The University of Chicago Genetic Services Laboratories LaboLaboratories 5841 S. Maryland Ave., Rm. G701, MC 0077, Chicago, Illinois 60637

CLIA #: 14D0917593 CAP #: 18827-49

Next Generation Sequencing Panel for Early Infantile Epileptic Encephalopathy

Clinical Features and Molecular Genetics: Early infantile epileptic encephalopathy (EIEE), also known as Ohtahara syndrome, is a severe form of characterized by frequent tonic spasms with onset in the first months of life. EEG reveals suppression-burst patterns, characterized by high- voltage bursts alternating with almost flat suppression phases. Seizures are medically intractable with evolution to West syndrome at 3-6 months of age and then Lennox-Gastaut syndrome at 1-3 years of age. EIEE represents approximately 1% of all occuring in children less than 15 years of age (1). Patients have severe developmental delay and poor prognosis. The diagnostic workup of EIEEs remains challenging because of frequent difficulties in defining etiologies. Acquired structural abnormalities like hypoxic-ischemic insults and isolated cortical malformations, which represent the most common causes of epileptic encelphalopathy in infancy should be excluded first (2).

Our EIEE Panel includes sequence analysis of all 30 listed below and deletion/duplication analysis of all 21 genes listed below in bold.

Early Infantile Epileptic Encephalopathy Panel

ALDH7A1 CLCN4 KCNT1 SCN2A STXBP1 ARFGEF2 EEF1A2 PCDH19 SCN8A SZT2 ARHGEF9 EFHC1 PLCB1 SLC25A22 ARHGEF15 GNAO1 PNKP SLC2A1 ARX GRIN2A PNPO SPTAN1 CDKL5 KCNH5 POLG ST3GAL3 CHD2 KCNQ2 SCN1A ST3GAL5

Gene Inheritance Clinical Features and Molecular Pathology ALDH7A1 AR Mutations in ALDH7A1 are associated with pyridoxine-dependent epilepsy (PDE), which [OMIM#107323] is characterized by EIEE that is resistant to antiepileptic drugs but responsive to large doses of pyridoxine (vitamin B6) (3). ARFGEF2 AR Homozygous splice-site mutations in the ubiquitously expressed ARFGEF2 were [OMIM#605371] identified in a consanguineous Pakistani kindred with multiple children who presented with West syndrome, evolving to Lennox–Gastaut syndrome with severe intellectual disability, microcephaly and associated with diffuse periventricular heterotopia (4). ARHGEF9 XL Shimojima et al. (2011) identified a nonsense mutation in ARHGEF9 in one out of 23 [OMIM#300607] males with severe intellectual disability and epilepsy (5). A missense mutation in ARHGEF9 in a male with severe intellectual disability, hyperekplexia (excessive startle) and refractory infantile-onset epilepsy has also previously been described (6). ARHGEF15 AD In a group of children with difficult to control epilepsy and developmental delay, Veeramah [OMIM#608504] et al. (2013) found a single patient with a de novo mutation in ARHGEF15 which codes for Ephexin5 (7). The degradation of Ephexin5 promotes synapse development and is mediated by the coded for by UBE3A (8). ARX XL Up to 10% of males with a clinical diagnosis of EIEE or West syndrome/cryptogenic [OMIM#300382] infantile spasms may have mutations in the ARX gene (1, 9). Carrier females of ARX mutations can be asymptomatic (10). CDKL5 XL Archer et al. (2006) identified CDKL5 mutations in 7/42 (17%) of females with severe [OMIM#300203] mental retardation and seizures in the first 6 months of life (11). The most common feature found in patients reported to date with CDKL5 mutations is the early onset of seizures. CDKL5 mutations have been reported in more female than male patients, however, Elia, et al (2008) reported CDKL5 mutations in 3/8 boys with severe mental retardation and early-onset seizures (12).

