ESHG Posters 19

R. Nabbout¹^,2, J. Prud'Homme², A. Hermann³, A. Brice³^,4, O. Dulac⁵, E. Leguern³^,4;
¹INSERM, Paris, FRANCE, ²Généthon, Evry, FRANCE, ³U289, INSERM, Paris, FRANCE, ⁴Departement de Génétique, Cytogénétique et Embryologie, Hôpital Pitié-Salpêtrière, Paris, FRANCE, ⁵Hôpital Saint Vincent de Paul, Paris, FRANCE.

We report a large multigenerational French family with a homogeneous phenotype consisting of isolated simple Febrile Seizures (FS). The FS trait did not show any linkage with the reported loci for FS and Generalised Epilepsy with Febrile Seizures (GEFS+). After a genome scan, a new locus for FS was identified.
Patients and methods: Clinical study: Our family consisted of 166 individuals on 5 generations. Affected members presented a history of simple FS that segregates as autosomosal dominant trait. Genotyping and linkage analysis: After exclusion of reported loci for FS and GEFS+, a genome-wide search was performed with 380 markers.
Results: All affected members presented FS that fulfil the criteria of simple FS. All FS ceased before 5 years of age and no later afebrile seizures or epilepsy were reported. In the oldest generation (II), the status of all members was considered as unknown for the reliability of the genetic analysis. The genome wide search allowed the identification of a new candidate region (Zmax=3.31 at q=0.00). Multipoint analysis and haplotypes construction confirmed the genetic linkage of this region to simple FS trait.
Conclusion: Our family presents a homogeneous phenotype consisting of isolated simple FS, the commonest form of FS. A new locus is identified in this family and the sequence analysis of a potential candidate gene is in progress.

Clinical and Genetic Analysis of a New Multigenerational Pedigree with GEFS+ (Generalized Epilepsy with Febrile Seizures Plus)

P. Roll¹, S. Pereira¹, F. Gerard², S. Jamali¹, A. Lemainque³, A. Robaglia-Schlupp¹, P. Genton², M. G. Lathrop³, P. Szepetowski¹;
¹INSERM U491, Marseille, FRANCE, ²Centre St Paul, Marseille, FRANCE, ³CNG, Evry, FRANCE.

Febrile seizures (FS) affect 3% of all children under six years of age. A small proportion of children with FS later develops epilepsy. While most FS show complex inheritance, a small proportion of FS is autosomal dominant. Two FS genes have been localized at 8q14-q21 and 19p. The syndrome of generalized epilepsy with febrile seizures plus (GEFS+) is a heterogeneous disorder characterized by febrile seizures that may persist beyond the age of six, and non-febrile seizures. GEFS+ is an autosomal dominant disorder and three genes (SCN1A, SCN1B, GABRG2) have been identified to date at 2q34, 19q13, and 5q34, respectively, while a fourth GEFS+ locus (5q14-q15) has been suggested.
A large multigenerational GEFS+ family was collected in France. All affected members had typical FS. Among them, seven had other types of seizures including FS after the age of 6 years, non-febrile generalized seizures, or partial seizures later in life.
Exclusion study of candidate genes and loci was performed with penetrance at 0.9 and phenocopy rates at 0.02 or 0.03. Multipoint LOD scores < -2 were obtained at 5q14-q15, 8q14-q21 and 19p. In addition, the genomic areas surrounding the SCN1A, SCN1B and GABRG2, were also excluded.
Our data provide further evidence for the high level of genetic heterogeneity associated with familial febrile seizures and GEFS+, and prove the existence of a new GEFS+ gene situated elsewhere in the genome. Genome-wide scan is now underway in order to localize this new GEFS+ gene and preliminary data will be presented.

