 1109 e e e e
a Only variants with QUAL >500 with population allele frequency <1% were counted (Strom et al., 2014). b Variants also homozygous in the parent(s) or control individuals were not included.
Table 1. Two variants of interest have been identified and the data summary is presented in Table 2. Hemizygous and compound heterozygous variants were inherited from the parents and involved only non-clinical genes. Characteristics of the variants are summarized in Tables 3 and 4. 5. Discussion Over the past decade, remarkable advances have been made in our understanding of the genetic causes of many genetic syndromes associated with seizures, and mutations in ion channel and neuroreceptor genes have been identified. EIEE presents shortly after birth with frequent, severe seizures, a burst-suppression EEG pattern, and progressive disturbance of cerebral function. This group of disorders is genetically heterogenous. In individuals with EIEE, mutations have been identified in several genes including sodium channel, neuronal type a1 subunit (SCN1A) (Marini et al., 2009), syntaxin binding protein 1 (STXBP1) (Saitsu et al., 2008), Aristaless related homeobox (ARX) (Kato et al., 2007), cyclindependent kinase-like 5 (CDKL5) (Nemos et al., 2009), solute carrier family 25 member 22 (SLC25A22) (Molinari et al., 2009), and others (Tavyev Asher and Scaglia, 2012). Recently, mutations in KCNQ2 as a cause of EIEE have received attention (Kato et al., 2007). In many cases of EIEE genetic cause remains unknown. This is the first case of EIEE associated with a de novo heterozygous variant in ZEB2 that was identified by clinical exome sequencing. ZEB2, the zinc finger E-box binding homeobox 2 gene (previously annotated as ZFHX1B or SIP1) is a known disease-causing gene linked to MowateWilson syndrome (MWS) which is characterized by a distinctive facial appearance, usually severe intellectual disability, and variable associated features including agenesis of the corpus callosum, hypospadias, cardiac defects, and Hirschsprung
disease (Mowat et al., 2003; da Paz et al., 2015). Epilepsy is one of the main features of MWS, with a prevalence of 70e75%. The electroclinical phenotype of individuals with MWS has been described. In these patients, epilepsy is usually characterized by frontal lobe and atypical absence seizures with age-dependent EEG pattern with frequent diffuse frontal dominant spike and wave discharges during awake state and a near to continuous spike and wave activity during slow sleep (Cordelli et al., 2013). In our case, the patient presented with EIEE and profound developmental delay, but his EEG showed more severe epileptiform abnormalities in the right posterior distribution. Moreover, he lacked the typical facial gestalt of MWS, agenesis of the corpus callosum, conotruncal heart defects, urogenital malformations or Hirschsprung disease to support the diagnosis of MWS. In individuals with MWS the majority of mutations lead to haploinsufficiency through premature stop codons, small or large gene deletions (Dastot-Le Moal et al., 2007). Although more than 220 different ZEB2 mutations have been identified (Dastot-Le Moal et al., 2007; Garavelli et al., 2009), to date, only six individuals with de novo heterozygous missense ZEB2 mutations have been reported. In three patients, missense mutations were in the C-ZF cluster of ZEB2: c.3134A>G p.His1045Arg was located in the second C-ZF domain, and c3164A>G p.Tyr1055Cys and c.3211T>C p.Ser1071Pro were located in the third C-ZF domain. These patients' phenotype included the facial gestalt of MWS and moderate intellectual disability, but no microcephaly, heart defects or Hirschsprung disease. Functional studies confirmed mutation-dependent, embryo