Linkage mapping of benign familial infantile convulsions (BFIC) to chromosome 19q

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1 99 Oxford University Press Human Molecular Genetics, 99, Vol. 6, No. - Linkage mapping of benign familial infantile convulsions (BFC) to chromosome 9q Michel Guipponi, Frangois Rivier, Federico Vigevano, Corinne Beck, Arielle Crespel, Bernard Echenne, Pierpaolo Lucchini, Rosella Sebastianelli, Michel Baldy-Moulinier and Alain Malafosse - * laboratory of Experimental Medicine, CNRS UPR 9008, NSERM U9, Montpellier, France, Department of Neuropediatrics, Gui-de-Chauliac Hospital, Montpellier, France, Section of Neurophysiology, Bambino Gesu Children's Hospital, Rome, taly, General Hospital, Treviso, taly and Division of Neuropsychiatry, University Hospital of Geneva, Geneva, Switzerland Received November, 996; Revised and Accepted December, 996 Benign familial infantile convulsions (BFC) are an autosomal-dominant epileptic syndrome characterized by an age of onset within the first year of life. Although they were first reported in families of talian descent, BFC have also been described in non-talian families. We have mapped the BFC gene to chromosome 9 by linkage analysis in five talian families with a maximum two-point lod score of 6.6 at D9S; maximum multipoint lod scores >8 were obtained for the interval D9S0-D9S. BFC are therefore the third idiopathic partial epileptic syndrome to be mapped on the human genome. NTRODUCTON Epilepsy is one of the most common neurological disorders with a cumulative incidence of -% in the general population by age 0 (). However, despite intense research, its basic pathogenetic mechanisms remain poorly known. As several lines of evidence suggest an important genetic contribution to the etiology of the epilepsies, gene mapping is potentially an important experimental approach for understanding the molecular basis of these disorders (). Gene-mapping studies, however, work best when the phenotype can be determined accurately and when the disease gene is highly penetrant (). Consequently, genetic linkage analysis of epileptic syndromes with a Mendelian mode of inheritance, as a first step in order to clone the responsible genes and define their protein product, is now one of the most promising approaches in identifying the pathogenetic mechanisms underlying epilepsies. Mendelian epileptic diseases are rare in human. To date only one syndrome with an autosomal-dominant mode of inheritance has been recognized among the idiopathic epilepsies in the nternational Classification of Epilepsies (): the benign familial neonatal convulsions (BFNC). Linkage of BFNC to chromosome 0q has been initially reported (EBN;,) and evidence for non-allelic genetic heterogeneity has been demonstrated by linkage mapping of a second BFNC locus to chromosome 8q (EBN; 6). Mapping of the other most common idiopathic epilepsies is difficult because of age-dependent penetrance, spontaneous remission and clinical heterogeneity within the same pedigree. These difficulties have been responsible for the discrepancies between the linkage studies of juvenile myoclonic epilepsy (JME) (). Benign familial infantile convulsions (BFC) is a recently recognized idiopathic epileptic syndrome with an autosomaldominant mode of transmission (8). BFC was originally described in families of talian ancestry (8). The disease has also been reported in other talian (9) and non-talian families, in France (0,), Singapore (), Sweden (Liiovigson, personal communication), Germany (Dr Kurlemann, personal communication) and the United States of America (Dr Ryan, personal communication). BFC presents with onset between. and months, seizures of partial type in most cases (according to ictal EEG recordings), no etiological factors, normal psychomotor development, and normal interictal electroencephalograms (EEGs) (8). BFC is inherited in a manner consistent with an autosomal dominant disorder (8). To initiate studies that should lead to the identification of the gene defect responsible for BFC, genetic linkage analysis was performed in five kindreds with this disorder. As a first step, we have used a candidate gene approach in performing linkage analysis using microsatellite polymorphisms flanking the previously mapped epilepsy genes. Due to some similar clinical features between BFNC and BFC (epileptic syndrome with no etiological factors, normal psychomotor development, normal interictal electroencephalograms, and autosomal dominant mode of inheritance) wefirstanalysed the EBN region. This region has been excluded () as well as all other candidate regions so far analysed (unpublished data). Consequently, a systematic approach using microsatellite polymorphisms lying in nonexcluded regions has been used. We now report that the gene for BFC maps to the long arm of chromosome 9 in our five pedigrees. *To whom correspondence should be addressed at: Division de Neuropsychiatrie, Hopital Belle-dee, Chemin du Petit-Bel-Air, Chene-Bourg, Switzerland

2 Human Molecular Genetics, 99, Vol. 6, No. RESULTS Over 0 microsatellites lying in non-excluded regions were genotyped before linkage was detected. Two-point maximum likelihood calculations were performed after each complete genotyping with one marker. We detected one microsatellite polymorphism giving a maximum two-point lod score of.6 at 0 centimorgan from the marker locus DJ9S0. This prompted us to study chromosome 9q with additional markers. Based on their position reported by Weber et al. (), five other microsatellite markers (DJ9S9, D9SJ, D9S9, D9S and D9S) on chromosome 9 were studied. The results of two-point maximum likelihood calculations between the disease phenotype and each of the marker loci are shown in Table, and the pedigree genotyping data for these markers are summarized in Figure. Haplotype analysis (Figs and ), two-point lod scores (Table ), and multipoint analyses (maximum multipoint lod scores >8 obtained for the interval D9S0-DJ9S) suggested that BFC is linked to chromosome 9 in the five pedigrees. Moreover, haplotype analysis showed that there may be a common haplotype in Families \-A (Fig. ). Combined with the fact that these 9q-linked families represent a homogeneous group of pedigrees, since they are from talian descent and seizures recorded in all probands are of partial type (8), this result may indicate the existence of a founder effect. Table. Cumulative and pedigree-specific two-point lod scores for benign familial infantile convulsions versus chromosome 9 markers Locus Recombination 0.00 fraction Zmax D9S9 -co OO D9S D9S D9S D9S ' D9S

3 Human Molecular Genetics, 99, Vol. 6, No. talian family D9S9 D9S0 D9S D9S9 D9SS D9S talian family 0-T-0 : D9S9 f D9S0 D9S D9S9 D9S D9S L O-ri 6 J iri } l LJ i D9S9 D9SO D9S D9S9 D9SS D9SS --0 r- ' im J ih ' UL D9S9 D9S0 D9SU D9S9 D9SS D9S u jl ns Lj w D9S9 V D9SSO D9S D9S9 D9S D9SS D9S9 D9SS0 D9S D9S9 D9S D9S : m V ifir < U.if " - J rim JL t D9S9 nf D9SS0 D9S D9S9 D9SS D9S <UL D9S9 D9S0 D9S D9S9 D9S D9S D9S9 D9S0 D9S D9S9 D9S L D9S L _r J H : f f " LJi r< ) 6 i. -Li. _ J 6 i 6 JU. m± talian family D9S9 D9SS0 D9S D9S9 D9SS D9S O-ri «fl> L J J talian family D9S9 D9S0 D9S D9S 9 D9S D9ffi J D9S9 " : D9S0 D9S D9S9 D9S D9S L LJ : D9S9 ~ J D9S0 D9SU M D9S9 D9S D9S LJ ( >T -0' H 6 J is : 6 l_ J D9S9 D9S0 D9S D9S9 D9S D9S Symbol dchnllions Uaa fretted AfTecled D O Figure. The observed alleles with markers D9S9, D9S0, D9S, D9S9, D9S and D9S in five BFC families. The phasing of haplotypes was inferred as most likely using all available genotype data for the families. The segment of conserved disease-bearing chromosome inherited by a member is contained within the rectangle. The pedigree designations for all families are consistent with those used previously (). (A.O.C.O: apparent obligate cross-over). DSCUSSON We mapped BFC to chromosome 9q, with no evidence of heterogeneity within our family sample. The main clinical features of BFC (afebrile seizures between. and months of age, normal neurodevelopmental status, normal interictal electroencephalogram and no demonstrable underlying pathology) led us to consider this syndrome as an idiopathic epilepsy. Thus BFC follows autosomal dominant nocturnal frontal epilepsy () and a partial epileptic syndrome with auditory features (6) as the

