- Email: [email protected]
PCDH19-related epilepsy in a male with Klinefelter syndrome: Additional evidence supporting PCDH19 cellular interference disease mechanism
Epilepsy Research 145 (2018) 89–92
Contents lists available at ScienceDirect
Epilepsy Research journal homepage: www.elsevier.com/locate/epilepsyres
PCDH19-related epilepsy in a male with Klinefelter syndrome: Additional evidence supporting PCDH19 cellular interference disease mechanism
T
Edward J. Romaskoa, Elizabeth T. DeChenea, Jorune Balciunienea, Gozde T. Akgumusa, Ingo Helbigb,c, Jennifer M. Tarpiniand, Beth A. Keenad, Maria G. Vogiatzic,e, Elaine H. Zackaic,d, ⁎ ⁎ Kosuke Izumia,d, Shavonne L. Masseyb,c, , Ahmad N. Abou Tayouna,f, ,1 a
Division of Genomic Diagnostics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States c Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, United States d Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States e Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Philadelphia, PA, United States f Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States b
A R T I C LE I N FO
A B S T R A C T
Keywords: PCDH19 Klinefelter syndrome Epilepsy Molecular diagnostics
Heterozygous de novo or inherited pathogenic variants in the PCDH19 gene cause a spectrum of neurodevelopmental features including developmental delay and seizures. PCDH19 epilepsy was previously known as “epilepsy and mental retardation limited to females”, since the condition almost exclusively affects females. It is hypothesized that the co-existence of two populations of neurons, some with and some without PCDH19 protein expression, results in pathologically abnormal interactions between these neurons, a mechanism also referred to as cellular interference. Consequently, PCDH19-related epilepsies are inherited in an atypical X-linked pattern, such that hemizygous, non-mosaic, 46,XY males are typically unaffected, while individuals with a diseasecausing PCDH19 variant, mainly heterozygous females and mosaic males, are affected. As a corollary to this hypothesis, an individual with Klinefelter syndrome (KS) (47,XXY) who has a heterozygous disease-causing PCDH19 variant should develop PCDH19-related epilepsy. Here, we report such evidence: - a male child with KS and PCDH19-related epilepsy – supporting the PCDH19 cellular interference disease hypothesis.
1. Introduction PCDH19-related epilepsy, caused by pathogenic variants in the PCDH19 gene, has a highly variable clinical presentation often characterized by infantile or early childhood onset intractable seizures that cluster and are fever-sensitive. Seizure types vary but include generalized tonic, clonic or tonic-clonic, myoclonic and/or focal seizures. Seizure frequency decreases with age, and patients may become seizure-free during childhood or adolescence. Most affected individuals have developmental delay, mild to severe intellectual disability, autism, and/or behavioral issues; however, some patients have normal cognition. The phenotype, including the degree of intellectual and behavioral problems, can overlap with Dravet syndrome, however, PCDH19-related epilepsy is associated with later seizure onset, lack of photosensitivity, increased seizure cluster frequency, and good response to high-dose steroid treatment for clusters of seizures (Depienne and LeGuern, 2012; Depienne et al., 2011; Higurashi et al., 2015; Steel ⁎
1
et al., 2017; Trivisano et al., 2016; van Harssel et al., 2013). The PCDH19 gene on chromosome Xq22.3 encodes protocadherin 19, which is postulated to play a role in mediating calcium-dependent cell–cell adhesion and regulating the organization of the developing brain (Depienne and LeGuern, 2012). It is hypothesized that co-existence of two neuron populations, one expressing PCDH19 while the other is deficient for this protein, results in abnormal interactions between these neurons, a process referred to as cellular interference (Depienne et al., 2009; Depienne and LeGuern, 2012). Although unproven, this hypothesis could explain why PCDH19 variants are pathogenic in individuals with two different populations of neurons (e.g., females with random X-inactivation and mosaic males) but not in hemizygous males who only have one population (de Lange et al., 2017; Depienne et al., 2009). Similarly, this hypothesis would suggest that an individual with a sex chromosome abnormality leading to multiple X chromosomes (e.g. Klinefelter syndrome (KS) (47,XXY) and triple X syndrome (47,XXX)) with disease-causing PCDH19 variants would also
Corresponding authors at: 3401 Civic Center Blvd., Philadelphia, PA 19104-4399, United States. E-mail addresses: [email protected] (S.L. Massey), [email protected] (A.N.A. Tayoun). Current Address: Al Jalila Children's Specialty Hospital, Dubai, UAE.
https://doi.org/10.1016/j.eplepsyres.2018.06.008 Received 12 January 2018; Received in revised form 7 April 2018; Accepted 14 June 2018 0920-1211/ © 2018 Elsevier B.V. All rights reserved.