rescue, correlating with the severity of the patients' phenotype (Ghoumid et al., 2013). Three other missense mutations (c.1597G>C p.Val533Leu (DastotLe Moal et al., 2007), c. 2857A>G p.Arg953Gly (Gregory-Evans et al., 2004), and c.3356A>G p.Gln1119Arg (Heinritz et al., 2006)) were localized outside a known functional domain of ZEB2. In all three cases, these patients had Hirschsprung disease, the first patient died at 3 years of age, the second also had Down syndrome and the third had additional features including cleft lip, cleft palate and brachytelephalangy. In our patient his missense variant also falls outside the known functional domains, but his phenotype is very different from reported individuals with ZEB2 mutations (Fig. 2). ZEB2 encodes the Smad Interacting Protein 1, which is involved in the TGF-b/BMP/Smad signaling cascade (Verschueren et al., 1999; Postigo et al., 2003). ZEB2 mRNA is expressed during early embryogenesis in most human tissues and plays an important role in neural crest cell migration (Van de Putte et al., 2003). More recent data demonstrate involvement of ZEB2 in the regulation of corticogenesis (Seuntjens et al., 2009). The mechanism of epilepsy in individuals with ZEB2 mutations is not well understood. Usually,
Table 2 Variants of interests. Genomic position (hg19)
Chr2:145154114
Chr7:147336360
Gene Reference allele (REF) Alternate allele (ALT) No. reads with REF in proband No. reads with ALT in proband No. reads with REF in mother No. reads with ALT in mother No. reads with REF in father No. reads with ALT in father Mutation type DNA change Protein change Prediction Sanger verification
ZEB2 C A 103 101 176 0 196 0 missense NM_014795.3: c.2932G>T p.Asp978Tyr Likely pathogenic Yes
CNTNAP2 C T 77 79 132 0 142 0 missense NM_014141.5:c.2060C>T p.Ser687Phe Variant of uncertain significance (VUS) Yes
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Table 3 Hemizygous variants. Genomic position (hg19)
X:40523654
X:47088020
Gene Reference allele (REF) Alternate allele (ALT) No. reads with REF in proband No. reads with ALT in proband No. reads with REF in mother No. reads with ALT in mother No. reads with REF in father No. reads with ALT in father Mutation type DNA change Protein change Prediction Genomic position (hg19)
MED14 G T 0 28 54 37 37 0 missense NM_004229.3: c.3353C>A p.Pro1118His VUS 3:184102470
CDK16 G C 0 78 84 62 88 0 missense NM_033018.3:c.1448G>C p.Arg483Pro VUS 3:184103913
Table 4 Compound heterozygous variants. Genomic position (hg19)
3:184102470
3:184103913
4:110763634
4:110765244
Gene Reference allele (REF) Alternate allele (ALT) No. reads with REF in proband No. reads with ALT in proband No. reads with REF in mother No. reads with ALT in mother No. reads with REF in father No. reads with ALT in father Mutation type DNA change Protein change Prediction
CHRD C T 41 52 30 31 78 0 missense NM_003741.2:c.1586C>T p.Ala529Val VUS
CHRD C T 37 31 89 0 42 38 missense NM_003741.2:c.1898C>T p.Thr633Ile VUS
RRH AT A 111 76 225 0 104 65 frameshift NM_006583.2:c.731delT p.Ile244ThrfsX2 VUS
RRH G A 137 94 146 104 250 0 missense NM_006583.2:c.905G>A p.Arg302Gln VUS
Fig. 2. Schematic representation of ZEB2 protein structure. The functional domains of ZEB2 and the corresponding amino acid positions (in brackets) are represented in the scheme: NIM (nucleosome remodeling and deacetylase-interaction motif), N-ZF (Nterminal zinc-finger cluster), SBD (Smad-binding domain), HD (homeodomain), CID (CtBP-interacting domain) and C-ZF (C-terminal zinc-finger cluster). Star indicates the localization of the missense mutation in our patient. (Hs) is the human amino acid sequence with the corresponding positions indicated in brackets (modified Ghoumid et al., 2013).
