Iris E. Martínez-Juárez

Hyperglycosylation and Reduced GABA Currents of Mutated GABRB3 Polypeptide in Remitting Childhood Absence Epilepsy

Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.

Novel mutations in Myoclonin1/EFHC1 in sporadic and familial juvenile myoclonic epilepsy

Background: Juvenile myoclonic epilepsy (JME) accounts for 3 to 12% of all epilepsies. In 2004, the GENESS Consortium demonstrated four missense mutations in Myoclonin1/EFHC1 of chromosome 6p12.1 segregating in 20% of Hispanic families with JME. Objective: To examine what percentage of consecutive JME clinic cases have mutations in Myoclonin1/EFHC1. Methods: We screened 44 consecutive patients from Mexico and Honduras and 67 patients from Japan using heteroduplex analysis and direct sequencing. Results: We found five novel mutations in transcripts A and B of Myoclonin1/EFHC1. Two novel heterozygous missense mutations (c.755C>A and c.1523C>G) in transcript A occurred in both a singleton from Mexico and another singleton from Japan. A deletion/frameshift (C.789del.AV264fsx280) in transcript B was present in a mother and daughter from Mexico. A nonsense mutation (c.829C>T) in transcript B segregated in four clinically and seven epileptiform-EEG affected members of a large Honduran family. The same nonsense mutation (c.829C>T) occurred as a de novo mutation in a sporadic case. Finally, we found a three-base deletion (−364○%–362del.GAT) in the promoter region in a family from Japan. Conclusion: Nine percent of consecutive juvenile myoclonic epilepsy cases from Mexico and Honduras clinics and 3% of clinic patients from Japan carry mutations in Myoclonin1/EFCH1. These results represent the highest number and percentage of mutations found for a juvenile myoclonic epilepsy causing gene of any population group. GLOSSARY: CAE = childhood absence epilepsy; FS = febrile seizures in infancy/childhood; GM = grand mal tonic clonic seizure; JME = Juvenile myoclonic epilepsy; PSW = 3–6 Hz polyspike and slow wave complexes; SW = single spike and slow wave complex.

Seizures of Idiopathic Generalized Epilepsies

Summary:  Idiopathic generalized epilepsies (IGEs) comprise at least 40% of epilepsies in the United States, 20% in Mexico, and 8% in Central America. Here, we review seizure phenotypes across IGE syndromes, their response to treatment and advances in molecular genetics that influence nosology. Our review included the Medline database from 1945 to 2005 and our prospectively collected Genetic Epilepsy Studies (GENESS) Consortium database. Generalized seizures occur with different and similar semiologies, frequencies, and patterns, ages at onset, and outcomes in different IGEs, suggesting common neuroanatomical pathways for seizure phenotypes. However, the same seizure phenotypes respond differently to the same treatments in different IGEs, suggesting different molecular defects across syndromes. De novo mutations in SCN1A in sporadic Dravet syndrome and germline mutations in SCN1A, SCN1B, and SCN2A in generalized epilepsies with febrile seizures plus have unraveled the heterogenous myoclonic epilepsies of infancy and early childhood. Mutations in GABRA1, GABRG2, and GABRB3 are associated with absence seizures, while mutations in CLCN2 and myoclonin/EFHC1 substantiate juvenile myoclonic epilepsy as a clinical entity. Refined understanding of seizure phenotypes, their semiology, frequencies, and patterns together with the identification of molecular lesions in IGEs continue to accelerate the development of molecular epileptology.

Novel Myoclonin1/EFHC1 mutations in Mexican patients with juvenile myoclonic epilepsy

PURPOSE The purpose of this study was to identify the prevalence of mutations in the Myoclonin1/EFHC1 gene in Mexican patients with juvenile myoclonic epilepsy (JME). METHOD We studied forty-one patients at the National Institute of Neurology and Neurosurgery in 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. RESULTS We found three new putative mutations, all of which were heterozygous missense mutations located in exon 3. The first identified mutation, 352C>T, produces a R118C change in the protein and cosegregated in the patients affected father and brother. The second identified mutation, 544C>T, produces a R182L change in the protein and was found in the patients asymptomatic father. The third identified mutation, 458>A, produces a R153Q change in the protein and was also found in the patients 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.