Epilepsy Research 145 (2018) 89–92
E.J. Romasko et al.
EEG activity demonstrated generalized diffuse background slowing for age. No interictal abnormalities were present. Ictal activity demonstrated electroclinical seizures with right temporal and/or right parietal-temporal onset which evolved into high amplitude, rhythmic spike and wave discharges over the entire right hemisphere. Postictal slowing was often seen most prominently over the right hemisphere. Clinically, these seizures were characterized by staring and unresponsiveness. Subclinical seizures of similar right temporal and right parietal-temporal onset were also captured in sleep. Specific seizure semiologies included generalized tonic-clonic, generalized atonic, generalized tonic and focal non-motor seizures with secondary generalization characterized by staring with head version to either direction followed by whole body limpness, drooling, and generalized clonic activity. Generally, the patient's seizures were triggered by fever and illness, including two episodes of status epilepticus. His seizures had a clustering pattern with a 72 h cluster of intermittent seizures of all semiologies described above, occurring every 3–4 months. Seizures have been stable with last breakthrough seizure occurring in the setting of an illness at age 5.5 years.
develop PCDH19-related epilepsy. Here, we report a non-mosaic XXY male child with KS and PCDH19-related epilepsy. 2. Materials and methods We reviewed medical charts to determine the clinical and previous testing data for the patient, who was examined by K.I., E.H.Z., S.L.M., and M.G.V. Informed consent was obtained from the individual and legal guardians. Genomic DNA was extracted from peripheral blood and sequencing was performed by capture of the coding regions and splice sites ( ± 15 bp) of targeted genes using the Agilent SureSelect XT Clinical Research Exome kit (per manufacturer’s instructions) followed by sequencing on the Illumina HiSeq 2500 platform with 100bp pairedend reads at an average sequencing depth of 100×. Sequence variant calls were generated using a custom-built bioinformatics pipeline in 100 genes known to cause monogenic forms of epilepsy (Supplemental Table 1). Exon-level copy number variants were also called using ExomeDepth (Plagnol et al., 2012) with our internal exome cohort as a normalizing reference. Rare variants were classified according to the ACMG/AMP variant interpretation framework (Richards et al., 2015).
3.2. Genetic findings 3. Results After sequence variant calling and filtration, eight rare variants were interpreted. No copy number changes were detected. Seven of these were classified as likely benign or benign, because they were synonymous or intronic and not predicted to affect splicing, or had a minor allele frequency in the general population inconsistent with disease prevalence and penetrance. The NM_001184880.1(PCDH19):c.706C > T, p.Pro236Ser variant was detected heterozygously on the X chromosome, confirming the XXY karyotype, with read fractions of 165 and 198 for the variant (45%) and reference (55%) alleles, respectively (Fig. 2A). This variant had been previously reported as de novo in a female with epilepsy and autistic features (Specchio et al., 2011). Two variants affecting the same codon (p.Pro236Leu and p.Pro236Arg) had also been reported as de novo in a female with early infantile epileptic encephalopathy (Trump et al., 2016) and in another patient without phenotypic information [ClinVar, RCV000480604.1]. Furthermore, nearby missense variants (p.Asp230Asn, p.Asn232Ser, p.Asn234Ser) have been reported in affected individuals (Depienne and LeGuern, 2012; Trump et al., 2016). The c.706C > T p.Pro236Ser variant was not observed in the gnomAD (Genome
3.1. Clinical history A five year old male presented with intractable epilepsy and microcephaly of unknown etiology (Fig. 1). Prenatal history was unremarkable except for maternal cervical incompetence. The proband was born at 35 weeks via vaginal delivery weighing 5 lbs 15 oz (75th percentile for 35 weeks gestation) and went to the well-baby nursery after delivery. Developmental history included sitting at normal time and walking by 12 months. Speech and language development were delayed, and he began receiving occupational therapy, physical therapy, and speech services from 18 months old. Physical exam was normal except for microcephaly (OFC 48 cm (< 5%), 50% for 16 months) and epicanthi (Fig. 1). SNP microarray previously performed at an outside hospital identified 47,XXY consistent with KS. At age 16 months he presented with both clinical and subclinical seizures in the setting of an influenza infection. Before age 2 years, additional generalized and focal seizures types developed. Background
Fig. 1. Clinical features of the proband. Portrait and profile photographs of the proband at age 6 years. 90
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Fig. 2. Sequencing results for PCDH19 variant. A) IGV screenshot of PCDH19 variant region in the proband. B) Sanger sequencing chromatograms of the proband and mother. IGV, Integrative Genomics Viewer.