there is no evidence of cortical malformations on the brain imaging, but agenesis of the corpus callosum is quite frequent (Yamada et al., 2014). Studies by McKinsey and Van den Berghe revealed the influence of ZEB2 on the neurogenesis of cortical g-aminobutyric acid (GABA)ergic interneurons. Lack of ZEB2 prevents the repression of NKX2-1 homeobox transcription factor, the expression of which induces the differentiation of progenitor cells into striatal interneurons rather than cortical neurons (McKinsey et al., 2013; Van den Berghe et al., 2013). Deficit of GABAergic inhibition is thought to result in focal and absence seizures (Yalcin, 2012). In this context, this hypothesis might be an explanation for the mechanism of seizures in individuals with ZEB2 mutations. Our patient also had a heterozygous de novo variant in CNTNAP2 gene. Defects in CNTNAP2 gene are reported to cause cortical dysplasia-focal epilepsy syndrome (Strauss et al., 2006) with an
autosomal recessive mode of inheritance and may influence sus~ agarikano et al., ceptibility to autism (Bakkaloglu et al., 2008; Pen 2011). Considering that the CNTNAP2 variant found in this patient was heterozygous, with no evidence of cortical dysplasia, the de novo variant found in ZEB2 was predicted to be the causal variant. However, the possibility of a synergistic effect of heterozygous mutations in ZEB2 and CNTNAP2 variants could not be excluded. Ion channel gene mutations in SCN1A can lead to a range of severity in the epileptic phenotype ranging from GEFSþ to Dravet syndrome, depending upon the extent of loss of function in the GABAergic interneurons. Recent findings of KCNQ2 associated severe EIEE phenotype, similarly, contrasts with our association of the mild loss of function mutations with benign familial neonatal convulsions. The possible involvement of GABAergic deficit as an etiological factor for EIEE in our case can be compared to epileptic encephalopathies associated with ARX mutations that affect, among other things, tangential migration of GABAergic interneurons. While these explanations center around the inhibitory role of GABA, in the early developing brain GABA provides for the fast excitation that is important in driving neuronal migration, among other developmental cues. It is not clear if our patient may also have widespread cortical dysplasia that is too subtle to be discerned in imaging studies, nevertheless contributing to EIEE phenotype. In conclusion, this unique case expands the range of phenotypes associated with variants in ZEB2, and further studies are needed to confirm this observation, to determine the genotype/phenotype correlations, and to identify the mechanism for EIEE. Functional studies are needed to confirm the causality of the reported variant, to determine the mechanism, and to delineate possible genotypeephenotype correlation. From a practical standpoint, improved
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understanding of the mechanism of epilepsy in individuals with ZEB2 mutations might guide the pharmacotherapy. References Bakkaloglu, B., O'Roak, B.J., Louvi, A., Gupta, A.R., Abelson, J.F., Morgan, T.M., et al., 2008. Molecular cytogenetic analysis and resequencing of contactin associated protein-like 2 in autism spectrum disorders. Am. J. Hum. Genet. 82 (1), 165e173. Van den Berghe, V., Stappers, E., Vandesande, B., Dimidschstein, J., Kroes, R., Francis, A., et al., 2013. Directed migration of cortical interneurons depends on the cell-autonomous action of Sip1. Neuron 77, 70e82. Cordelli, D.M., Garavelli, L., Savasta, S., Guerra, A., Pellicciari, A., Giordano, L., et al., 2013. Epilepsy in MowateWilson syndrome: delineation of the electroclinical phenotype. Am. J. Med. Genet. 161A, 273e284. Dastot-Le Moal, F., Wilson, M., Mowat, D., Collot, N., Niel, F., Goossens, M., 2007. ZFHX1B mutations in patients with MowateWilson syndrome. Hum. Mutat. 28, 313e321. Garavelli, L., Zollino, M., Mainardi, P.C., Gurrieri, F., Rivieri, F., Soli, F., et al., 2009. MowateWilson syndrome: facial phenotype changing with age: study of 19 Italian patients and review of the literature. Am. J. Med. Genet. 149A, 417e426. Ghoumid, J., Drevillon, L., Alavi-Naini, S.M., Bondurand, N., Rio, M., BriandSuleau, A., et al., 2013. ZEB2 zinc-finger missense mutations lead to hypomorphic alleles and a mild MowateWilson syndrome, 1 Hum. Mol. Genet. 22 (13), 2652e2661. Gregory-Evans, C.Y., Vieira, H., Dalton, R., Adams, G.G., Salt, A., Gregory-Evans, K., 2004. Ocular coloboma and high myopia with Hirschsprung disease associated with a novel ZFHX1B missense mutation and trisomy 21. Am. J. Med. Genet. 131, 86e90. Heinritz, W., Zweier, C., Froster, U.G., Strenge, S., Kujat, A., Syrbe, S., et al., 2006. A missense mutation in the ZFHX1B gene associated with an atypical MowateWilson syndrome phenotype, 1 Am. J. Med. Genet. A 140 (11), 1223e1227. Kato, M., Saitoh, S., Kamei, A., Shiraishi, H., Ueda, Y., Akasaka, M., et al., 2007. A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome). Am. J. Hum. Genet. 81 (2), 361e366. Marini, C., Scheffer, I.E., Nabbout, R., Mei, D., Cox, K., Dibbens, L.M., et al., 2009. SCN1A duplications and deletions detected in Dravet syndrome: implications for molecular diagnosis. Epilepsia 50 (7), 1670e1678. McKinsey, G.L., Lindtner, S., Trzcinski, B., Visel, A., Pennacchio, L.A., Huylebroeck, D., et al., 2013. Dlx1&2-dependent expression of Zfhx1b (Sip1, Zeb2) regulates the fate switch between cortical and striatal interneurons. Neuron 77, 83e98. Molinari, F., Kaminska, A., Fiermonte, G., Boddaert, N., Raas-Rothschild, A., Plouin, P., et al., 2009. Mutations in the mitochondrial glutamate carrier SLC25A22 in neonatal epileptic encephalopathy with suppression bursts. Clin. Genet. 76 (2), 188e194. Mowat, D.R., Wilson, M.J., Goossens, M., 2003. MowateWilson syndrome. Med. Genet. 40, 305e310.