R. L. Touraine, A. Combes, F. Prieur, B. de Freminville, B. Lauras;
CHU de SAINT ETIENNE, Saint Etienne, FRANCE.

Familial episodic ataxia are rare genetically heterogeneous neurological disorders. Type 1, or myokymia with periodic ataxia (OMIM 160120) is characterized by short-lasting attacks of ataxia, sometimes accompanied by jerking movements, episodes of dizziness and permanent tremor of the head and hands (myokymia). This disorder has been ascribed to alteration of a potassium channel subunit, encoded by the KCNA1 gene, localized in 12p13.
We describe a large French family with 11 affected patients over 3 generations. The propositus, a young lady, is particularly handicapped by myokymia. She was previously diagnosed as having a psychiatric disturbance.
Direct DNA sequencing of the KCNA1 gene disclosed a novel heterozygous misense mutation, leading to a Leucine to Phenylalanine substitution at position 305, in the 4th transmembrane domain. The mutation was confirmed by enzymatic restriction analysis with the disappearance of an AluI cutting site. This Leucine residue is highly conserved through evolution, from drosophila to human. All the studied affected family members carried this mutation that is absent in 100 unrelated and unaffected individuals. Therefore, it is likely that this Leu to Phe substitution at position 305 is pathogenic. Only speculations can be made regarding the possible consequences of the Leu to Phe substitution for the K+ channel function.
This autosomal dominant disorder is important to recognize, not only to avoid a erroneous diagnosis of psychiatric disturbance that frequently mislabel these patients, but also to offer specific therapies.

C. M. E. Tallaksen¹^,2, E. Guichard-Gomez², V. Hahn-Barma², J. Hazan³, B. Fontaine², B. Dubois², A. Dürr¹^,4;
¹Inserm U289, Paris, FRANCE, ²Fédération de Neurologie, Paris, FRANCE, ³Génoscope, Evry, FRANCE, ⁴Département de Génétique, Cytogénétique et Embryologie, Paris, FRANCE.

Sixty five individuals from 10 French families with identified mutations of the spastin gene (SPG4) participated in a study on cognitive function. The study was conducted to verify previous reports on the association of cognitive impairment and hereditary pure spastic paraparesis. Thirty six carriers (24 female, 12 male) including 5 asymptomatic at the time of the study as well as 29 of their non-carriers siblings (18 female, 11 male) were included in the study. The Cambridge Cognitive Examination test (CAMCOG) was used in all subjects, supplemented by additional neuropsychological tests for 30 individuals to evaluate executive functions, memory and visual form discrimination.
There was no overall significant difference between carriers and non-carriers. However, in the group with age over 50 years, a significant cognitive impairment was found in carriers compared to non-carriers, with lower scores in several parameters of executive functions. Similar results have been reported previously in Irish families with spastic paraparesis. These findings suggest a wider involvement of subcortical and, may be, cortical functions even in pure spastic paraparesis after 50 years.

S. Adinolfi¹, S. Martin², A. Pastore²;
¹National Institute of Medical, London, UNITED KINGDOM, ²National Institute of Medical Research, London, UNITED KINGDOM.

Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative disease, is caused by low expression of frataxin: a small mitochondrial protein, encoded into the nucleus. At biochemical level the lack of frataxin leads to disregulation of mitochondrial iron homeostasis and oxidative damage which eventually causes neuronal death. Recently, it has been reported the Yfh1 (yeast frataxin homologue) shows a ferritin-like behaviour in vitro: the protein was shown to form large aggregates able to sequester iron form solution in the presence of an iron excess (1). No direct iron binding was however shown for human frataxin under similar conditions (2). We have carried out an exhaustive study on three frataxin orthologues from E. coli, yeast and from human with the aim of further testing the working hypothesis of a direct involvement of frataxin in iron binding. Using
these three proteins, selected as representatives of different evolution steps, we have characterised their fold and thermodynamic stability, compared their ion binding specificity and their tendencies to aggregate. A number of mutants was produced to identify the protein surface involved in these functions. Our work leads us to a more complex and complete picture of the binding and aggregative properties of frataxins.
References
1) Adamec J, Rusnak F, Owen WG, Naylor S, Benson LM, Gacy AM,Isaya G.
Am J Hum Genet. 2000 Sep;67(3):549-62.
2) Musco G, Stier G, Kolmerer B, Adinolfi S, Martin S, Frenkiel T, Gibson T, Pastore A
Structure Fold Des. 2000 Jul 15;8(7):695-707.

Spastin, the protein mutated in autosomal dominant hereditary spastic paraplegia, is involved in microtubule dynamics

A. Errico¹, P. Claudiani¹, A. Ballabio¹^,2, E. I. Rugarli¹;
¹Telethon Institute of Genetics and Medicine, Napoli, ITALY, ²II University of Naples, Napoli, ITALY.