DNA variants in coding region of EFHC1: SNPs do not associate with juvenile myoclonic epilepsy

Purpose:  Juvenile myoclonic epilepsy (JME) accounts for 3 to 12% of all epilepsies. In 2004, we identified a mutation‐harboring Mendelian gene that encodes a protein with one EF‐hand motif (EFHC1) in chromosome 6p12. We observed one doubly heterozygous and three heterozygous missense mutations in EFHC1 segregating as an autosomal dominant gene with 21 affected members of six Hispanic JME families from California and Mexico. In 2006, similar and three novel missense mutations were reported in sporadic and familial Caucasian JME from Italy and Austria. In this study, we asked if coding single nucleotide polymorphisms (SNPs) of EFHC1 also contribute as susceptibility alleles to JME with complex genetics.

EFHC1 variants in juvenile myoclonic epilepsy: reanalysis according to NHGRI and ACMG guidelines for assigning disease causality

Purpose:EFHC1 variants are the most common mutations in inherited myoclonic and grand mal clonic-tonic-clonic (CTC) convulsions of juvenile myoclonic epilepsy (JME). We reanalyzed 54 EFHC1 variants associated with epilepsy from 17 cohorts based on National Human Genome Research Institute (NHGRI) and American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants.Methods:We calculated Bayesian LOD scores for variants in coinheritance, unconditional exact tests and odds ratios (OR) in case–control associations, allele frequencies in genome databases, and predictions for conservation/pathogenicity. We reviewed whether variants damage EFHC1 functions, whether efhc1−/− KO mice recapitulate CTC convulsions and “microdysgenesis” neuropathology, and whether supernumerary synaptic and dendritic phenotypes can be rescued in the fly model when EFHC1 is overexpressed. We rated strengths of evidence and applied ACMG combinatorial criteria for classifying variants.Results:Nine variants were classified as “pathogenic,” 14 as “likely pathogenic,” 9 as “benign,” and 2 as “likely benign.” Twenty variants of unknown significance had an insufficient number of ancestry-matched controls, but ORs exceeded 5 when compared with racial/ethnic-matched Exome Aggregation Consortium (ExAC) controls.Conclusions:NHGRI gene-level evidence and variant-level evidence establish EFHC1 as the first non–ion channel microtubule–associated protein whose mutations disturb R-type VDCC and TRPM2 calcium currents in overgrown synapses and dendrites within abnormally migrated dislocated neurons, thus explaining CTC convulsions and “microdysgenesis” neuropathology of JME.Genet Med 19 2, 144–156.

Late onset Lafora disease and novel EPM2A mutations: Breaking paradigms

Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy with classic adolescent onset of stimuli sensitive seizures. Patients typically deteriorate rapidly with dementia, ataxia, vegetative failure and death by 25 years of age. LD is caused by homozygous mutations in EPM2A or EPM2B genes. We found four novel mutations in EPM2A - three in exon 4 (Q247X, H265R G279C) and one in exon 1 (Y86D) - and a previously described mutation in exon 4 (R241X). These five EPM2A mutations were found in four index cases and affected relatives. Patient 1 with classic LD was doubly heterozygous for H265R and R241X in exon 4; while Patient 2, who also had classic LD, was homozygous for Q247X in exon 4. Patient 3 with classic LD was homozygous for Y86D in exon 1, but the same mutation in his affected brother manifested an atypical earlier childhood onset. For the first time, we describe a later onset and slower progression of EPM2A-deficient LD seen in Patient 4 and her three sisters who were doubly heterozygous for R241X and G279C in exon 4. In these sisters, seizures started later at 21 to 28 years of age and progressed slowly with patients living beyond 30 years of age. Our observations suggest that variations in phenotypes of EPM2A-deficient LD, like an earlier childhood or adolescent or later adult onset with a rapid or slower course, depend on a second modifying factor separate from pathogenicity or exon location of EPM2A mutations. A modifying gene amongst the patients genetic background or environmental factors may condition age of onset and rapid or slow progression of LD.

