The University of Chicago Genetic Services Laboratories

5841 S. Maryland Ave., Rm. G701, MC 0077, Chicago, Illinois 60637 Toll Free: (888) UC GENES (888) 824 3637 Local: (773) 834 0555 FAX: (773) 702 9130 [email protected] dnatesting.uchicago.edu. 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 epilepsy 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 epilepsies 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 15 genes listed below, and deletion/duplication analysis of the 12 genes listed in bold below.

Early Infantile Epileptic Encephalopathy Panel

ARHGEF9 KCNQ2 PNKP SLC2A1 ARX MAGI2 SCN1A SPTAN1 CDKL5 PCDH19 SCN2A STXBP1 GRIN2A PLCB1 SLC25A22

Gene / Condition Clinical Features and Molecular Pathology ARHGEF9 The ARHGEF9 gene encodes the protein collybistin, which is a brain-specific protein involved in EIEE8 inhibitory synaptogenesis (3). Shimojima et al (2011) identified a nonsense mutation in ARHGEF9 in [OMIM#300607] one out of 23 males with severe intellectual disability and epilepsy (4). 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 (3).

ARX ARX encodes a transcription factor expressed primarily in fetal and adult brain and skeletal muscle EIEE1 and is important for the maintenance of specific neuronal subtypes in the cerebral cortex and axonal [OMIM#300382] guidance in the floor plate. Up to 10% of males with a clinical diagnosis of EIEE or West syndrome/cryptogenic infantile spasms may have mutations in the ARX gene (1, 5).

CDKL5 CDKL5 contains a serine/threonine kinase domain and is implicated in MeCP2 modification in vitro EIEE2 (6). The most common feature found in patients reported to date with CDKL5 mutations is the early [OMIM#300203] onset of seizures. Archer et al (2006) identified CDKL5 mutations in 7/42 (17%) of females with severe mental retardation and seizures in the first 6 months of life (7). 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 (8).

GRIN2A GRIN2A encodes a protein that is incorporated as a subunit of N-methyl-D-aspartate (NMDA) Epilepsy with receptors, which are neurotransmitter-gated ion channels involved in the regulation of synaptic neurodevelopmental function in the central nervous system (9). Endele et al (2010) found heterozygous GRIN2A defects mutations in two out of 127 individuals with a history of idiopathic epilepsy and/or abnormal EEG [OMIM#613971] findings and a variable degree of intellectual disability (9). 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 early-onset epileptic encephalopathy (9).

KCNQ2 Weckhuysen et al. (2012) identified heterozygous KCNQ2 mutations in 8 out of 80 patients with EIEE7 EIEE, 6 of which were confirmed to be de novo. KCNQ2 encodes a subunit of a voltage-gated [OMIM#602235] potassium channel. 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 (10).

2/13 PCDH19 EIEE9 has been associated with mutations in the PCDH19 gene, which encodes for protocadherin- EIEE9 19. Protocadherins form a subfamily of calcium-dependent cell-cell adhesion molecules in the [OMIM#300088] cadherin superfamily, and protocadherin-19 has been found to be expressed in the central nervous system, including the hippocampus and cortex, suggesting a role in cognitive function. Marini et al. (2010) 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 (11).

MAGI2 The MAGI2 gene encodes for the scaffolding protein membrane-associated guanylate kinase Infantile spasms and inverted-2, which interacts with N-methyl-D-aspartic acid (NMDA) receptors at excitatory synapses in intellectual disability the brain, as well as with many other proteins pre- and postsynaptically and at both excitatory and [OMIM#606382] inhibitory synapses (12). Marshall et al (2008) found that in individuals with a 7q11.23-q21.1 deletion associated with infantile spasms in addition to a Williams-Beuren syndrome phenotype, 15 out of 16 had a full or partial deletion of MAGI2, whereas only one of 11 eleven individuals with a chromosome 7 deletion and no seizure history was missing any part of this gene (12).

PLCB1 Kurian et al. (2010) identified a homozygous deletion of the promoter element and exons 1-3 of the EIEE12 PLCB1 gene in a child with EIEE from a consanguineous family of Bangladeshi descent. The deletion [OMIM#607120] was associated with complete loss of expression of the PLCB1 gene (13). The PLCB1 protein is involved in mediation of a wide variety of extracellular signals transduced across the cell membrane (13). PNKP Mutations in the PNKP gene are associated with early-onset intractable epilepsy, microcephaly, EIEE10 developmental delay and behavioral abnormalities (EIEE10) (14). Both homozygous and compound [OMIM#613402] heterozygous mutations have been reported. The PNKP protein is involved in DNA repair of both double and single-stranded breaks. 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 (14, 15). At this time no features typically associated with DNA repair defects, such as cancer predisposition or immunological abnormalities have been reported in affected individuals (14).

SCN1A Mutations in the SCN1A gene cause EIEE6, which is more commonly known as Dravet syndrome. EIEE6 SCN1A encodes for the sodium channel neuronal type 1α subunit, which associates with two smaller [OMIM#607208] β-subunits to form the voltage-gated sodium channel (14). Mutations in SCN1A can cause either gain or loss of protein function, which predispose the central nervous system to abnormal excitability (14, 15). 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 (16). 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 (16). SCN2A The SCN2A gene encodes a voltage-gated sodium channel α-subunit. Ogiwara et al. (2009) EIEE11 identified 2 de novo mutations in the SCN2A gene in a cohort of 116 patients with intractable [OMIM#182390] childhood epilepsies (17). SLC25A22 Homozygous mutations in SLC25A22 have been described in case reports of consanguineous EIEE3 families with EIEE (17, 18). The SLC25 gene family encodes mitochondrial carriers that transport a [OMIM#609302] variety of metabolites across the inner mitochondrial membrane. SLC25A22 is specifically expressed in the brain, within areas proposed to contribute to the genesis and control of myoclonic seizures.