expected PCDH19-related epilepsy phenotype, i.e.: recurrent clusters of brief seizures, fever sensitivity, tonic seizures, and focal seizures often with subsequent generalization. While it is clear the PCHD19 variant, and not increased X chromosome gene dosage inherent in KS is causative for our patient’s seizures, it is unclear what proportion of KS individuals have a known genetic etiology for their epilepsy, and how significant the atypical PCDH19 X-linked inheritance pattern and mechanism may underlie seizures in this sex chromosomal disorder. The co-existence of neurons with and without PCDH19, by virtue of tissue mosaicism in XY males or random X-inactivation in XX females, is predicted to result in divergent cell sorting and migration leading to abnormal neuronal networks (de Lange et al., 2017; Depienne et al., 2009). Mosaicism in fibroblasts from a XY male with PCDH19-related epilepsy has been observed, however, human brain tissue mosaicism or a mosaic mouse model has not yet been reported (Depienne et al., 2009). Compagni et al. confirmed cellular interference as the mechanism of pathogenicity in X-linked EFNB1-related craniofrontonasal syndrome using an analogous mouse model, wherein female mice heterozygous for EFNB1 mutations had mosaic expression resulting in ectopic interactions between Ephrin B1 ligand and EphB receptors that were sufficient to induce skeletal defects (Compagni et al., 2003; Wieland et al., 2004). Such experimental evidence is still lacking for PCDH19 cellular interference disease mechanism. Our current report of an affected male heterozygous for a PCDH19 pathogenic variant is
Aggregation Database) population database (Lek et al., 2016). The variant was confirmed by Sanger sequencing and reported as likely pathogenic for PCDH19-related epilepsy, thus explaining the seizures in this patient (Fig. 2B). Additional testing did not identify the PCDH19 p.Pro236Ser variant in DNA obtained from maternal whole blood.A paternal sample was unavailable for testing. Therefore, it is possible that the PCDH19 variant either arose de novo or was inherited from an unaffected hemizygous father who contributed an XY sperm to his son mostly due to nondisjunction in meiosis II during gametogenesis(Martin, 2005). 4. Discussion An association between Klinefelter syndrome (KS) and epilepsy has been previously reported, but the proportion of KS patients with this phenotype is unclear (Tatum et al., 1998). Case reports and small cohort studies have identified up to 54% of KS individuals with epilepsy, but these studies are biased and non-representative of the general KS population (Elia et al., 1995; Mazen et al., 2010; Tatum et al., 1998). A Danish register study examining comorbidities in hospital discharge diagnoses for KS patients and randomly selected, age-matched control males found epilepsy was significantly more frequent among KS patients (hazard ratio 4.28; 95% CI 2.86–6.41) (Bojesen et al., 2006). The phenotype of the described patient significantly overlaps with the 91
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consistent with this mechanism, where two genetically distinct cell populations cause the PCDH19 epilepsy phenotype. In summary, the case presented here further supports the postulated PCDH19 disease mechanism.
infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females. PLoS Genet. 5, e1000381. Depienne, C., Trouillard, O., Bouteiller, D., Gourfinkel-An, I., Poirier, K., Rivier, F., Berquin, P., Nabbout, R., Chaigne, D., Steschenko, D., Gautier, A., HoffmanZacharska, D., Lannuzel, A., Lackmy-Port-Lis, M., Maurey, H., Dusser, A., Bru, M., Gilbert-Dussardier, B., Roubertie, A., Kaminska, A., Whalen, S., Mignot, C., Baulac, S., Lesca, G., Arzimanoglou, A., LeGuern, E., 2011. Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females. Hum. Mutat. 32, E1959–1975. Elia, M., Musumeci, S.A., Ferri, R., Scuderi, C., Del Gracco, S., Stefanini, M.C., 1995. Seizures in Klinefelter’s syndrome: a clinical and EEG study of five patients. Ital. J. Neurol. Sci. 16, 231–238. Higurashi, N., Takahashi, Y., Kashimada, A., Sugawara, Y., Sakuma, H., Tomonoh, Y., Inoue, T., Hoshina, M., Satomi, R., Ohfu, M., Itomi, K., Takano, K., Kirino, T., Hirose, S., 2015. Immediate suppression of seizure clusters by corticosteroids in PCDH19 female epilepsy. Seizure 27, 1–5. Lek, M., Karczewski, K.J., Minikel, E.V., Samocha, K.E., Banks, E., Fennell, T., O’DonnellLuria, A.H., Ware, J.S., Hill, A.J., Cummings, B.B., Tukiainen, T., Birnbaum, D.P., Kosmicki, J.A., Duncan, L.E., Estrada, K., Zhao, F., Zou, J., Pierce-Hoffman, E., Berghout, J., Cooper, D.N., Deflaux, N., DePristo, M., Do, R., Flannick, J., Fromer, M., Gauthier, L., Goldstein, J., Gupta, N., Howrigan, D., Kiezun, A., Kurki, M.I., Moonshine, A.L., Natarajan, P., Orozco, L., Peloso, G.M., Poplin, R., Rivas, M.A., Ruano-Rubio, V., Rose, S.A., Ruderfer, D.M., Shakir, K., Stenson, P.D., Stevens, C., Thomas, B.P., Tiao, G., Tusie-Luna, M.T., Weisburd, B., Won, H.H., Yu, D., Altshuler, D.M., Ardissino, D., Boehnke, M., Danesh, J., Donnelly, S., Elosua, R., Florez, J.C., Gabriel, S.B., Getz, G., Glatt, S.J., Hultman, C.M., Kathiresan, S., Laakso, M., McCarroll, S., McCarthy, M.I., McGovern, D., McPherson, R., Neale, B.M., Palotie, A., Purcell, S.M., Saleheen, D., Scharf, J.M., Sklar, P., Sullivan, P.F., Tuomilehto, J., Tsuang, M.T., Watkins, H.C., Wilson, J.G., Daly, M.J., MacArthur, D.G., Exome Aggregation, C., 2016. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291. Martin, R.H., 2005. Mechanisms of nondisjunction in human spermatogenesis. Cytogenet. Genome Res. 111, 245–249. Mazen, I., El-Ruby, M., El-Bassyouni, H.T., 2010. Variable associations of Klinefelter syndrome in children. J. Pediatr. Endocrinol. Metab. 23, 985–989. Plagnol, V., Curtis, J., Epstein, M., Mok, K.Y., Stebbings, E., Grigoriadou, S., Wood, N.W., Hambleton, S., Burns, S.O., Thrasher, A.J., Kumararatne, D., Doffinger, R., Nejentsev, S., 2012. A robust model for read count data in exome sequencing experiments and implications for copy number variant calling. Bioinformatics 28, 2747–2754. Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W.W., Hegde, M., Lyon, E., Spector, E., Voelkerding, K., Rehm, H.L., Committee, A.L.Q.A., 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–424. Specchio, N., Marini, C., Terracciano, A., Mei, D., Trivisano, M., Sicca, F., Fusco, L., Cusmai, R., Darra, F., Bernardina, B.D., Bertini, E., Guerrini, R., Vigevano, F., 2011. Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations. Epilepsia 52, 1251–1257. Steel, D., Symonds, J.D., Zuberi, S.M., Brunklaus, A., 2017. Dravet syndrome and its mimics: beyond SCN1A. Epilepsia 58, 1807–1816. Tatum, W.Ot., Passaro, E.A., Elia, M., Guerrini, R., Gieron, M., Genton, P., 1998. Seizures in Klinefelter’s syndrome. Pediatr. Neurol. 19, 275–278. Trivisano, M., Pietrafusa, N., Ciommo, V., Cappelletti, S., Palma, L., Terracciano, A., Bertini, E., Vigevano, F., Specchio, N., 2016. PCDH19-related epilepsy and Dravet syndrome: face-off between two early-onset epilepsies with fever sensitivity. Epilepsy Res. 125, 32–36. Trump, N., McTague, A., Brittain, H., Papandreou, A., Meyer, E., Ngoh, A., Palmer, R., Morrogh, D., Boustred, C., Hurst, J.A., Jenkins, L., Kurian, M.A., Scott, R.H., 2016. Improving diagnosis and broadening the phenotypes in early-onset seizure and severe developmental delay disorders through gene panel analysis. J. Med. Genet. 53, 310–317. van Harssel, J.J., Weckhuysen, S., van Kempen, M.J., Hardies, K., Verbeek, N.E., de Kovel, C.G., Gunning, W.B., van Daalen, E., de Jonge, M.V., Jansen, A.C., Vermeulen, R.J., Arts, W.F., Verhelst, H., Fogarasi, A., de Rijk-van Andel, J.F., Kelemen, A., Lindhout, D., De Jonghe, P., Koeleman, B.P., Suls, A., Brilstra, E.H., 2013. Clinical and genetic aspects of PCDH19-related epilepsy syndromes and the possible role of PCDH19 mutations in males with autism spectrum disorders. Neurogenetics 14, 23–34. Wieland, I., Jakubiczka, S., Muschke, P., Cohen, M., Thiele, H., Gerlach, K.L., Adams, R.H., Wieacker, P., 2004. Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome. Am. J. Hum. Genet. 74, 1209–1215.