Nemos, C., Lambert, L., Giuliano, F., Doray, B., Roubertie, A., Goldenberg, A., et al., 2009. Mutational spectrum of CDKL5 in early-onset encephalopathies: a study of a large collection of French patients and review of the literature. Clin. Genet. 76 (4), 357e371. Noh, G.J., Tavyev Asher, Y., Graham Jr., J.M., 2012. Clinical review of genetic epileptic encephalopathies. Eur. J. Med. Genet. 55 (5), 281e298. Pal, D.K., Pong, A.W., Chung, W.K., 2010. Genetic evaluation and counseling for epilepsy. Nature Reviews. Neurol. 6 (8), 445e453. da Paz, J.A., Kim, C.A., Goossens, M., Giurgea, I., Marques-Dias, M.J., 2015 Jan. Mowat-Wilson syndrome: neurological and molecular study in seven patients. Arq. Neuropsiquiatr. 73 (1), 12e17. ~ agarikano, O., Abrahams, B.S., Herman, E.I., Winden, K.D., Gdalyahu, A., Dong, H., Pen et al., 2011. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits. Cell 147 (1), 235e246. Postigo, A.A., Depp, J.L., Taylor, J.J., Kroll, K.L., 2003. Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. EMBO J. 22, 2453e2462. Van de Putte, T., Maruhashi, M., Francis, A., Nelles, L., Kondoh, H., Huylebroeck, D., Higashi, Y., 2003. Mice lacking ZFHX1B, the gene that codes for Smadinteracting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental retardation syndrome. Am. J. Hum. Genet. 72, 465e470. Saitsu, H., Kato, M., Mizuguchi, T., Hamada, K., Osaka, H., Tohyama, J., et al., 2008. De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy. Nat. Genet. 40 (6), 782e788. Seuntjens, E., Nityanandam, A., Miquelajauregui, A., Debruyn, J., Stryjewska, A., Goebbels, S., et al., 2009. Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors. Nat. Neurosci. 12, 1373e1380. Strauss, K.A., Puffenberger, E.G., Huentelman, M.J., Gottlieb, S., Dobrin, S.E., Parod, J.M., et al., 2006. Recessive symptomatic focal epilepsy and mutant contactin-associated protein-like 2, 30 N. Engl. J. Med. 354 (13), 1370e1377. Strom, S.P., Lee, H., Das, K., Vilain, E., Nelson, S.F., Grody, W.W., Deignan, J.L., 2014. Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory. Genet. Med. 16 (7), 510e515. Tavyev Asher, Y.J., Scaglia, F., 2012 May. Molecular bases and clinical spectrum of early infantile epileptic encephalopathies. Eur. J. Med. Genet. 55 (5), 299e306. http://dx.doi.org/10.1016/j.ejmg.2012.04.002. Verschueren, K., Remacle, J.E., Collart, C., Kraft, H., Baker, B.S., Tylzanowski, P., et al., 1999. SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5-CACCT sequences in candidate target genes. J. Biol. Chem. 274, 20489e20498. Yalcın, O., 2012. Genes and molecular mechanisms involved in the epileptogenesis of idiopathic absence epilepsies. Seizure 21, 79e86. Yamada, Y., Nomura, N., Yamada, K., Matsuo, M., Suzuki, Y., Sameshima, K., et al., 2014. The spectrum of ZEB2 mutations causing the MowateWilson syndrome in Japanese populations. Am. J. Med. Genet. A. http://dx.doi.org/10.1002/ ajmg.a.36551.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Early Infantile Epileptic Encephalopathy with a de novo variant in ZEB2 identified by exome sequencing Natalia Babkina a, b, *, Joshua L. Deignan c, Hane Lee c, Eric Vilain b, d, Raman Sankar e, Irina Giurgea f, David Mowat g, John M. Graham Jr. a, b, h a
Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA Department of Pediatrics, Division of Medical Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA d Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA e Department of Neurology, Pediatrics and Children's Discovery and Innovation Institute at Mattel Children's Hospital, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA f ^pital Henri Mondor, 94000 Cr Service de Biochimie G en etique, INSERM U955 Equipe 11, Ho eteil, France g Department of Medical Genetics, Sydney Children's Hospital, School of Women's and Children's Health, University of New South Wales, Australia h Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA 90502, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2015 Received in revised form 8 November 2015 Accepted 19 December 2015 Available online 22 December 2015