12/14 CHD2 AD In a cohort of 500 patients with epileptic encephalopathy, 1.2% were found to have de [OMIM#602119] novo mutations in CHD2 (13). Reported features include: Dravet syndrome (14), myoclonic seizures, absence seizures, photosensitivity, and moderate to severe intellectual disability (13). Mutations leading to protein truncation, as well as missense mutations have been reported. CLCN4 XL In a group of children with difficult to control epilepsy and developmental delay, Veeramah [OMIM#302910] et al (2013) found a single patient with a hemizygous de novo missense mutation in CLNC4 (7). In addition to epilepsy, this child had microcephaly, hypotonia, myotonia, and intellectual disability. EEF1A2 AD In 2012, de Ligt et al. identified a de novo missense mutation in a patient with a history of [OMIM#602959] neonatal hypotonia and seizures at 4 months and intellectual disability, autistic features, and aggressive behavior (15). Subsequently, in a group of children with difficult to control epilepsy and developmental delay, Veeramah et al (2013) found a single patient with a de novo missense mutation in EEF1A2 (7). EFHC1 AD/AR Berger et al. (2012) described a family with primary intractable epilepsy in infancy with a [OMIM#608815] homozygous missense mutation in EFHC1 (16). The affected children died at 18-36 months of age. Heterozygous mutations in EFHC1 have been associated with increased susceptibility to junvenile myoclonic epilepsy (17). GNAO1 AD Nakamura et al. (2013) identified de novo mutations in four individuals with EIEE, three of [OMIM#139311] whom were described as having Ohtahara syndrome, and two of whom had involuntary movements (18). One of the patients had somatic mosaicism, with only one third to half of various cell types harboring the mutation. Missense mutations and a small in-frame deletion were reported. GRIN2A AD Endele et a.l (2010) found heterozygous GRIN2A mutations in two out of 127 individuals [OMIM#613971] with a history of idiopathic epilepsy and/or abnormal EEG findings and a variable degree of intellectual disability (19). The most consistent clinical feature seen in individuals with GRIN2A mutations is epilepsy, which varies in severity between individuals. The most severe cases are associated with EIEE (19). KCNH5 AD In a group of children with difficult to control epilepsy and developmental delay, Veeramah [OMIM#605716] et al. (2013) found a single patient, whose presentation was consistent with West syndrome, with a de novo heterozygous mutation in KCNH5 (7). KCNQ2 AD Weckhuysen et al. (2012) identified heterozygous KCNQ2 mutations in 8 out of 80 [OMIM#602235] patients with EIEE, 6 of which were confirmed to be de novo. Mutations in KCNQ2 can also be associated with other phenotypes including benign familial neonatal seizures (BFNS), which is an autosomal dominant seizure disorder typically associated with a good prognosis (20). Parental mosaicism has been described in one family with a mutation in KCNQ2 (20, 21). KCNT1 AD Barcia et al. (2012) identified de novo heterozygous gain-of-function mutations in KCNT1 [OMIM#608167] in six patients with a subtype of EIEE known as malignant migrating partial seizures of infancy (22). Missense mutations in KCNT1 have also been reported in families with autosomal dominant nocturnal frontal lobe epilepsy-5 (23) PCDH19 XL Mutations in the PCDH19 gene have been associated with EIEE. Marini et al. (2010) [OMIM#300088] identified 13 different mutations in the PCDH19 gene in 13 (11%) of 117 female patients with febrile seizures and a wide spectrum of epilepsy phenotypes (24). PCDH19 mutations are X-linked, with the phenotype being restricted to females. Males with hemizygous mutations are apparently unaffected with normal cognitive functions. This unusual mode of inheritance is likely to be due to cellular interference, a mechanism assuming that only the co-existence of PCDH19 positive and negative cells, as a result of random X inactivation in females, is pathogenic (11). PLCB1 AR Kurian et al. (2010) identified a homozygous deletion of the promoter element and exons [OMIM#607120] 1-3 of the PLCB1 gene in a child with EIEE from a consanguineous family of Bangladeshi descent. PNKP AR Mutations in the PNKP gene are associated with early-onset intractable epilepsy, [OMIM#613402] microcephaly, developmental delay and behavioral abnormalities (25). Missense and frameshift mutations identified in PNKP have been associated with severe encephalopathy, whereas an intronic deletion identified in one individual, that was predicted to disrupt proper mRNA splicing, was associated with a milder phenotype (25, 26). The PNKP protein is involved in DNA repair of both double and single-stranded breaks, however at this time no features typically associated with DNA repair defects, such as cancer predisposition or immunological abnormalities have been reported in affected individuals (25). PNPO AR Mills et al (2005) found homozygous mutations in PNPO in 3 individuals with early [OMIM#603287] infantile epileptic encephalopathy and biochemical changes in the CSF, indicative of reduced activity of aromatic L-amino acid decarboxylase (AADC) (27). This was found to