Hereditary spastic paraplegia (HSP) is a neurodegenerative disease characterised by weakness and spasticity of the lower limbs due to degeneration of the corticospinal tracts. The gene responsible for the most frequent form of autosomal dominant HSP (SPG4) encodes spastin, an ATPase belonging to the AAA proteins family. Interestingly, almost all the missense mutations found in HSP patients fall into the AAA functional domain.
The cellular pathways in which spastin operates and its role in causing degeneration of motor axons are still unclear. By expressing wild-type or ATPase-defective spastin in several cell types, we show that spastin interacts dynamically with microtubules. This association is mediated by the N-terminal region of the protein and regulated through the ATPase activity of the AAA domain. Expression of missense mutations falling into the AAA cassette leads to constitutive binding to microtubules in transfected cells and induces the disappearance of the aster and the formation of thick perinuclear bundles, suggesting a role of spastin in microtubule dynamics. Moreover, overexpression of wild-type spastin seems to promote microtubule disassembly in transfected cells. These data suggest that the degeneration of corticospinal axons, in HSP patients, could be due to impairment of fine regulation of the microtubule cytoskeleton.
To better understand spastin localization, specific antibodies have been produced and experiments to determine the endogenous protein localization are in progress. Furthermore, a recombinant spastin obtained with the baculovirus system will be used in microtubule severing assay in order to confirm the hypothesized role of spastin in microtubule disassembly.

Mice lacking paraplegin, a mitochondrial AAA protease involved in hereditary spastic paraplegia, show axonal degeneration and abnormal mitochondria

F. Ferreirinha¹, A. Quattrini², V. Valsecchi¹, M. Pirozzi¹, A. Ballabio¹^,3, E. I. Rugarli¹;
¹Telethon Institute of Genetics and Medicine, Napoli, ITALY, ²San Raffaele Hospital, Milano, ITALY, ³II University of Naples, Napoli, ITALY.

Hereditary spastic paraplegia (HSP) is a progressive neurological disorder characterized by degeneration of the corticospinal tracts. The gene responsible for the autosomal recessive form linked to chromosome 16q (SPG7) encodes paraplegin, a mitochondrial ATPase involved in protein quality control in the inner mitochondrial membrane. In order to study the pathogenesis of HSP due to lack of paraplegin, we have generated a mouse model by inactivation of the Spg7 gene. Paraplegin deficient mice are born at the expected mendelian ratio, are viable, and fertile. At approximately 6 months of age homozygous Spg7 -/- animals start displaying an impaired performance on the rotarod apparatus. Semithin sections of the spinal cord of 7-months-old animals show a variable number of focal axonal swellings in the distal axons of the fasciculus gracilis and of descending spinal tracts, consistent with a retrograde axonopathy. This phenotype is slowly progressive, with signs of axonal degeneration becoming prominent at 12 month of age. Mice older than one year show additional phenotypes, such as axonal swelling and degeneration in the optic and sciatic nerves, and muscle abnormalities. Electron microscopy analysis of affected spinal cords demonstrates that, long before degeneration, axons are filled with mitochondria with abnormal morphology, indicating that mitochondrial dysfunction is likely the cause for the disease. Swollen axons contain accumulated organelles and neurofilaments, suggesting that axonal degeneration may be due to impaired axonal transport. We are currently analyzing whether defective ATP production, oxidative stress or apoptosis are important determinants of pathology in our HSP animal model.

The transcription factor SOX10 regulates PLP expression in the central nervous system

M. Girard¹, N. Bondurand¹, N. Lemort¹, V. Pingault¹^,2, M. Goossens¹^,2;
¹INSERM U468, Creteil, FRANCE, ²Laboratoire de Biochimie et Génétique Moléculaire, AP-HP, Creteil, FRANCE.

SOX10 is an essential factor for the enteric nervous system (ENS), melanocytes and glial cells development. Mutations in the SOX10 gene were described in several cases of Shah-Waardenburg syndrome, a neurocristopathy characterized by the association of Hirschsprung disease (intestinal aganglionosis) and Waardenburg syndrome (pigmentation defects and sensorineural deafness). In accordance, it was shown that SOX10 controls expression of MITF and RET, which play important roles during melanocytes and ENS development, respectively. Some patients also present with myelination defects of the central (CNS) and peripheral nervous system (PNS), which is in agreement with the demonstration that P0 and Cx32, two major proteins of the PNS, are controlled by SOX10. Nevertheless, these findings cannot explain the defects of the CNS, consistent with Pelizaeus-Merzbacher disease (PMD), observed in one patient. This suggests that SOX10 may regulate other genes involved in the myelination process of the CNS.
To test this hypothesis, we sought the possible involvement of SOX10 in the regulation of expression of PLP and its alternative transcript DM20. Both proteins are major components of myelin in the CNS, and mutations of the PLP gene are associated with PMD. Here we show that SOX10 activates expression of the PLP gene in transfection assays, and that EGR2, another major regulator in the CNS, is also able to regulate the PLP promoter. These results were further confirmed by the study of a cell line expressing SOX10 in an inducible manner, where PLP and DM20 expression is upregulated when SOX10 is induced.