Early Infantile Epileptic Encephalopathy (EIEE) presents shortly after birth with frequent, severe seizures, a burst-suppression EEG pattern, and progressive disturbance of cerebral function. We present a case of EIEE associated with a de novo missense variant in ZEB2. Heterozygous truncating mutations or deletions in ZEB2 are known to cause Mowat-Wilson syndrome (MWS), which is characterized by seizures with onset in the second year of life, distinctive dysmorphic facial features and malformations that were absent in this patient. This unique case expands the range of phenotypes associated with variants in ZEB2 and indicates that this gene should be included in the molecular investigation of EIEE cases. © 2016 Published by Elsevier Masson SAS.
Keywords: Infantile Epileptic Encephalopathy (EIEE) ZEB2 MowateWilson syndrome (MWS) Seizures Infantile spasms Burst-suppression EEG pattern Developmental delay Cortical gray and white matter atrophy Clinical exome sequencing
1. Introduction Seizure disorder is a frequent finding in individuals seeking a medical genetics consultation. The etiology of seizures is very diverse and more than half of epilepsies reportedly have some genetic basis (Pal et al., 2010). EIEE is a group of disorders that present shortly after birth with frequent and severe seizures, a burst-suppression EEG pattern, and progressive disturbance of cerebral function. EIEE includes Dravet, Ohtahara, and West syndromes, but in many cases, the genetic cause remains unknown
* Corresponding author. Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. E-mail address: [email protected] (N. Babkina). http://dx.doi.org/10.1016/j.ejmg.2015.12.006 1769-7212/© 2016 Published by Elsevier Masson SAS.
(Noh et al., 2012). Identification of the genetic cause is valuable not only for diagnosis but also for guiding the therapy. Application of exome sequencing has enabled identification of genetic causes of many new syndromes and has expanded the phenotypes of known genetic disorders. We present a case of EIEE associated with a de novo heterozygous variant in ZEB2 that was identified by clinical exome sequencing. 2. Clinical description We describe a case of an eight-year-old Ashkenazi Jewish boy with severe EIEE and profound developmental delay. The patient was born at 39 weeks to a 42-year-old mother and a 36-year-old father. His growth and head circumference were appropriate for gestational age. The family history was negative for any known
N. Babkina et al. / European Journal of Medical Genetics 59 (2016) 70e74
genetic disorders, intellectual disability or seizures. The pregnancy was uncomplicated, prenatal serum screening was negative, chorionic villus sampling revealed normal male karyotype, and second trimester anatomy ultrasound was negative for structural abnormalities. Shortly after birth this neonate developed seizures and was diagnosed with EIEE at the age of 5 weeks, followed by infantile spasms with a burst-suppression EEG pattern. His physical examination was significant for postnatal-onset microcephaly (10th centile at birth and 4SD by age 4 months), short stature (6SD at 8.5 years), coarse facial features due to prolonged use of various antiepileptic drugs, and lack of distinctive dysmorphic features (Fig. 1). The patient had severe developmental delay, feeding problems and failure to thrive. Seizures were refractory to treatment and multiple antiepileptic medications were required. EEG was significant for background slowing with predominant delta frequencies and more significant involvement of the right hemisphere. MRI showed significant cortical gray and white matter atrophy and hypomyelination in the supratentorial regions, but no abnormalities of the corpus callosum. MR spectroscopy revealed increased glutamine/glutamate complex, decreased NAA/creatine ratio, decreased choline/creatine ratio consistent with hypomyelination, normal myoinositol peak, and no definite lactate peak. There was no evidence of focal cortical dysplasia. Metabolic evaluation included plasma amino acids, urine organic acids, plasma acylcarnitine profile, biotinidase, long chain fatty acids, CSF neurotransmitters, and enzyme essays for GM1 gangliosidosis, beta mannosidosis, Tay-Sachs, and Krabbe and these studies were all negative. Conjunctival biopsy was negative for storage. SNP microarray was performed using the Affymetrix Cytoscan HD using 743,000 SNP probes and 1,953,000 NPCN probes with a median spacing of 0.86 kb within genes. Microarray was negative for copy number variants or regions of homozygosity to suggest consanguinity. GLRA1 and MECP2 sequence analyses were normal.
