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Antimyotonic effects of enantiomers of mexiletine-like drugs
S19
EP2
EP3
ANTIMYOTONIC EFFECTS OF ENANTIOMERS OF MEXILETINE-LIKE DRUGS.
HOMOZYGOUS MYOTONIC CIANICAL AND M ~ THREE UNRELATED CASES.
A. De Luca, F. Natuzzi, S. Piemo, H. Jockusch°, A. Duranti*, G. Lenfini*, F. Franchini*, V. Tcftorella* and D. Conte Camerino. Dept. of Pharmacobiology and *Dept. of Medicinal Chemistry, University of Bari, Italy and °Developmental Biol. Unit., University of Bielefeld, Germany. Mexiletine (Mex), a first choice drug for the myotonic syndromes, stereoselectively blocks skeletal muscle Na" currents (INa). TO search for safer anlimyotonic drugs, the enantiom~rs of ,an homolog (homo-Mex) with an increased hindrance on the chiral carbon atom (methylenamino vs. amino group) were compared with those of Mex in their ability to solve in vitro the hyperexcitabflity of intercostal muscle fibers of myotonic (ADR) mouse, with the two microeleclrode technique. 50BM of S-(+) and R-(-) homo-Mex and of R-(-) Mex reduced the myotonic repetitive firing by 6%, 32% and 30%, respectively. To clarify the structural rextuirement of the stereospecific receptor, the enantiomers of both compounds were tested on INs of frog semitendinosus muscle fibers by voltage clamp recordings (holding potential, h.p.f-100mV). The ICso values for tonically blocking the maximum INa (single test pulse to -20 mV) were 100p.M for R-(-) and 200gM for S-(+) homo-Mex vs. 75gM and 120BM for the R-(-) and S-(+) Mex, respectively. Each compound produced a stereosefective usedependent block of INa after 30s of a 2Hz stimulation, that led to a 2.5 and 1.5 fold lowering of the IC50 values for homo-Mex and Mex enantiomers, respectively. Thus a slight increase of the hindrance on the chiral carbon atom does not alter the stereoselectivity of the compounds. A change in the pKa of the molecule (charged/uncharged ratio), can explain the higher usedependency of homo-Mex with respect to Mex and the comparable antimyotonic action of both drugs (Telethon-Italy, project # 579).
L.Martorell, J.Rosell*, MJ.Sedan&, J.Benitez$, M.Cornet, I.Illa, M.Baiget. Hospital Sant Pan, Barcelona; *Hospital Son Dureta, Mallorca; &Hospital General Yagiie, Burgos; $Fundaci6n Jimenez Diaz, Madrid. We report the clinical and molecular studies of tree unrelated myotonic dystrophy homozygous patients, a very unusual state previously reported only in two asymptomatic sisters and in a congenitally affected man born from an incestuous mating. In the first family, the homozygous patient, shows the classical form of the disease, with a very different expansion sizes in the two alleles (1000 and 60 repeats). In the second family, the homozygous patient is mildly affected and carries a minimally expanded allele (64 repeats) and a "normal predisposed" allele (38 repeats) that increases in size when transmitted. Such an intergenerational expansion of an allele in this range of repeats has not been reported to date. The third homozygous case is mildly affected and she has two minimally expanded alleles (51/120 repeats) that show different intergenerational enlargement during transmission to the next generation.
EP5
EP4 GENOTYPE-PHENOTYPE
DYSTROPHY: STUDIES OF
CORRELATION
AND
M. Gennarelli, G. Novel[i, M. Pavoni, F. Andreasi-Bassi, L. Martorrell, M. Cornet, E. Manegazzo, M.L. Mostaceiuolo, J.M. Martinez, C. Ansclini, A. Pizzuti, M. Baiget, B. I)allapiccola. Catt. di Genetica Umana, Univ. Tof Vm-gata, Rtmaa; IsL dl Fisica, Univ. Cattolica Roma; Ist. Neurologia Univ. di Padova e Univ. di Milano;~e CSS, S. Giovanni Rotoudo Italia. Unit. de Cametica Molecular, Servei de Nmn-ologia, Barcellona, Spagna. A genotyI~phanotype correlation study, based on the clinical symptom in 465 patients with myotonic dystrophy (DM) frtxn Italy and Spain, was carried out to assess the [CTG] repeat number as a Imalictive test of the disease severity. This analysis showed that the DM subtypes defined by strict clinical criteria, including muscular disability, ¢adocrinological dysfimctions, cardiac abnormalities and cognitive functions, fall into three diffca'ent major classes with a log-normal distribution. These cltstrilmfions will allow to calc~at¢ the post~ior Wol~gMlities P(Cln) of em:h patient to belong to a specific phanotypic class (C), on the basis of the [CTG] triplet number (n), using Bayes theccem
P(C]n)=P(C)P(nIC)I~'~P(C)P(rdC)whm: the class f~equancy ol~a'ved in the sample was utilized as marginal probability P(C), ami best fit distribution ftmction of eacla specific class at n as conditional ~ i t y P(nIC). This study dcmoustrates that meammmamt of the lynphocytes' triplet expamious is highly acctwate and valuable for patieats' prognostic ~ e n t . Wo*k supported by Tclethou (Grant. N.511) and Italian Ministry of Health.