High-dose versus low-dose valproate for the treatment of juvenile myoclonic epilepsy: Going from low to high.

Juvenile myoclonic epilepsy (JME) is a genetic generalized epilepsy accounting for 3-12% of adult cases of epilepsy. Valproate has proven to be the first-choice drug in JME for controlling the most common seizure types: myoclonic, absence, and generalized tonic-clonic (GTC). In this retrospective study, we analyzed seizure outcome in patients with JME using valproate monotherapy for a minimum period of one year. Low valproate dose was considered to be 1000mg/day or lower, while serum levels were considered to be low if they were at or below 50mcg/dl. One hundred three patients met the inclusion criteria. Fifty-six patients (54.4%) were female. The current average age was 28.4±7.4years, while the age of epilepsy onset was 13.6±2.9years. Most patients corresponded to the subsyndrome of classic JME. Forty-six (44.7%) patients were free from all seizure types, and 76 (73.7%) patients were free from GTC seizures. No significant difference was found in seizure freedom among patients using a low dose of valproate versus a high dose (p=0.535) or among patients with low blood levels versus high blood levels (p=0.69). In patients with JME, it seems appropriate to use low doses of valproate (500mg to 1000mg) for initial treatment and then to determine if freedom from seizures was attained.

Variant Intestinal-Cell Kinase in Juvenile Myoclonic Epilepsy

BACKGROUND In juvenile myoclonic epilepsy, data are limited on the genetic basis of networks promoting convulsions with diffuse polyspikes on electroencephalography (EEG) and the subtle microscopic brain dysplasia called microdysgenesis. METHODS Using Sanger sequencing, we sequenced the exomes of six members of a large family affected with juvenile myoclonic epilepsy and confirmed cosegregation in all 37 family members. We screened an additional 310 patients with this disorder for variants on DNA melting‐curve analysis and targeted real‐time DNA sequencing of the gene encoding intestinal‐cell kinase (ICK). We calculated Bayesian logarithm of the odds (LOD) scores for cosegregating variants, odds ratios in case–control associations, and allele frequencies in the Genome Aggregation Database. We performed functional tests of the effects of variants on mitosis, apoptosis, and radial neuroblast migration in vitro and conducted video‐EEG studies in mice lacking a copy of Ick. RESULTS A variant, K305T (c.914A→C), cosegregated with epilepsy or polyspikes on EEG in 12 members of the family affected with juvenile myoclonic epilepsy. We identified 21 pathogenic ICK variants in 22 of 310 additional patients (7%). Four strongly linked variants (K220E, K305T, A615T, and R632X) impaired mitosis, cell‐cycle exit, and radial neuroblast migration while promoting apoptosis. Tonic–clonic convulsions and polyspikes on EEG resembling seizures in human juvenile myoclonic epilepsy occurred more often in knockout heterozygous mice than in wild‐type mice (P=0.02) during light sleep with isoflurane anesthesia. CONCLUSIONS Our data provide evidence that heterozygous variants in ICK caused juvenile myoclonic epilepsy in 7% of the patients included in our analysis. Variant ICK affects cell processes that help explain microdysgenesis and polyspike networks observed on EEG in juvenile myoclonic epilepsy. (Funded by the National Institutes of Health and others.)

A comparison of waiting times for assessment and epilepsy surgery between a Canadian and a Mexican referral center

To provide a comprehensive transnational overview of wait times for epilepsy surgery in Canada and Mexico.