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3. Methods Clinical Exome Sequencing (CES) was performed at the UCLA Clinical Genomics Center using clinically validated protocols from DNA extraction to variant analysis (Strom et al., 2014) and interpretation. The proband and both parents were sequenced simultaneously. Genomic DNA was extracted from blood specimen using standard methods. CES was performed using customized Agilent SureSelect Human All Exon 50 Mb kit for exome capture and an Illumina Hiseq2000 for sequencing as 50 bp paired end run. In total ~15 Gb of sequence was generated and uniquely aligned to the human reference genome, generating a mean coverage of 165 per base within Refseq protein coding bases of the human genome. All variants were annotated using annotation tool Variant Annotator X that retrieves information from databases such as OMIM, UniProt, NHLBI Exome Sequencing Project (ESP), SIFT and PolyPhen2 for each variant. All genes harboring de novo, homozygous or compound heterozygous variants with allele frequencies <1% in the general population [estimated using data from the 1000 Genomes Project (1 Kg), ESP, NIEHS project and HapMap] were evaluated by a Genomics Data Board consisting of physicians, pathologists, clinical geneticists, laboratory directors, genetic counselors, and informatics specialists. Clinically significant variants were confirmed using Sanger sequencing of the proband and both parents. 4. Results In total 22,024 DNA variants were identified across the exome, including 20,883 single nucleotide substitutions and 1141 small deletions/insertions (1e10 bp). The data were consistent with a high quality genomic sequence and fell within normal human genomic variation quality parameters. It was estimated that about 95% of the known disease-causing bases were reliably sequenced with at least 10 coverage. CES data summary is presented in
Fig. 1. Patient's facial characteristics from infancy till 8 years of age.
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Table 1 Next-generation sequencing summary for the trio.
RefSeq coding regions ± 2 bp average coverage RefSeq coding regions ± 2 bp with coverage 10 Total number of SNPs Total number of INDELs No. de novo variantsa No. homozygous variantsa,b No. genes with compound heterozygous variantsa No. hemizygous variantsa
Proband
Father
Mother
165 95% 20,883 1141 2 0 2 2
170 95% 20,861 1124 e e e e
153 95% 21,019 1109 e e e e
a Only variants with QUAL >500 with population allele frequency <1% were counted (Strom et al., 2014). b Variants also homozygous in the parent(s) or control individuals were not included.
