Novel Myoclonin1/EFHC1 Mutations in Mexican Patients with Juvenile

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Short communication

Novel Myoclonin1/EFHC1 mutations in Mexican patients with juvenile

myoclonic epilepsy

a b a a

Aurelio Jara-Prado , Iris E. Martı´nez-Jua´rez , Adriana Ochoa , Vı´ctor M. Gonza´lez ,

c d e

Marı´a del Carmen Ferna´ndez-Gonza´lez-Arago´ n , Minerva Lo´ pez-Ruiz , Marco T. Medina ,

f f a,

Julia N. Bailey , Antonio V. Delgado-Escueta , Marı´a Elisa Alonso *

Neurogenetics and Molecular Biology Department, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Col. La Fama, Tlalpan, Me´xico D.F. 14269, Mexico

Epilepsy Clinic, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Col. La Fama, Tlalpan, Me´xico D.F. 14269, Mexico

Clinical Neurophysiology Department, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Col. La Fama, Tlalpan, Me´xico D.F. 14269, Mexico

Neurology and Neurosurgery Unit, Mexico General Hospital, Dr. Balmis 161, Col. Doctores, Mexico D.F, Mexico

National Autonomous University of Honduras, Tegucigalpa, Honduras

Epilepsy Genetics/Genomics Laboratories and Epilepsy Center of Excellence, Neurology and Research Services, VA GLAHS and David Geffen School of Medicine at UCLA, Los Angeles, CA

90073, USA

A R T I C L E I N F O A B S T R A C T

Article history: Purpose: The purpose of this study was to identify the prevalence of mutations in the Myoclonin1/EFHC1

Received 1 February 2012

gene in Mexican patients with juvenile myoclonic epilepsy (JME).

Received in revised form 28 May 2012

Method: We studied forty-one patients at the National Institute of Neurology and Neurosurgery in

Accepted 29 May 2012

Mexico City and 100 healthy controls. DNA was extracted from the peripheral venous blood of all

participants. The exons of EFHC1 were then ampliﬁed and sequenced.

Keywords:

Results: We found three new putative mutations, all of which were heterozygous missense mutations

Juvenile myoclonic epilepsy

located in exon 3. The ﬁrst identiﬁed mutation, 352C>T, produces a R118C change in the protein and

EFHC1 gene

cosegregated in the patient’s affected father and brother. The second identiﬁed mutation, 544C>T,

Mutations

produces a R182L change in the protein and was found in the patient’s asymptomatic father. The third

identiﬁed mutation, 458>A, produces a R153Q change in the protein and was also found in the patient’s

father. These mutations were not found in controls.

Conclusions: The frequency of Myoclonin1/EFHC1 mutations in our sample is 7.3%. Thus, we conclude that

mutations in the Myoclonin1/EFHC1 gene are an important cause of JME in Mexican patients.

1. Introduction Honduras and Mexico and 3% of JME patients from Japan were found

to carry heterozygous missense, nonsense and deletion frameshift

Juvenile myoclonic epilepsy (JME) is one of the most common mutations in the EFHC1 gene. Here, we report the presence of three

types of genetic generalized epilepsy. JME is characterized by additional novel EFHC1 heterozygous missense mutations in a new

adolescent onset, infrequent absence seizures, awakening myoclon- cohort of forty-one Mexican JME patients. We also compared the

ic seizures and generalized tonic–clonic (GTC) or clonic–tonic– presence of the JME subsyndromes and the persistence of seizures in

clonic (CTC) seizures. Two JME-causing genes have been identiﬁed JME patients with and without EFHC1 mutations.

and their associations with JME have been replicated. These genes

2,3

include GABRA1 on chromosome 5q34q35 and EFHC1 on 2. Methods

chromosome 6p12. In another study, 9% of JME patients from

2.1. Patient and family samples

* Corresponding author at: Insurgentes Sur #3877, Col. La Fama, Del. Tlalpan, Forty-one Mexican JME patients were recruited for the present

Mexico City 14269, Mexico. Tel.: +52 55 56063822x2022; fax: +52 55 54240808.

study from the National Institute of Neurology and Neurosurgery

E-mail addresses: [email protected] (A. Jara-Prado),

(NINN) in Mexico City. The diagnosis of JME was established by two

[email protected] (I.E. Martı´nez-Jua´rez), [email protected]

epileptologists using the following inclusion criteria. (a) The onset

(M.d.C. Ferna´ndez-Gonza´lez-Arago´ n), [email protected]