5. Conclusions We report a boy with Klinefelter syndrome (KS) and PCDH19-related epilepsy, with a novel genetic makeup supporting the cellular interference disease mechanism underlying PCDH19-related disorder, as seen in female children with a heterozygous disease-causing variant and in 46,XY males with somatic mosaicism. To our knowledge, this is the first report of a boy with Klinefelter syndrome and PDCH19-related epilepsy. Declarations of interest None. Consent We confirm that informed consent was obtained for the patient described in this manuscript. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgments We thank the patient and his family for their participation and support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.eplepsyres.2018.06. 008. References Bojesen, A., Juul, S., Birkebaek, N.H., Gravholt, C.H., 2006. Morbidity in Klinefelter syndrome: a Danish register study based on hospital discharge diagnoses. J. Clin. Endocrinol. Metab. 91, 1254–1260. Compagni, A., Logan, M., Klein, R., Adams, R.H., 2003. Control of skeletal patterning by ephrinB1-EphB interactions. Dev. Cell 5, 217–230. de Lange, I.M., Rump, P., Neuteboom, R.F., Augustijn, P.B., Hodges, K., Kistemaker, A.I., Brouwer, O.F., Mancini, G.M.S., Newman, H.A., Vos, Y.J., Helbig, K.L., PeetersScholte, C., Kriek, M., Knoers, N.V., Lindhout, D., Koeleman, B.P.C., van Kempen, M.J.A., Brilstra, E.H., 2017. Male patients affected by mosaic PCDH19 mutations: five new cases. Neurogenetics 18, 147–153. Depienne, C., LeGuern, E., 2012. PCDH19-related infantile epileptic encephalopathy: an unusual X-linked inheritance disorder. Hum. Mutat. 33, 627–634. Depienne, C., Bouteiller, D., Keren, B., Cheuret, E., Poirier, K., Trouillard, O., Benyahia, B., Quelin, C., Carpentier, W., Julia, S., Afenjar, A., Gautier, A., Rivier, F., Meyer, S., Berquin, P., Helias, M., Py, I., Rivera, S., Bahi-Buisson, N., Gourfinkel-An, I., Cazeneuve, C., Ruberg, M., Brice, A., Nabbout, R., Leguern, E., 2009. Sporadic
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Contents lists available at ScienceDirect
Epilepsy Research journal homepage: www.elsevier.com/locate/epilepsyres
PCDH19-related epilepsy in a male with Klinefelter syndrome: Additional evidence supporting PCDH19 cellular interference disease mechanism
T
Edward J. Romaskoa, Elizabeth T. DeChenea, Jorune Balciunienea, Gozde T. Akgumusa, Ingo Helbigb,c, Jennifer M. Tarpiniand, Beth A. Keenad, Maria G. Vogiatzic,e, Elaine H. Zackaic,d, ⁎ ⁎ Kosuke Izumia,d, Shavonne L. Masseyb,c, , Ahmad N. Abou Tayouna,f, ,1 a
Division of Genomic Diagnostics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States c Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, United States d Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States e Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Philadelphia, PA, United States f Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States b
A R T I C LE I N FO
A B S T R A C T
Keywords: PCDH19 Klinefelter syndrome Epilepsy Molecular diagnostics
Heterozygous de novo or inherited pathogenic variants in the PCDH19 gene cause a spectrum of neurodevelopmental features including developmental delay and seizures. PCDH19 epilepsy was previously known as “epilepsy and mental retardation limited to females”, since the condition almost exclusively affects females. It is hypothesized that the co-existence of two populations of neurons, some with and some without PCDH19 protein expression, results in pathologically abnormal interactions between these neurons, a mechanism also referred to as cellular interference. Consequently, PCDH19-related epilepsies are inherited in an atypical X-linked pattern, such that hemizygous, non-mosaic, 46,XY males are typically unaffected, while individuals with a diseasecausing PCDH19 variant, mainly heterozygous females and mosaic males, are affected. As a corollary to this hypothesis, an individual with Klinefelter syndrome (KS) (47,XXY) who has a heterozygous disease-causing PCDH19 variant should develop PCDH19-related epilepsy. Here, we report such evidence: - a male child with KS and PCDH19-related epilepsy – supporting the PCDH19 cellular interference disease hypothesis.