Table 1. Two variants of interest have been identified and the data summary is presented in Table 2. Hemizygous and compound heterozygous variants were inherited from the parents and involved only non-clinical genes. Characteristics of the variants are summarized in Tables 3 and 4. 5. Discussion Over the past decade, remarkable advances have been made in our understanding of the genetic causes of many genetic syndromes associated with seizures, and mutations in ion channel and neuroreceptor genes have been identified. EIEE presents shortly after birth with frequent, severe seizures, a burst-suppression EEG pattern, and progressive disturbance of cerebral function. This group of disorders is genetically heterogenous. In individuals with EIEE, mutations have been identified in several genes including sodium channel, neuronal type a1 subunit (SCN1A) (Marini et al., 2009), syntaxin binding protein 1 (STXBP1) (Saitsu et al., 2008), Aristaless related homeobox (ARX) (Kato et al., 2007), cyclindependent kinase-like 5 (CDKL5) (Nemos et al., 2009), solute carrier family 25 member 22 (SLC25A22) (Molinari et al., 2009), and others (Tavyev Asher and Scaglia, 2012). Recently, mutations in KCNQ2 as a cause of EIEE have received attention (Kato et al., 2007). In many cases of EIEE genetic cause remains unknown. This is the first case of EIEE associated with a de novo heterozygous variant in ZEB2 that was identified by clinical exome sequencing. ZEB2, the zinc finger E-box binding homeobox 2 gene (previously annotated as ZFHX1B or SIP1) is a known disease-causing gene linked to MowateWilson syndrome (MWS) which is characterized by a distinctive facial appearance, usually severe intellectual disability, and variable associated features including agenesis of the corpus callosum, hypospadias, cardiac defects, and Hirschsprung
disease (Mowat et al., 2003; da Paz et al., 2015). Epilepsy is one of the main features of MWS, with a prevalence of 70e75%. The electroclinical phenotype of individuals with MWS has been described. In these patients, epilepsy is usually characterized by frontal lobe and atypical absence seizures with age-dependent EEG pattern with frequent diffuse frontal dominant spike and wave discharges during awake state and a near to continuous spike and wave activity during slow sleep (Cordelli et al., 2013). In our case, the patient presented with EIEE and profound developmental delay, but his EEG showed more severe epileptiform abnormalities in the right posterior distribution. Moreover, he lacked the typical facial gestalt of MWS, agenesis of the corpus callosum, conotruncal heart defects, urogenital malformations or Hirschsprung disease to support the diagnosis of MWS. In individuals with MWS the majority of mutations lead to haploinsufficiency through premature stop codons, small or large gene deletions (Dastot-Le Moal et al., 2007). Although more than 220 different ZEB2 mutations have been identified (Dastot-Le Moal et al., 2007; Garavelli et al., 2009), to date, only six individuals with de novo heterozygous missense ZEB2 mutations have been reported. In three patients, missense mutations were in the C-ZF cluster of ZEB2: c.3134A>G p.His1045Arg was located in the second C-ZF domain, and c3164A>G p.Tyr1055Cys and c.3211T>C p.Ser1071Pro were located in the third C-ZF domain. These patients' phenotype included the facial gestalt of MWS and moderate intellectual disability, but no microcephaly, heart defects or Hirschsprung disease. Functional studies confirmed mutation-dependent, embryo rescue, correlating with the severity of the patients' phenotype (Ghoumid et al., 2013). Three other missense mutations (c.1597G>C p.Val533Leu (DastotLe Moal et al., 2007), c. 2857A>G p.Arg953Gly (Gregory-Evans et al., 2004), and c.3356A>G p.Gln1119Arg (Heinritz et al., 2006)) were localized outside a known functional domain of ZEB2. In all three cases, these patients had Hirschsprung disease, the first patient died at 3 years of age, the second also had Down syndrome and the third had additional features including cleft lip, cleft palate and brachytelephalangy. In our patient his missense variant also falls outside the known functional domains, but his phenotype is very different from reported individuals with ZEB2 mutations (Fig. 2). ZEB2 encodes the Smad Interacting Protein 1, which is involved in the TGF-b/BMP/Smad signaling cascade (Verschueren et al., 1999; Postigo et al., 2003). ZEB2 mRNA is expressed during early embryogenesis in most human tissues and plays an important role in neural crest cell migration (Van de Putte et al., 2003). More recent data demonstrate involvement of ZEB2 in the regulation of corticogenesis (Seuntjens et al., 2009). The mechanism of epilepsy in individuals with ZEB2 mutations is not well understood. Usually,
Table 2 Variants of interests. Genomic position (hg19)
Chr2:145154114
Chr7:147336360
Gene Reference allele (REF) Alternate allele (ALT) No. reads with REF in proband No. reads with ALT in proband No. reads with REF in mother No. reads with ALT in mother No. reads with REF in father No. reads with ALT in father Mutation type DNA change Protein change Prediction Sanger verification