(M. Lo´ pez-Ruiz), [email protected] (M.T. Medina), [email protected] of myoclonic seizures presented between 8 and 20 years of age. The

(A.V. Delgado-Escueta), [email protected] (M.E. Alonso). myoclonic seizures experienced by the patient were characterized

A. Jara-Prado et al. / Seizure 21 (2012) 550–554 551

by short, bilateral, symmetrical or asymmetrical muscular contrac- JME phenotypes and the persistence of seizures between patients

tions. These seizures predominantly manifested in the shoulders with and without EFHC1 mutations were made using x or Fisher’s

and arms and were less frequently observed in the legs. There was no exact test. The data were analyzed using SPSS version 17.

evidence of loss of consciousness with the seizures. (b) Interictal EEG

of the patient showed diffuse, bilateral, symmetrical and synchro- 2.2. DNA sequencing

nous 3–6 Hz spike, polyspike and slow wave complexes. (c) The

presence of GTC or CTC seizures at the onset or over the course of the Peripheral venous blood was drawn from forty-one JME patients

disease. (d) The patient was required to have a normal neurological and 100 healthy controls. Blood was also drawn from seven family

exam. (e) The patient was required to have normal brain members of the probands with EFHC1 mutations. DNA was extracted

neuroimaging studies, such as computed tomography and/or from each blood sample using the QIAmp DNA blood Mini Kit

magnetic resonance imaging, at the onset of symptoms. (QIAGEN, Valencia, CA, USA). The exons of EFHC1 were ampliﬁed

The exclusion criteria for patients in the study included: (a) using the primers and polymerase chain reaction (PCR) conditions

history of symptomatic seizures; (b) family history of progressive described by Suzuki et al. The PCR products were then puriﬁed and

myoclonic epilepsy; (c) evidence of a progressive neurological sequenced using the Big Dye Terminator sequencing reactions in an

disease that could cause epilepsy and (d) presence of simple partial automated DNA sequencer (ABI PRISM 3130 Applied Biosystems,

or complex seizures. When EFHC1 mutations were identiﬁed in Foster City, CA). The identiﬁed variants were sequenced at least

the patients, blood was drawn from the proband’s family members twice in both the forward and reverse directions.

for DNA analysis. The family members also underwent EEG.

Informed consent was obtained from all participants, and the study 3. Results

was approved by the Bioethics Committee of the NINN. We also

obtained the donated blood samples of 100 anonymous individuals 3.1. Clinical characteristics

without a history of seizures from the NINN blood bank. These

samples were tested as ethnically matched healthy controls. Next, In our cohort of 41 JME patients, 25 (61%) were female. The average

we categorized each of the forty-one JME patients into one of the age at onset of epilepsy was 14.24 years (range 5–20 years). Twenty-

four previously described JME subsyndromes. Comparisons of the one subjects (51.2%) had a positive family history of epilepsy. The

Fig. 1. Families with novel EFHC1 mutations. Brief description: Circles: female; Square: male; Circles and square ﬁlled: affected subjects; Circles and square empty: non

affected subjects; M: mutated; N: no mutated. *Normal EEG. **Abnormal EEG.

552 A. Jara-Prado et al. / Seizure 21 (2012) 550–554

Fig. 2.

majority (33 or 80%) of the patients met the criteria for classic JME, also found in the patient’s affected father and brother (Fig. 1A). The

four patients (10%) had CAE that evolved into JME, two patients (5%) second mutation, 544C>T, substitutes lysine for arginine at

met the criteria for JME with adolescent-onset pyknoleptic and two position 182 of the protein (R182L). The patient’s asymptomatic

patients (5%) met the criteria for JME with astatic seizures. father, who had a normal EEG, also carried the same mutation

(Fig. 1B). The third mutation, 458G>A, replaces an arginine with

3.2. Mutation analysis glutamine at position 153 of the protein (R153Q). The patient’s

asymptomatic father also carried this mutation (Fig. 1C). None of

The three new mutations (7.3%) identiﬁed in this cohort of JME these mutations were found in the controls.