1. Introduction PCDH19-related epilepsy, caused by pathogenic variants in the PCDH19 gene, has a highly variable clinical presentation often characterized by infantile or early childhood onset intractable seizures that cluster and are fever-sensitive. Seizure types vary but include generalized tonic, clonic or tonic-clonic, myoclonic and/or focal seizures. Seizure frequency decreases with age, and patients may become seizure-free during childhood or adolescence. Most affected individuals have developmental delay, mild to severe intellectual disability, autism, and/or behavioral issues; however, some patients have normal cognition. The phenotype, including the degree of intellectual and behavioral problems, can overlap with Dravet syndrome, however, PCDH19-related epilepsy is associated with later seizure onset, lack of photosensitivity, increased seizure cluster frequency, and good response to high-dose steroid treatment for clusters of seizures (Depienne and LeGuern, 2012; Depienne et al., 2011; Higurashi et al., 2015; Steel ⁎
1
et al., 2017; Trivisano et al., 2016; van Harssel et al., 2013). The PCDH19 gene on chromosome Xq22.3 encodes protocadherin 19, which is postulated to play a role in mediating calcium-dependent cell–cell adhesion and regulating the organization of the developing brain (Depienne and LeGuern, 2012). It is hypothesized that co-existence of two neuron populations, one expressing PCDH19 while the other is deficient for this protein, results in abnormal interactions between these neurons, a process referred to as cellular interference (Depienne et al., 2009; Depienne and LeGuern, 2012). Although unproven, this hypothesis could explain why PCDH19 variants are pathogenic in individuals with two different populations of neurons (e.g., females with random X-inactivation and mosaic males) but not in hemizygous males who only have one population (de Lange et al., 2017; Depienne et al., 2009). Similarly, this hypothesis would suggest that an individual with a sex chromosome abnormality leading to multiple X chromosomes (e.g. Klinefelter syndrome (KS) (47,XXY) and triple X syndrome (47,XXX)) with disease-causing PCDH19 variants would also
Corresponding authors at: 3401 Civic Center Blvd., Philadelphia, PA 19104-4399, United States. E-mail addresses: [email protected] (S.L. Massey), [email protected] (A.N.A. Tayoun). Current Address: Al Jalila Children's Specialty Hospital, Dubai, UAE.
https://doi.org/10.1016/j.eplepsyres.2018.06.008 Received 12 January 2018; Received in revised form 7 April 2018; Accepted 14 June 2018 0920-1211/ © 2018 Elsevier B.V. All rights reserved.
Epilepsy Research 145 (2018) 89–92
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EEG activity demonstrated generalized diffuse background slowing for age. No interictal abnormalities were present. Ictal activity demonstrated electroclinical seizures with right temporal and/or right parietal-temporal onset which evolved into high amplitude, rhythmic spike and wave discharges over the entire right hemisphere. Postictal slowing was often seen most prominently over the right hemisphere. Clinically, these seizures were characterized by staring and unresponsiveness. Subclinical seizures of similar right temporal and right parietal-temporal onset were also captured in sleep. Specific seizure semiologies included generalized tonic-clonic, generalized atonic, generalized tonic and focal non-motor seizures with secondary generalization characterized by staring with head version to either direction followed by whole body limpness, drooling, and generalized clonic activity. Generally, the patient's seizures were triggered by fever and illness, including two episodes of status epilepticus. His seizures had a clustering pattern with a 72 h cluster of intermittent seizures of all semiologies described above, occurring every 3–4 months. Seizures have been stable with last breakthrough seizure occurring in the setting of an illness at age 5.5 years.