4 6 Human Molecular Genetics, 99, Vol. 6, No. Families D9S9 D9S0 D9S D9S9 D9S D9S? Figure. Haplotypes on benign familial infantile convulsions chromosomes for D9S9, D9S0, DJ9S, D9S9, DJ9S and D9S. third form of inherited idiopathic epilepsy that has had its disease locus genetically mapped. These results confirm the genetic basis, the autosomal inheritance and the accuracy of the clinical diagnostic criteria of BFC. Moreover, the mapping of the BFC gene on a different chromosome from BFNC definitively demonstrates that these two idiopathic epilepsies are not allelic, and thus confirm our previous results () which indicated that this epileptic syndrome is distinct from 0q-linked BFNC. The existence of a founder effect in these talian 9q-linked BFC families is suggested by the common haplotype found in our pedigrees (Fig. ). Haplotypes in pedigrees and may indicate that the BFC gene is located between D9S and D9S9. However, to confirm this hypothesis a larger number of talian families and more genealogical information from the pedigrees is required. Clinical characteristics may indicate that BFC is a localisation-related epilepsy. The implication of this locus in other inherited PE remains to be tested. Because of the occipital discharges observed in 9q-l inked BFC, benign occipital epilepsy, which is characterized by an age of onset between and 8 years and an autosomal dominant mode of inheritance (), should represent a good candidate for such studies. t is currently impossible to determine whether the different kinds of seizures in BFNC and BFC are directly caused by the responsible genes or are in fact the result of interactions between them and different developmental factors. Conversely, the specific ages of onset of BFNC and BFC may indicate that the corresponding genes play a role in the maturation of regulatory processes of cerebral excitability. The next step will be to narrow down the BFC region, and identify candidate genes from these regions. Thus, the identification of the gene defect responsible for BFC may provide new and unsuspected clues into the mechanisms by which neuronal excitability is regulated. Strategies in the search for linkage and genetic marker typing EDTA blood samples (0 ml) were collected and DNA was prepared from isolated lymphocytes by SDS lysis, proteinase K digestion, phenol/chloroform extraction, ethanol precipitation and Tris-EDTA resuspension. Using the polymerase chain reaction (PCR), hypervariable microsatellites flanking loci responsible for BFNC (,6), JME () and progressive myoclonus epilepsies (8) were assessed in a first attempt to localize the BFC gene. Due to the exclusion of these candidate regions, other microsatellites from the Genethon collection (9) and developed by other authors were further investigated. Polymorphism analysis was performed by PCR amplifications in il containing 0-00 ng of genomic DNA,. mm MgCl,00 pmols of each primer,. mm of each dntps, 0. u.ci [oc- P]dCTP and 0. U Taq polymerase (Amersham). Amplification conditions were 9 C for 0 min followed by 0 cycles at 9 C for 0 s, specific annealing temperature and duration for each primers, C for 0 s and a final extension of 0 min at C. Amplified DNA ( \i\) was mixed with (il formamide and 0. ju,l loading buffer (xylene cyanol/bromophenol blue/glycerol), electrophoresed at 00 V for h in 6% denaturing polyacrylamide gels. The dried gel was exposed to X-ray film for -8 h at -0 C. Linkage analysis Linkage analysis was performed using the M-LNK and LNKMAP options of the. version of the LNKAGE program (0). LOD score values were calculated under the assumption of single-gene autosomal dominant inheritance with a complete penetrance and a gene frequency of 0.00 for the BFC allele, in view of the low prevalence of this condition. Distances between marker loci used in the multipoint linkage analysis were those estimated by Weber et al. (). ACKNOWLEDGEMENTS We thank the families for their kind cooperation, and Genethon for technical assitance. This work was supported by grants from the Association Franchise contre les Myopathies, the Groupement d'etude et de Recherche sur les Genomes, the Centre National de la Recherche Scientifique, the nstitut National de la Recherche Medicale, and the Fond National Suisse pour la Recherche. C.B. received a fellowship from the Association Franc,aise contre les Myopathies. F.R. and A.C. received a fellowship from the Fondation pour la Recherche Medicale. MATERALS AND METHODS Clinical description The five pedigrees included in the present study (Fig. ) have been previously described (). Family from Malafosse et al. () has not been included because of insufficient data and the impossibility of obtaining additional information. 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