12/14 be due to deficiency of pyridoxal phosphate (PLP), which is a co-factor of AADC and is synthesized by the enzyme encoded for by the PNPO gene. Of the 3 affected children, only one was treated with PLP and survived the neonatal period, however he continued to have symptoms such as seizures, severe developmental delays and dystonic spasms (27). POLG AR In a study of 213 children with early or juvenile onset nonsyndromic intractable epilepsy, [OMIM#174763] Uusimaa et al. (2013) identified 5 (2.3%) with compound heterozygous or homozygous mutations in the POLG gene (28). The majority of patients had elevated cerebrospinal fluid lactate. A proportion of affected individuals may develop liver failure, particularly if their seizures are being treated with sodium valproate (28). SCN1A AD Mutations in the SCN1A gene can cause EIEE6, which is more commonly known as [OMIM#607208] Dravet syndrome. EIEE6 is characterized by onset of seizures in the first year of life, often triggered by fever, photostimulation, or modest hyperthermia, which usually evolve to include myoclonic seizures over time (29). Mutations in SCN1A associated with EIEE6 are typically de novo (1). Of those with a clinical diagnosis of EIEE6, 85% have a mutation in SCN1A (29). SCN2A AD Ogiwara et al. (2009) identified 2 de novo mutations in the SCN2A gene in a cohort of 116 [OMIM#182390] patients with intractable childhood epilepsies (30). Mutations in the SCN2A gene can also cause benign familial neonatal seizures (BFNS). SCN8A AD Veeramah et al. (2012) identified a de novo mutation in SCN8A in a female with EIEE [OMIM#600702] (31). Symptoms started at 6 months of age with refractory generalized seizures, and the patient died suddenly at age 15 years (31). SLC25A22 AR Homozygous mutations in SLC25A22 have been described in case reports of [OMIM#609302] consanguineous families with EIEE (30, 32). SLC2A1 AD Glucose transporter-1 (GLUT1) deficiency syndrome is caused by heterozygous [OMIM#606777] mutations in the SLC2A1 gene, which lead to impaired glucose transport in the brain. The classic GLUT-1 deficiency syndrome presentation is drug-resistant infantile-onset seizures, developmental delay, acquired microcephaly, hypotonia, spasticity, ataxia and dystonia (32). Seizures are typically refractory and worsen during periods of fasting (33). The majority of reported cases are due to de novo mutations (34). SPTAN1 AD Saitsu et al (2010) identified de-novo heterozygous mutations in 2 unrelated Japanese [OMIM#182810] patients with EIEE (35).

ST3GAL3 AR A homozygous mutation in ST3GAL3 was identified in a consanguineous Palestinian [OMIM#606494] family with four individuals affected by severe early infantile epileptic encephalopathy (36). Mutations in ST3GAL3 have also been described in patients with mild to moderate non-syndromic intellectual disability (36). ST3GAL5 AR Mutations in ST3GAL5 are associated with infantile onset of refractory and recurrent [OMIM#604402] seizures, associated with profoundly delayed psychomotor development, abnormal movements, and vision loss (37). A founder mutation is present in the Amish community (37). A homozygous mutation was also found in an affected child of French ancestry. STXBP1 AD Sequencing of STXBP1 detected mutations in 4 out of 106 patients with EIEE (38). Earlier [OMIM#602926] reports identified 4 heterozygous missense mutations in 13 patients with EIEE(39). Parental mosaicism has been described in one family with a mutation in STXBP1 (20, 21).

SZT2 AR In 2 unrelated patients with EIEE, Basel-Vanagaite et al. (2013) identified biallelic [OMIM#615463] truncating mutations in the SZT2 gene (40). The phenotype was characterized by lack of psychomotor development apparent from birth, dysmorphic facial features, early onset of refractory seizures, and thick corpus callosum and persistent cavum septum pellucidum on brain imaging.