develop PCDH19-related epilepsy. Here, we report a non-mosaic XXY male child with KS and PCDH19-related epilepsy. 2. Materials and methods We reviewed medical charts to determine the clinical and previous testing data for the patient, who was examined by K.I., E.H.Z., S.L.M., and M.G.V. Informed consent was obtained from the individual and legal guardians. Genomic DNA was extracted from peripheral blood and sequencing was performed by capture of the coding regions and splice sites ( ± 15 bp) of targeted genes using the Agilent SureSelect XT Clinical Research Exome kit (per manufacturer’s instructions) followed by sequencing on the Illumina HiSeq 2500 platform with 100bp pairedend reads at an average sequencing depth of 100×. Sequence variant calls were generated using a custom-built bioinformatics pipeline in 100 genes known to cause monogenic forms of epilepsy (Supplemental Table 1). Exon-level copy number variants were also called using ExomeDepth (Plagnol et al., 2012) with our internal exome cohort as a normalizing reference. Rare variants were classified according to the ACMG/AMP variant interpretation framework (Richards et al., 2015).
3.2. Genetic findings 3. Results After sequence variant calling and filtration, eight rare variants were interpreted. No copy number changes were detected. Seven of these were classified as likely benign or benign, because they were synonymous or intronic and not predicted to affect splicing, or had a minor allele frequency in the general population inconsistent with disease prevalence and penetrance. The NM_001184880.1(PCDH19):c.706C > T, p.Pro236Ser variant was detected heterozygously on the X chromosome, confirming the XXY karyotype, with read fractions of 165 and 198 for the variant (45%) and reference (55%) alleles, respectively (Fig. 2A). This variant had been previously reported as de novo in a female with epilepsy and autistic features (Specchio et al., 2011). Two variants affecting the same codon (p.Pro236Leu and p.Pro236Arg) had also been reported as de novo in a female with early infantile epileptic encephalopathy (Trump et al., 2016) and in another patient without phenotypic information [ClinVar, RCV000480604.1]. Furthermore, nearby missense variants (p.Asp230Asn, p.Asn232Ser, p.Asn234Ser) have been reported in affected individuals (Depienne and LeGuern, 2012; Trump et al., 2016). The c.706C > T p.Pro236Ser variant was not observed in the gnomAD (Genome
3.1. Clinical history A five year old male presented with intractable epilepsy and microcephaly of unknown etiology (Fig. 1). Prenatal history was unremarkable except for maternal cervical incompetence. The proband was born at 35 weeks via vaginal delivery weighing 5 lbs 15 oz (75th percentile for 35 weeks gestation) and went to the well-baby nursery after delivery. Developmental history included sitting at normal time and walking by 12 months. Speech and language development were delayed, and he began receiving occupational therapy, physical therapy, and speech services from 18 months old. Physical exam was normal except for microcephaly (OFC 48 cm (< 5%), 50% for 16 months) and epicanthi (Fig. 1). SNP microarray previously performed at an outside hospital identified 47,XXY consistent with KS. At age 16 months he presented with both clinical and subclinical seizures in the setting of an influenza infection. Before age 2 years, additional generalized and focal seizures types developed. Background
Fig. 1. Clinical features of the proband. Portrait and profile photographs of the proband at age 6 years. 90
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Fig. 2. Sequencing results for PCDH19 variant. A) IGV screenshot of PCDH19 variant region in the proband. B) Sanger sequencing chromatograms of the proband and mother. IGV, Integrative Genomics Viewer.