patients were heterozygous missense mutations. They have not The three identiﬁed missense mutations, R118C, R153Q and

been previously reported in the single nucleotide polymorphism R182C, are found in exon 3. In silico analysis shows that the affected

database (SNPs). All three of the novel mutations are located in region includes part of the ﬁrst DM10 domain (position 93–198)

exon 3 of the EFHC1 gene. The ﬁrst identiﬁed mutation, 352C>T, (Figs. 2 and 3) and resides in an evolutionary conserved amino acid

changes the Myoclonin1 protein by substituting cysteine for sequence (EMBL Clustal W2). These mutations were predicted to

arginine at amino acid position 118 (R118C). This mutation was have damaging effects on function by the Polyphen program. The Protein 1 300 640

DM1 DM1 DM1 EF

93-198 239-359 416-520 579-639

Gene

rs804506 rs3804505 rs1266787 rs17851770

1 2 3 4 5 6 7 8 9 10 11 P77T I174 D210N F229L T252K D253Y C259Y A394S N606N

R221 R118C R153Q R182L

New mutationsare shown in bold type

Fig. 3. Diagram of genomic organization of the EFHC1 gene. New mutations are shown in bold type.

A. Jara-Prado et al. / Seizure 21 (2012) 550–554 553

three mutations received PSIC scores of 3.241, 2.278, and 2.124, additional Mexican family in which the R118C mutation segregat-

respectively. ed with the three affected members in two different generations.

The following previously described polymorphisms were also The two other mutations that we have identiﬁed, R182L and

identiﬁed: G545A was found in three patients; G475T was found in R152Q, were transmitted by unaffected parents to 2 unrelated

four patients; G881A was found in one patient; and F1058C and singletons. These mutations can be added to the 12 currently

A1855C were found in two patients. None of these polymorphisms known singleton EFHC1 mutations in the literature. The heterozy-

appear to predispose individuals to JME. gous missense mutations reported here should be considered

putative mutations because functional studies of the effects of

4. Discussion these speciﬁc mutations have not yet been performed. We

originally screened 41 new JME patients to conﬁrm the frequency

In total, 674 SNPs variants have been identiﬁed in the EFHC1 gene. of Myoclonin1/EFHC1 mutations in Mexican families. We wished

They can be found in the human genome database (GENOME 1000). to perform this analysis in our own genetics laboratory, indepen-

Of these variants, 132 are located in exon regions. Prior to our dent of the Los Angeles epilepsy genomics/genetics laboratories.

present report, 22 of these exon variants (18 missense variants, one It is too early to tell how many of the remaining 119 EFHC1

nonsense variant, one three-base deletion variant in the promoter exonic variants reported in GENOME 1000 are pathogenic and how

region, one variant in the 3 UTR and one deletion/frameshift variant) frequently these mutations occur in various populations. However,

were considered to be heterozygous pathogenic mutations that as described previously and in this report, EFHC1 mutations were

were transmitted as autosomal dominant traits. This assumption present in 7% to 9% of JME patients that presented to the

was based on several pieces of evidence. (1) Some studies have Neurology/Epilepsy Clinics of Mexico, Los Angeles and Hon-

4,5

reported the segregation of EFHC1 mutations with the incidence of duras. To date, the GENESS consortium has described fourteen

epilepsy and EEG polyspike waves. These studies were based on different EFHC1 mutations in unrelated JME families, including the

analyses of affected family members across 2–4 generations of 13 three mutations described in this paper. Most of these mutations

JME families from Mexico, Honduras, Japan and Italy. These ﬁndings were also found in our Mexican sample. This result suggests that

suggest that EFHC1 is a Mendelian gene that strongly predisposes EFHC1 mutations are frequently present in Mexican patients with

4–6 4,5

individuals to epilepsy in these rare families. (2) Additionally, JME. In addition to the mutations found in the Mexican JME

EFHC1 mutations were found to be transmitted by unaffected families, heterozygous missense EFHC1 mutations have also been

6 7

parents to 12 JME singletons in studies conducted in Mexico, Austria, described in Caucasian JME families from Italy, Austria, and

Honduras, Tennessee (USA) and Japan. These ﬁndings support the Tennessee, USA. Interestingly, EFHC1 mutations have been

view that these mutations constitute an autosomal dominant trait identiﬁed in individuals with different idiopathic epilepsies and

