Epilepsy with Auditory Features a Heterogeneous Clinico-Molecular Disease

Epilepsy with auditory features A heterogeneous clinico-molecular disease Tommaso Pippucci, PhD ABSTRACT Laura Licchetta, MD Objective: To identify novel genes implicated in epilepsy with auditory features (EAF) in phenotyp- Sara Baldassari, BSc ically heterogeneous families with unknown molecular basis. Flavia Palombo, BSc Methods: We identified 15 probands with EAF in whom an LGI1 mutation had been excluded. We Veronica Menghi, MD performed electroclinical phenotyping on all probands and available affected relatives. We used Romina D’Aurizio, PhD whole-exome sequencing (WES) in 20 individuals with EAF (including all the probands and 5 rela- Chiara Leta, MD tives) to identify single nucleotide variants, small insertions/deletions, and copy number variants. Carlotta Stipa, MD Giovanni Boero, MD Results: WES revealed likely pathogenic variants in genes that had not been previously associated CNTNAP2 DEPDC5 Giuseppe d’Orsi, MD with EAF: a intragenic deletion, 2 truncating mutations of , and a missense SCN1A Alberto Magi, PhD change. Ingrid Scheffer, PhD, MD Conclusions: EAF is a clinically and molecularly heterogeneous disease. The association of EAF Marco Seri, MD with CNTNAP2, DEPDC5,andSCN1A mutations widens the phenotypic spectrum related to Paolo Tinuper, MD these genes. CNTNAP2 encodes CASPR2, a member of the voltage-gated potassium channel Francesca Bisulli, PhD, complex in which LGI1 plays a role. The finding of a CNTNAP2 deletion emphasizes the impor- MD tance of this complex in EAF and shows biological convergence. Neurol Genet 2015;1:e5; doi: 10.1212/NXG.0000000000000005 Correspondence to GLOSSARY Dr. Pippucci: ADEAF 5 autosomal dominant EAF; CADD 5 Combined Annotation-Dependent Depletion; CNV 5 copy number variant; [email protected] EAF 5 epilepsy with auditory features; FS 5 febrile seizure; GEFS1 5 generalized epilepsy with febrile seizures plus; InDels 5 insertions and deletions; OMIM 5 Online Mendelian Inheritance in Man; RVIS 5 Residual Variation Intolerance Score; SNV 5 single nucleotide variant; VGKC 5 voltage-gated potassium channel; WES 5 whole-exome sequencing. Epilepsy with auditory features (EAF) is an uncommon presentation of focal epilepsy character- ized by focal seizures with auditory auras and symptoms suggesting lateral temporal onset.1 EAF is the hallmark of autosomal dominant EAF (ADEAF),1–3 a familial epilepsy syndrome due to mutations of the leucine-rich, glioma-inactivated 1 (LGI1) gene (Online Mendelian Inheritance in Man [OMIM] *604619)4 in 50% of families.5 In ADEAF, affected members typically have EAF, although to make the diagnosis only 2 family members need to have EAF, irrespective of their degree of relatedness and the total number of individuals with epilepsy in the family.5,6 EAF also occurs in sporadic cases or in pedigrees with no LGI1 mutation. In these pedigrees, EAF is indistinguishable from LGI1-positive ADEAF. However, the pattern of inheritance may be unclear, there may be phenotypic heterogeneity and EAF may occur in only a minority of cases. Such families do not usually fulfill the criteria for any specific familial epilepsy syndrome. Supplemental data at Neurology.org/ng From the U.O. Medical Genetics (T.P., M.S.), Polyclinic Sant’Orsola-Malpighi, Bologna, Italy; Department of Medical and Surgical Sciences (S.B., F.P., M.S.) and Department of Biomedical and Neuromotor Sciences (L.L., V.M., C.L., C.S., P.T., F.B.), University of Bologna, Bologna, Italy; IRCCS Istituto delle Scienze Neurologiche di Bologna (L.L., V.M., C.L., C.S., P.T., F.B.), Bologna, Italy; Laboratory of Integrative Systems Medicine (LISM) (R.D’.A.), Institute of Informatics and Telematics and Institute of Clinical Physiology, National Research Council, Pisa, Italy; S.C. of Neurology (G.B.), SS. Annunziata Hospital, Taranto, Italy; Epilepsy Centre (G.d’.O.), Clinic of Nervous System Diseases, University of Foggia, Riuniti Hospital, Foggia, Italy; Department of Clinical and Experimental Medicine (A.M.), University of Florence, Florence, Italy; and Florey Institute (I.S.), University of Melbourne, Austin Health and Royal Children’s Hospital, Melbourne, Australia. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was paid by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-Noncommercial No Derivative 4.0 License, which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially. Neurology.org/ng © 2015 American Academy of Neurology 1 ª 2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. The small size, clinical heterogeneity, and were not observed to occur in regions known to be involved , & incomplete penetrance displayed by such fam- in segmental duplications or were rare (allele frequency 1 ) in the Database of Genomic Variants (http://dgv.tcag.ca/dgv/ ilies with EAF may have hampered gene map- app/home) by manual inspection. When WES of 2 affected ping through traditional approaches, such as members of the family (table 1) had been performed, only the linkage analysis. No mutation in genes other variants shared by the 2 were retained. than LGI1 has been identified so far in Validation and segregation analysis of candidate variants and CNVs were performed using Sanger sequencing and quantitative 7–10 EAF. The advent of next-generation PCR analyses, respectively, following standard protocols. sequencing has made the identification of causal variants possible even in small families. RESULTS We included 12 consecutive probands of In this study, we describe a comprehensive Italian ancestry attending the Epilepsy Center of our Institute in Bologna. Three additional cases were whole-exome sequencing (WES) approach referred by 2 other epilepsy centers in Italy. A muta- that enabled us to identify likely pathogenic tion or CNV of LGI116 was excluded in all 15 pro- variants in EAF probands and their affected bands with EAF. Pedigree analysis revealed a total of relatives, demonstrating genetic heterogeneity 34 affected relatives: 6 with EAF (in the 5 families A, underlying this epilepsy phenotype. D, G, H, and I in table 1) and 28 with other epilepsy (table 1). Two families have been previously METHODS Families. From 2001 to 2014, we recruited con- reported,6,17 while the remaining 13 pedigrees are secutive probands with EAF and family history of epilepsy (at described here for the first time. least 1 relative within 3 degrees of kinship affected with EAF or other epilepsy). Molecular genetic findings in 344 “epilepsy genes.” To All affected family members underwent phenotyping, includ- analyze WES data, we initially focused on a set of ing medical and seizure history, neurologic examination, EEG 344 genes (table e-1 at Neurology.org/ng) implicated recordings, and brain MRI. Previous medical records were in monogenic disorders including epilepsy as a obtained. phenotypic feature based on previously published Standard protocol approvals, registrations, and patient work18 and the OMIM database (www.omim.org). consents. We received approval from institutional ethical stand- ards committee on human experimentation, and written We identified 7 candidate variants of 5 genes, each informed consent was obtained from all patients participating in one of 7 families. These variants were validated in in the study or their parents in the case of minors. every individual who underwent exome testing. Molecular genetic analysis. WES was performed on 20 indi- We then tested segregation in all the available viduals: 15 probands and 5 relatives with EAF. DNA was family members, both unaffected and affected by extracted from peripheral blood using the QIAamp DNA Blood EAF or other epilepsies, who had no WES Mini Kit (Qiagen, Venlo, the Netherlands) following the manufac- performed. After testing segregation, 6 variants turer’s protocol. Whole-exome DNA was captured using the remained: 1 CNV in CNTNAP2 (contactin- NimbleGenSeqCap EZ Exome v3 array (Nimblegen Inc., associated protein-like 2; OMIM *604569), 1 SNV Madison, WI) and sequenced as 91-bp paired-end reads on the and 1 InDel in DEPDC5 (DEP domain-containing Illumina HiSeq2000 platform (Illumina Inc., Santa Clara, CA) at * Beijing Genomic Institute (Shenzhen, China). Reads were processed protein 5; OMIM 614191), 1 SNV in SCN1A following a general analysis pipeline described elsewhere.11 (sodiumchannel,voltage-gated,type1,alpha Single nucleotide variants (SNVs) and small insertions and subunit; OMIM *182389), 1 SNV in CACNA1H deletions (InDels) were called with the Genome Analysis (calcium channel, voltage-dependent, T type, 12 ToolKit, and copy number variants (CNVs) were called from alpha-1H subunit; OMIM *607904), and 1 SNV WES data using EXCAVATOR.13 Among SNVs and InDels, in RELN (REELIN, OMIM *600514). nonsynonymous SNVs, splice-site substitutions, and coding small insertions/deletions with a population allele frequency Likely pathogenic variants. CNTNAP2. An intragenic ,1& in public databases (1000 Genomes, http://www. deletion of CNTNAP2 (p.Gln33Argfs*7) was identi- 1000genomes.org; Exome Variant Server, http://evs.gs.wash- fied in both the affected members of family A ington.edu/EVS/; ExAC, http://exac.broadinstitute.org) and (figure 1). The proband (A:IV:1), aged 47 years, pre- absent from approximately 200 patients without epilepsy in our internal exome database were considered

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