expected PCDH19-related epilepsy phenotype, i.e.: recurrent clusters of brief seizures, fever sensitivity, tonic seizures, and focal seizures often with subsequent generalization. While it is clear the PCHD19 variant, and not increased X chromosome gene dosage inherent in KS is causative for our patient’s seizures, it is unclear what proportion of KS individuals have a known genetic etiology for their epilepsy, and how significant the atypical PCDH19 X-linked inheritance pattern and mechanism may underlie seizures in this sex chromosomal disorder. The co-existence of neurons with and without PCDH19, by virtue of tissue mosaicism in XY males or random X-inactivation in XX females, is predicted to result in divergent cell sorting and migration leading to abnormal neuronal networks (de Lange et al., 2017; Depienne et al., 2009). Mosaicism in fibroblasts from a XY male with PCDH19-related epilepsy has been observed, however, human brain tissue mosaicism or a mosaic mouse model has not yet been reported (Depienne et al., 2009). Compagni et al. confirmed cellular interference as the mechanism of pathogenicity in X-linked EFNB1-related craniofrontonasal syndrome using an analogous mouse model, wherein female mice heterozygous for EFNB1 mutations had mosaic expression resulting in ectopic interactions between Ephrin B1 ligand and EphB receptors that were sufficient to induce skeletal defects (Compagni et al., 2003; Wieland et al., 2004). Such experimental evidence is still lacking for PCDH19 cellular interference disease mechanism. Our current report of an affected male heterozygous for a PCDH19 pathogenic variant is
Aggregation Database) population database (Lek et al., 2016). The variant was confirmed by Sanger sequencing and reported as likely pathogenic for PCDH19-related epilepsy, thus explaining the seizures in this patient (Fig. 2B). Additional testing did not identify the PCDH19 p.Pro236Ser variant in DNA obtained from maternal whole blood.A paternal sample was unavailable for testing. Therefore, it is possible that the PCDH19 variant either arose de novo or was inherited from an unaffected hemizygous father who contributed an XY sperm to his son mostly due to nondisjunction in meiosis II during gametogenesis(Martin, 2005). 4. Discussion An association between Klinefelter syndrome (KS) and epilepsy has been previously reported, but the proportion of KS patients with this phenotype is unclear (Tatum et al., 1998). Case reports and small cohort studies have identified up to 54% of KS individuals with epilepsy, but these studies are biased and non-representative of the general KS population (Elia et al., 1995; Mazen et al., 2010; Tatum et al., 1998). A Danish register study examining comorbidities in hospital discharge diagnoses for KS patients and randomly selected, age-matched control males found epilepsy was significantly more frequent among KS patients (hazard ratio 4.28; 95% CI 2.86–6.41) (Bojesen et al., 2006). The phenotype of the described patient significantly overlaps with the 91
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consistent with this mechanism, where two genetically distinct cell populations cause the PCDH19 epilepsy phenotype. In summary, the case presented here further supports the postulated PCDH19 disease mechanism.
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5. Conclusions We report a boy with Klinefelter syndrome (KS) and PCDH19-related epilepsy, with a novel genetic makeup supporting the cellular interference disease mechanism underlying PCDH19-related disorder, as seen in female children with a heterozygous disease-causing variant and in 46,XY males with somatic mosaicism. To our knowledge, this is the first report of a boy with Klinefelter syndrome and PDCH19-related epilepsy. Declarations of interest None. Consent We confirm that informed consent was obtained for the patient described in this manuscript. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgments We thank the patient and his family for their participation and support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.eplepsyres.2018.06. 008. References Bojesen, A., Juul, S., Birkebaek, N.H., Gravholt, C.H., 2006. Morbidity in Klinefelter syndrome: a Danish register study based on hospital discharge diagnoses. J. Clin. Endocrinol. Metab. 91, 1254–1260. Compagni, A., Logan, M., Klein, R., Adams, R.H., 2003. Control of skeletal patterning by ephrinB1-EphB interactions. Dev. Cell 5, 217–230. de Lange, I.M., Rump, P., Neuteboom, R.F., Augustijn, P.B., Hodges, K., Kistemaker, A.I., Brouwer, O.F., Mancini, G.M.S., Newman, H.A., Vos, Y.J., Helbig, K.L., PeetersScholte, C., Kriek, M., Knoers, N.V., Lindhout, D., Koeleman, B.P.C., van Kempen, M.J.A., Brilstra, E.H., 2017. Male patients affected by mosaic PCDH19 mutations: five new cases. Neurogenetics 18, 147–153. Depienne, C., LeGuern, E., 2012. PCDH19-related infantile epileptic encephalopathy: an unusual X-linked inheritance disorder. Hum. Mutat. 33, 627–634. Depienne, C., Bouteiller, D., Keren, B., Cheuret, E., Poirier, K., Trouillard, O., Benyahia, B., Quelin, C., Carpentier, W., Julia, S., Afenjar, A., Gautier, A., Rivier, F., Meyer, S., Berquin, P., Helias, M., Py, I., Rivera, S., Bahi-Buisson, N., Gourfinkel-An, I., Cazeneuve, C., Ruberg, M., Brice, A., Nabbout, R., Leguern, E., 2009. Sporadic
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