Test methods: Comprehensive sequence coverage of the coding regions and splice junctions of all genes in this panel is performed. Targets of interests are enriched and prepared for sequencing using the Agilent SureSelect system. Sequencing is performed using Illumina technology and reads are aligned to the reference sequence. Variants are identified and evaluated using a custom collection of bioinformatic tools and comprehensively interpreted by our team of directors and genetic counselors. All novel and/or potentially pathogenic variants are confirmed by Sanger sequencing. The technical sensitivity of this test is estimated to be >99% for single nucleotide changes and insertions and deletions of less than 20 bp.

Deletion/duplication analysis of the panel genes is performed by oligonucleotide array-CGH. Partial exonic copy number changes and rearrangements of less than 400 bp may not be detected by array-CGH. Array-CGH will not detect low-level mosaicism, balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype. The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

12/14 Our Infantile and Childhood Epilepsy Panel is also available (see website). In addition, sequencing and deletion duplication analysis of individual genes is offered separately for several of the genes on the EIEE panel, including ARX, CDKL5, PCDH19, PNKP, STXBP1, SLC25A22 and SPTAN1. Please see our website for more details regarding these other test options.

EIEE Next Generation Panel (sequence analysis of 30 genes and deletion/duplication analysis of 21 genes) Sample specifications: 3 to10 cc of blood in a purple top (EDTA) tube Cost: $4,500 CPT codes: 81479 Turn-around time: 8 weeks Note: We cannot bill insurance for the EIEE panel Note: The sensitivity of our assay may be reduced when DNA is extracted by an outside laboratory.

Results: Results, along with an interpretive report, are faxed to the referring physician as soon as they are completed. One report will be issued for the entire EIEE panel. All abnormal results are reported by telephone.