4–8

with incomplete penetrance. (3) Furthermore, the presence of one type of temporal lobe epilepsy. These results suggest that the

myoclonic and GTC seizures in efhc1/ mice has been reported and EFHC1 mutations may be pleiotropic and may be expressed as

replicated. This result proves that the loss of EFHC1 function alone is various epilepsy phenotypes. Currently, EFHC1 mutations have

sufﬁcient to cause epilepsy. been primarily identiﬁed in JME probands. They are rarely found in

Furthermore, the speciﬁc EFHC1 missense mutations of EFHC1 probands with juvenile absence and pure grand mal seizures.

that segregate in families and are found in singletons may However, EFHC1 mutations are found in the affected family

potentially be pathogenic. The changes of the domain sites at members of individuals with JME, pure grand mal seizures,

which EFHC1 mutations occur have potential deleterious func- childhood pyknoleptic absence seizures, juvenile absence seizures

4–7

tional effects on the encoded myoclonin1 protein, as predicted by and adolescent astatic seizures. The identiﬁed EFHC1 mutations

‘‘in silico analysis’’. Further, the identiﬁed EFHC1 mutations are include deletions, insertions and frameshifts that give rise to stop

often located in evolutionarily conserved domains. Finally, codons. However, genes that encode loss of function proteins are

functional studies of the effect of the individual missense present in normal healthy individuals. Approximately 100 genuine

mutations in vitro indicate that the mutations are pathogenic. loss of function variants and 20 completely inactivated genes have

For instance, the overexpression of EFHC1 in mouse hippocampal been estimated to exist in the human genome. Thus, the

primary neuronal cultures induced apoptosis. This apoptosis was identiﬁed mutations must undergo functional analysis before they

signiﬁcantly decreased in ﬁve types of hippocampal cells each with can be considered to cause epilepsy. This requirement is true for

a distinct EFHC1 missense mutation. These missense mutations the missense and stop codon mutations alike. Furthermore, the

were double heterozygous 229C>A and 662>A; 685T>C; 628G>A; presence of incomplete penetrance may explain the reason that the

757G>T; and 545G>A. In addition, patch clamp analysis of HEK patients’ fathers in families B and C remain asymptomatic and have

2+ 4

cell cultures revealed that EFHC1 speciﬁcally increased R-type Ca normal EEGs despite carrying EFHC1 mutations.

currents. These currents were also shown to increase apoptotic Several lines of evidence point to the mechanisms through

processes. The increased R-type Ca currents were partially which the impaired functions or loss of function mutations of

reversed by each of ﬁve tested EFHC1 missense mutations. EFHC1 may cause epilepsy. In vitro studies of mitosis in various cell

Analyses of the contribution of the EFHC1 variants to ‘‘epilepsy lines as well as in vivo studies in Drosophila melanogaster, show that

risk’’ have also been performed. Bai et al. asked if the coding SNPs EFHC1 is a microtubule-associated protein. EFHC1 co-localizes with

of EFHC1 contributed to the development of epilepsy as minor the mitotic spindle, especially with the spindle poles and

susceptibility alleles with small genetic displacements. This study midbody. In the in vitro experiments, the loss of EFHC1 function

tested an oligogenic or polygenic hypothesis of epilepsy suscepti- disrupts mitotic spindle organization, impairs M phase progres-

bility. Their study included 130 unrelated JME patients and 352 sion, prevents microtubule bundling and increases apoptosis.

family members. No statistically signiﬁcant differences between Experiments in the developing rat cortex have shown that the

the JME probands and controls were found in the association knockdown of EFHC1 by ex vivo and in utero electroporation with

analysis. No transmission disequilibrium was found for the four iRNA disrupts projection neuroblast radial migration. Several steps

tested SNPs (rs3804506, rs3804505, rs1266787, rs17851770) in of neurogenesis are affected to achieve this effect. The cell division,

the family-based association analysis. Thus, in this small cohort, mitosis and cell cycle exit of cortical progenitors are disrupted.

these four EFHC1 SNPs did not confer susceptibility for JME. To Additionally, the organization of the radial glia scaffold and the

date, the cosegregation of Myoclonin1/EFHC1 mutations with JME locomotion of postmitotic neurons in the intermediate zone are

has been reported in 13 JME families. We have identiﬁed an disturbed. The abnormalities in all of these steps contribute to the

554 A. Jara-Prado et al. / Seizure 21 (2012) 550–554

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