SLC2A1 Glucose transporter-1 (GLUT1) deficiency syndrome is caused by heterozygous mutations in the GLUT1 deficiency SLC2A1 gene, which lead to impaired glucose transport in the brain. The classic GLUT-1 deficiency syndrome syndrome presentation is drug-resistant infantile-onset seizures, developmental delay, acquired [OMIM#606777] microcephaly, hypotonia, spasticity, ataxia and dystonia (18). Seizures are typically refractory and worsen during periods of fasting (19). The majority of reported cases are due to de novo mutations (20).

SPTAN1 The protein encoded for by SPTAN1 belongs to the spectrin family, which are widely distributed EIEE5 filamentous cytoskeletal proteins whose functions include regulation of receptor binding and actin [OMIM#182810] crosslinking. Saitsu et al (2010) identified de-novo heterozygous mutations in 2 unrelated Japanese patients with EIEE5 (21). STXBP1 EIEE4 is caused by mutations in STXBP1, which encodes for syntaxin binding protein-1, more EIEE4 commonly referred to as MUNC18-1. Syntaxin binding protein-1 is a neuron specific protein of the [OMIM#602926] SEC1 family of membrane-trafficking proteins. MUNC18-1 is expressed throughout the brain and is a key component for calcium-dependent neurotransmitter release. Sequencing of STXBP1 detected mutations in 4 out of 106 patients with EIEE (22). Earlier reports identified 4 heterozygous missense mutations in 13 patients with EIEE(23).

2/13 Inheritance: ARX, CDKL5, ARHGEF9 and PCDH19 are inherited in an X-linked pattern. ARX mutations are more commonly associated with infantile spasms in males and carrier females can be asymptomatic (6). CDKL5 mutations appear to be more common in females than in males. 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 (7). Both inherited and de novo mutations have been described in these genes. Depending on parental carrier status, recurrence risk may be up to 50%.

SLC25A22 ,PNKP and PLCB1 mutations are inherited in an autosomal recessive pattern. Parents of an affected child are most likely obligate carriers. Recurrence risk for carrier parents is 25%.

STXBP1, SPTAN1, SCN1A, SCN2A, SLC2A1, GRIN2A MAGI2 and KCNQ2 mutations are inherited in an autosomal dominant pattern. Most cases described to date are de novo. Parental mosaicism has been described in two families (both paternal), one with a mutation in STXBP1, the other with a mutation in KCNQ2 (10, 24).

Test methods: Comprehensive sequence coverage of the coding regions and splice junctions of all genes in this panel is performed. Targets of interests are amplified using highly parallelized and multiplexed PCR reactions assembled with the Raindance System. DNA is sequenced 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. Please note that deletion/duplication analysis is not currently available for KCNQ2, PLCB1 and SCN2A. 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.

Sequencing and deletion duplication analysis of individual genes is also 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 individual tests.

EIEE Next Generation Panel (sequence analysis of 15 genes, deletion/duplication analysis of 12 genes) Sample specifications: 3 to10 cc of blood in a purple top (EDTA) tube Cost: $4500 CPT codes: 81407 Turn-around time: 8-10 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.

Testing for a known mutation in additional family members by sequence analysis Sample specifications: 3 to10 cc of blood in a purple top (EDTA) tube Cost: $390 CPT codes: 81403 Turn-around time: 3-4 weeks

Prenatal testing for a known mutation by sequence analysis Sample specifications: 2 T25 flasks of cultured cells from amniocentesis or CVS or 10 mL of amniotic fluid Cost: $540 CPT codes: 81403 Turn-around time: 1-2 weeks

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.

2/13 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. 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. 4. 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. 5. 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. 6. 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. 7. 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. 8. 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. 9. 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. 10. Weckhuysen S, Mandelstam S, Suls A et al. KCNQ2 encephalopathy: emerging phenotype of a neonatal epileptic encephalopathy. Ann Neurol 2012: 71: 15-25. 11. Marini C, Mei D, Parmeggiani L et al. Protocadherin 19 mutations in girls with infantile-onset epilepsy. Neurology 2010: 75: 646-653. 12. Marshall CR, Young EJ, Pani AM et al. Infantile spasms is associated with deletion of the MAGI2 gene on chromosome 7q11.23- q21.11. Am J Hum Genet 2008: 83: 106-111. 13. Kurian MA, Meyer E, Vassallo G et al. Phospholipase C beta 1 deficiency is associated with early-onset epileptic encephalopathy. Brain 2010: 133: 2964-2970. 14. Tavyev Asher YJ, Scaglia F. Molecular bases and clinical spectrum of early infantile epileptic encephalopathies. Eur J Med Genet 2012: 55: 299-306. 15. 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. 16. Escayg A, Goldin AL. Sodium channel SCN1A and epilepsy: mutations and mechanisms. Epilepsia 2010: 51: 1650-1658. 17. 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. 18. 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. 19. Brockmann K. The expanding phenotype of GLUT1-deficiency syndrome. Brain Dev 2009: 31: 545-552. 20. 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. 21. Byrne S, Kearns J, Carolan R et al. Refractory absence epilepsy associated with GLUT-1 deficiency syndrome. Epilepsia 2011: 52: 1021-1024. 22. 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. 23. Deprez L, Weckhuysen S, Holmgren P et al. Clinical spectrum of early-onset epileptic encephalopathies associated with STXBP1 mutations. Neurology 2010: 75: 1159-1165. 24. 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.

Committed to CUSTOMIZED DIAGNOSTICS, TRANSLATIONAL RESEARCH & YOUR PATIENTS’ NEEDS

2/13