References: 1. Deprez L, Jansen A, De Jonghe P. Genetics of epilepsy syndromes starting in the first year of life. Neurology 2009: 72: 273-281. 2. Mastrangelo M, Leuzzi V. Genes of early-onset epileptic encephalopathies: from genotype to phenotype. Pediatr Neurol 2012: 46: 24-31. 3. Salomons GS, Bok LA, Struys EA et al. An intriguing "silent" mutation and a founder effect in antiquitin (ALDH7A1). Ann Neurol 2007: 62: 414-418. 4. Banne E, Atawneh O, Henneke M et al. West syndrome, microcephaly, grey matter heterotopia and hypoplasia of corpus callosum due to a novel ARFGEF2 mutation. J Med Genet 2013: 50: 772-775. 5. Shimojima K, Sugawara M, Shichiji M et al. Loss-of-function mutation of collybistin is responsible for X-linked mental retardation associated with epilepsy. J Hum Genet 2011: 56: 561-565. 6. Harvey K, Duguid IC, Alldred MJ et al. The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering. J Neurosci 2004: 24: 5816-5826. 7. Veeramah KR, Johnstone L, Karafet TM et al. Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. Epilepsia 2013: 54: 1270-1281. 8. Margolis SS, Salogiannis J, Lipton DM et al. EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation. Cell 2010: 143: 442-455. 9. Strømme P, Mangelsdorf ME, Shaw MA et al. Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy. Nat Genet 2002: 30: 441-445. 10. Weaving LS, Christodoulou J, Williamson SL et al. Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation. Am J Hum Genet 2004: 75: 1079-1093. 11. Archer HL, Evans J, Edwards S et al. CDKL5 mutations cause infantile spasms, early onset seizures, and severe mental retardation in female patients. J Med Genet 2006: 43: 729-734. 12. Elia M, Falco M, Ferri R et al. CDKL5 mutations in boys with severe encephalopathy and early-onset intractable epilepsy. Neurology 2008: 71: 997- 999. 13. Carvill GL, Heavin SB, Yendle SC et al. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. Nat Genet 2013: 45: 825-830. 14. Suls A, Jaehn JA, Kecskes A et al. De novo loss-of-function mutations in CHD2 cause a fever-sensitive myoclonic epileptic encephalopathy sharing features with Dravet syndrome. Am J Hum Genet 2013: 93: 967-975. 15. de Ligt J, Willemsen MH, van Bon BW et al. Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med 2012: 367: 1921- 1929. 16. Berger I, Dor T, Halvardson J et al. Intractable epilepsy of infancy due to homozygous mutation in the EFHC1 gene. Epilepsia 2012: 53: 1436-1440. 17. de Nijs L, Wolkoff N, Coumans B et al. Mutations of EFHC1, linked to juvenile myoclonic epilepsy, disrupt radial and tangential migrations during brain development. Hum Mol Genet 2012: 21: 5106-5117. 18. Nakamura K, Kodera H, Akita T et al. De Novo mutations in GNAO1, encoding a Galphao subunit of heterotrimeric G , cause epileptic encephalopathy. Am J Hum Genet 2013: 93: 496-505. 19. Endele S, Rosenberger G, Geider K et al. Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes. Nat Genet 2010: 42: 1021-1026. 20. Weckhuysen S, Mandelstam S, Suls A et al. KCNQ2 encephalopathy: emerging phenotype of a neonatal epileptic encephalopathy. Ann Neurol 2012: 71: 15-25. 21. Saitsu H, Kato M, Mizuguchi T et al. De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy. Nat Genet 2008: 40: 782-788. 22. Barcia G, Fleming MR, Deligniere A et al. De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy. Nat Genet 2012: 44: 1255-1259. 23. Heron SE, Smith KR, Bahlo M et al. Missense mutations in the sodium-gated gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 2012: 44: 1188-1190. 24. Marini C, Mei D, Parmeggiani L et al. Protocadherin 19 mutations in girls with infantile-onset epilepsy. Neurology 2010: 75: 646-653. 25. Tavyev Asher YJ, Scaglia F. Molecular bases and clinical spectrum of early infantile epileptic encephalopathies. Eur J Med Genet 2012: 55: 299-306. 26. Shen J, Gilmore EC, Marshall CA et al. Mutations in PNKP cause microcephaly, seizures and defects in DNA repair. Nat Genet 2010: 42: 245-249. 27. Mills PB, Surtees RA, Champion MP et al. Neonatal epileptic encephalopathy caused by mutations in the PNPO gene encoding pyridox(am)ine 5'- phosphate oxidase. Hum Mol Genet 2005: 14: 1077-1086. 28. Uusimaa J, Gowda V, McShane A et al. Prospective study of POLG mutations presenting in children with intractable epilepsy-prevalence and clinical features. Epilepsia 2013. 29. Escayg A, Goldin AL. SCN1A and epilepsy: mutations and mechanisms. Epilepsia 2010: 51: 1650-1658. 30. Ogiwara I, Ito K, Sawaishi Y et al. De novo mutations of voltage-gated sodium channel alphaII gene SCN2A in intractable epilepsies. Neurology 2009: 73: 1046-1053. 31. Veeramah KR, O'Brien JE, Meisler MH et al. De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am J Hum Genet 2012: 90: 502-510. 32. Molinari F, Raas-Rothschild A, Rio M et al. Impaired mitochondrial glutamate transport in autosomal recessive neonatal myoclonic epilepsy. Am J Hum Genet 2005: 76: 334-339. 33. Brockmann K. The expanding phenotype of GLUT1-deficiency syndrome. Brain Dev 2009: 31: 545-552. 34. Molinari F, Kaminska A, Fiermonte G et al. Mutations in the mitochondrial glutamate carrier SLC25A22 in neonatal epileptic encephalopathy with suppression bursts. Clin Genet 2009: 76: 188-194. 35. Byrne S, Kearns J, Carolan R et al. Refractory absence epilepsy associated with GLUT-1 deficiency syndrome. Epilepsia 2011: 52: 1021-1024.

12/14 36. Edvardson S, Baumann AM, Mühlenhoff M et al. West syndrome caused by ST3Gal-III deficiency. Epilepsia 2013: 54: e24-27. 37. Simpson MA, Cross H, Proukakis C et al. Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet 2004: 36: 1225-1229. 38. Saitsu H, Tohyama J, Kumada T et al. Dominant-negative mutations in alpha-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic quadriplegia, and developmental delay. Am J Hum Genet 2010: 86: 881-891. 39. Deprez L, Weckhuysen S, Holmgren P et al. Clinical spectrum of early-onset epileptic encephalopathies associated with STXBP1 mutations. Neurology 2010: 75: 1159-1165. 40. Basel-Vanagaite L, Hershkovitz T, Heyman E et al. Biallelic SZT2 mutations cause infantile encephalopathy with epilepsy and dysmorphic corpus callosum. Am J Hum Genet 2013: 93: 524-529.

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