MUTATION ANALYSIS OF THE CLCN1 GENE IN I T A L I A N P A T I E N T S A F F E C T E D B Y THOMSEN MYOTONIA CONGENITA AND GENERALIZED BECKER MYOTONIA F. Sangiuolo, A. Botta, A. Mesoraca, L. Merlini, S. Servidei, M. Lo Monaco, P. Tonali, G. Fratta, G. Novelli, B. Dallapiccola Cattedra di Genetica Umana, Universi~ di Roma Tor Vergata, Servizio di Neurologia,/st. Rizzoli, Bologna, 1st. di Neurologia, Universit~ Cattolica del Sacro Cuore, Roma, e Ospedale C.S.S. San Giovanni Rotondo. Autosomal dominant Thomsen myotonia congenita (MC) and autosomal recessive generalized Becker myotonia (GM) are allelic non-dyslxophic disorders characterized by myotonia and muscle stiffness. MC and GM are due to mutation within the CLCN1 gene, the major skeletal muscle chloride channel, located on chromosome 7q35. The CLCNI gene was screened exon by exon in a panel of 23 single unrelated patients using direct sequencing of anomalous SSCA conformers. 12 different mutations was detected, including missense, frameshift and splicing types not described previously, with the exception of R894X. This nonsense mutation was detected in a GM patient.The heterozygous parents did not show clinical myotonia. This observation confirms the double "nature" (dominant or recessive) of the mutation depending on the genetic background of a particular subject. These mutations account for about 43% of CLCNI mutant chromosomes in the Ralian population and support the molecular heterogeneity of GM and MC disease. Work supported by grants from the Italian Ministry of Health and ASM, Milan.
EP2
EP3
ANTIMYOTONIC EFFECTS OF ENANTIOMERS OF MEXILETINE-LIKE DRUGS.
HOMOZYGOUS MYOTONIC CIANICAL AND M ~ THREE UNRELATED CASES.
A. De Luca, F. Natuzzi, S. Piemo, H. Jockusch°, A. Duranti*, G. Lenfini*, F. Franchini*, V. Tcftorella* and D. Conte Camerino. Dept. of Pharmacobiology and *Dept. of Medicinal Chemistry, University of Bari, Italy and °Developmental Biol. Unit., University of Bielefeld, Germany. Mexiletine (Mex), a first choice drug for the myotonic syndromes, stereoselectively blocks skeletal muscle Na" currents (INa). TO search for safer anlimyotonic drugs, the enantiom~rs of ,an homolog (homo-Mex) with an increased hindrance on the chiral carbon atom (methylenamino vs. amino group) were compared with those of Mex in their ability to solve in vitro the hyperexcitabflity of intercostal muscle fibers of myotonic (ADR) mouse, with the two microeleclrode technique. 50BM of S-(+) and R-(-) homo-Mex and of R-(-) Mex reduced the myotonic repetitive firing by 6%, 32% and 30%, respectively. To clarify the structural rextuirement of the stereospecific receptor, the enantiomers of both compounds were tested on INs of frog semitendinosus muscle fibers by voltage clamp recordings (holding potential, h.p.f-100mV). The ICso values for tonically blocking the maximum INa (single test pulse to -20 mV) were 100p.M for R-(-) and 200gM for S-(+) homo-Mex vs. 75gM and 120BM for the R-(-) and S-(+) Mex, respectively. Each compound produced a stereosefective usedependent block of INa after 30s of a 2Hz stimulation, that led to a 2.5 and 1.5 fold lowering of the IC50 values for homo-Mex and Mex enantiomers, respectively. Thus a slight increase of the hindrance on the chiral carbon atom does not alter the stereoselectivity of the compounds. A change in the pKa of the molecule (charged/uncharged ratio), can explain the higher usedependency of homo-Mex with respect to Mex and the comparable antimyotonic action of both drugs (Telethon-Italy, project # 579).