ZEB2 C A 103 101 176 0 196 0 missense NM_014795.3: c.2932G>T p.Asp978Tyr Likely pathogenic Yes
CNTNAP2 C T 77 79 132 0 142 0 missense NM_014141.5:c.2060C>T p.Ser687Phe Variant of uncertain significance (VUS) Yes
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Table 3 Hemizygous variants. Genomic position (hg19)
X:40523654
X:47088020
Gene Reference allele (REF) Alternate allele (ALT) No. reads with REF in proband No. reads with ALT in proband No. reads with REF in mother No. reads with ALT in mother No. reads with REF in father No. reads with ALT in father Mutation type DNA change Protein change Prediction Genomic position (hg19)
MED14 G T 0 28 54 37 37 0 missense NM_004229.3: c.3353C>A p.Pro1118His VUS 3:184102470
CDK16 G C 0 78 84 62 88 0 missense NM_033018.3:c.1448G>C p.Arg483Pro VUS 3:184103913
Table 4 Compound heterozygous variants. Genomic position (hg19)
3:184102470
3:184103913
4:110763634
4:110765244
Gene Reference allele (REF) Alternate allele (ALT) No. reads with REF in proband No. reads with ALT in proband No. reads with REF in mother No. reads with ALT in mother No. reads with REF in father No. reads with ALT in father Mutation type DNA change Protein change Prediction
CHRD C T 41 52 30 31 78 0 missense NM_003741.2:c.1586C>T p.Ala529Val VUS
CHRD C T 37 31 89 0 42 38 missense NM_003741.2:c.1898C>T p.Thr633Ile VUS
RRH AT A 111 76 225 0 104 65 frameshift NM_006583.2:c.731delT p.Ile244ThrfsX2 VUS
RRH G A 137 94 146 104 250 0 missense NM_006583.2:c.905G>A p.Arg302Gln VUS
Fig. 2. Schematic representation of ZEB2 protein structure. The functional domains of ZEB2 and the corresponding amino acid positions (in brackets) are represented in the scheme: NIM (nucleosome remodeling and deacetylase-interaction motif), N-ZF (Nterminal zinc-finger cluster), SBD (Smad-binding domain), HD (homeodomain), CID (CtBP-interacting domain) and C-ZF (C-terminal zinc-finger cluster). Star indicates the localization of the missense mutation in our patient. (Hs) is the human amino acid sequence with the corresponding positions indicated in brackets (modified Ghoumid et al., 2013).
there is no evidence of cortical malformations on the brain imaging, but agenesis of the corpus callosum is quite frequent (Yamada et al., 2014). Studies by McKinsey and Van den Berghe revealed the influence of ZEB2 on the neurogenesis of cortical g-aminobutyric acid (GABA)ergic interneurons. Lack of ZEB2 prevents the repression of NKX2-1 homeobox transcription factor, the expression of which induces the differentiation of progenitor cells into striatal interneurons rather than cortical neurons (McKinsey et al., 2013; Van den Berghe et al., 2013). Deficit of GABAergic inhibition is thought to result in focal and absence seizures (Yalcin, 2012). In this context, this hypothesis might be an explanation for the mechanism of seizures in individuals with ZEB2 mutations. Our patient also had a heterozygous de novo variant in CNTNAP2 gene. Defects in CNTNAP2 gene are reported to cause cortical dysplasia-focal epilepsy syndrome (Strauss et al., 2006) with an
autosomal recessive mode of inheritance and may influence sus~ agarikano et al., ceptibility to autism (Bakkaloglu et al., 2008; Pen 2011). Considering that the CNTNAP2 variant found in this patient was heterozygous, with no evidence of cortical dysplasia, the de novo variant found in ZEB2 was predicted to be the causal variant. However, the possibility of a synergistic effect of heterozygous mutations in ZEB2 and CNTNAP2 variants could not be excluded. Ion channel gene mutations in SCN1A can lead to a range of severity in the epileptic phenotype ranging from GEFSþ to Dravet syndrome, depending upon the extent of loss of function in the GABAergic interneurons. Recent findings of KCNQ2 associated severe EIEE phenotype, similarly, contrasts with our association of the mild loss of function mutations with benign familial neonatal convulsions. The possible involvement of GABAergic deficit as an etiological factor for EIEE in our case can be compared to epileptic encephalopathies associated with ARX mutations that affect, among other things, tangential migration of GABAergic interneurons. While these explanations center around the inhibitory role of GABA, in the early developing brain GABA provides for the fast excitation that is important in driving neuronal migration, among other developmental cues. It is not clear if our patient may also have widespread cortical dysplasia that is too subtle to be discerned in imaging studies, nevertheless contributing to EIEE phenotype. In conclusion, this unique case expands the range of phenotypes associated with variants in ZEB2, and further studies are needed to confirm this observation, to determine the genotype/phenotype correlations, and to identify the mechanism for EIEE. Functional studies are needed to confirm the causality of the reported variant, to determine the mechanism, and to delineate possible genotypeephenotype correlation. From a practical standpoint, improved
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