L.Martorell, J.Rosell*, MJ.Sedan&, J.Benitez$, M.Cornet, I.Illa, M.Baiget. Hospital Sant Pan, Barcelona; *Hospital Son Dureta, Mallorca; &Hospital General Yagiie, Burgos; $Fundaci6n Jimenez Diaz, Madrid. We report the clinical and molecular studies of tree unrelated myotonic dystrophy homozygous patients, a very unusual state previously reported only in two asymptomatic sisters and in a congenitally affected man born from an incestuous mating. In the first family, the homozygous patient, shows the classical form of the disease, with a very different expansion sizes in the two alleles (1000 and 60 repeats). In the second family, the homozygous patient is mildly affected and carries a minimally expanded allele (64 repeats) and a "normal predisposed" allele (38 repeats) that increases in size when transmitted. Such an intergenerational expansion of an allele in this range of repeats has not been reported to date. The third homozygous case is mildly affected and she has two minimally expanded alleles (51/120 repeats) that show different intergenerational enlargement during transmission to the next generation.
EP5
EP4 GENOTYPE-PHENOTYPE
DYSTROPHY: STUDIES OF
CORRELATION
AND
M. Gennarelli, G. Novel[i, M. Pavoni, F. Andreasi-Bassi, L. Martorrell, M. Cornet, E. Manegazzo, M.L. Mostaceiuolo, J.M. Martinez, C. Ansclini, A. Pizzuti, M. Baiget, B. I)allapiccola. Catt. di Genetica Umana, Univ. Tof Vm-gata, Rtmaa; IsL dl Fisica, Univ. Cattolica Roma; Ist. Neurologia Univ. di Padova e Univ. di Milano;~e CSS, S. Giovanni Rotoudo Italia. Unit. de Cametica Molecular, Servei de Nmn-ologia, Barcellona, Spagna. A genotyI~phanotype correlation study, based on the clinical symptom in 465 patients with myotonic dystrophy (DM) frtxn Italy and Spain, was carried out to assess the [CTG] repeat number as a Imalictive test of the disease severity. This analysis showed that the DM subtypes defined by strict clinical criteria, including muscular disability, ¢adocrinological dysfimctions, cardiac abnormalities and cognitive functions, fall into three diffca'ent major classes with a log-normal distribution. These cltstrilmfions will allow to calc~at¢ the post~ior Wol~gMlities P(Cln) of em:h patient to belong to a specific phanotypic class (C), on the basis of the [CTG] triplet number (n), using Bayes theccem
P(C]n)=P(C)P(nIC)I~'~P(C)P(rdC)whm: the class f~equancy ol~a'ved in the sample was utilized as marginal probability P(C), ami best fit distribution ftmction of eacla specific class at n as conditional ~ i t y P(nIC). This study dcmoustrates that meammmamt of the lynphocytes' triplet expamious is highly acctwate and valuable for patieats' prognostic ~ e n t . Wo*k supported by Tclethou (Grant. N.511) and Italian Ministry of Health.
MUTATION ANALYSIS OF THE CLCN1 GENE IN I T A L I A N P A T I E N T S A F F E C T E D B Y THOMSEN MYOTONIA CONGENITA AND GENERALIZED BECKER MYOTONIA F. Sangiuolo, A. Botta, A. Mesoraca, L. Merlini, S. Servidei, M. Lo Monaco, P. Tonali, G. Fratta, G. Novelli, B. Dallapiccola Cattedra di Genetica Umana, Universi~ di Roma Tor Vergata, Servizio di Neurologia,/st. Rizzoli, Bologna, 1st. di Neurologia, Universit~ Cattolica del Sacro Cuore, Roma, e Ospedale C.S.S. San Giovanni Rotondo. Autosomal dominant Thomsen myotonia congenita (MC) and autosomal recessive generalized Becker myotonia (GM) are allelic non-dyslxophic disorders characterized by myotonia and muscle stiffness. MC and GM are due to mutation within the CLCN1 gene, the major skeletal muscle chloride channel, located on chromosome 7q35. The CLCNI gene was screened exon by exon in a panel of 23 single unrelated patients using direct sequencing of anomalous SSCA conformers. 12 different mutations was detected, including missense, frameshift and splicing types not described previously, with the exception of R894X. This nonsense mutation was detected in a GM patient.The heterozygous parents did not show clinical myotonia. This observation confirms the double "nature" (dominant or recessive) of the mutation depending on the genetic background of a particular subject. These mutations account for about 43% of CLCNI mutant chromosomes in the Ralian population and support the molecular heterogeneity of GM and MC disease. Work supported by grants from the Italian Ministry of Health and ASM, Milan.