D. Simon¹, H. Puccio¹, N. Lagarde¹, L. Reutenauer¹, A. Gansmuller¹, P. Rustin², J. L. Mandel¹, M. Koenig¹;
¹IGBMC, Strasbourg, FRANCE, ²Hôpital Necker, Paris, FRANCE.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, associates degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes. FRDA is caused by severely reduced levels of frataxin, a mitochondrial protein of unknown function. Data from yeast and patients indicate that frataxin defect causes a specific iron-sulfur protein deficiency and mitochondrial iron accumulation, suggesting oxidative damage involvement.
As complete absence of frataxin in mouse was lethal early in development, we have chosen a conditional gene targeting approach, to generate first a heart frataxin-deficient line and a neuron/heart frataxin-deficient line. The heart line reproduces well the different steps of the human cardiac defects, but the neuron/heart line (generated to study the neurological defects) was very severe. Indeed, the mutant mice showed a short life expectancy (25 days), probably due to many additional lesions in brain and other tissues.
To better reproduce the natural evolution of the neurological symptoms, we have generated two inducible neuronal frataxin-deficient lines by using tamoxifen inducible recombinase. The deletion of frataxin is induced at 4 weeks of age. In one line the deletion occurs in the central and the peripheral nervous system and the other line presents a deletion more specific of the cerebellum.
The mice have a normal life expectancy but develop a progressive spinocerebellar degeneration revealed by histology, electron microscopy and behavioural studies to assess the evolution of the symptoms.
These models represent good models to evaluate treatment strategies for the neurological side of the human disease.

S. Kotov, O. Sidorova, V. Neretin, O. Evgrafov, A. Petrin, E. Dadali;
Moscow Regional Research Clinical Institute (MONIKI), Moscow, RUSSIAN FEDERATION.

Huhtington’s chorea is a severe hereditary neurodegenerative disease characterized by choreic hyperkinesis and progressing dementia. The object of the present study was creation of genetic register of the Huntington’s chorea in the Moscow Region in order to organize medico-genetic consulting patients using up-to-date diagnostic methods. Direct and indirect registration methods were applied. The total of 111 families suffering from the Huntington’s chorea was registered in the Moscow region. Molecular-genetic method was used to confirm the primary diagnosis. Diagnosis was considered confirmed if the number of repeating cytosine-adenine-guanine sequences in the disease gene molecule exceeded 37. Probands’ children underwent molecular-genetic investigation to determine disease gene carriers. Siblings were also examined if the disease in question was found in more than one generation. In cases when prenatal diagnosis revealed the disease gene carriage by probands’ children, the latter were prescribed to undergo DNA-investigation. Haloperidolum was used for treatment of the Huntington’s chorea patients.

Study of the normal CAG tract at the Huntington disease locus in the Portuguese population

M. C. Costa¹, L. Guimarães¹, F. Ferreirinha¹, A. Sousa¹^,2, P. Maciel¹^,2, J. Sequeiros¹^,2;
¹UnIGENe/IBMC, University of Porto, PORTUGAL, ²Dept. Estudos das Populações/ICBAS, University of Porto, PORTUGAL.

Huntington disease (HD) is a dominant disorder caused by the expansion of a (CAG)n localised on the first exon of the gene, which contains (1) 6-26 CAGs in normal stable alleles, (2) 27-35 CAGs in non-pathogenic but expandable alleles, (3) 36-39 CAGs in expanded alleles with reduced penetrance and (4) 40 CAGs in fully-penetrant alleles. Molecular diagnosis is based on the determination of the CAG repeat size by PCR.
Intermediate (class 2) alleles were present in 4.4%, while low-penetrance (class 3) alleles were present in 2.0%, among all 'control' chromosomes (n=249) from our routine genetic testing. This high frequency of unstable alleles led us to study a large control sample (n=1772 chromosomes) from the Portuguese population (50 Guthrie cards from each district in mainland Portugal and the islands of Madeira and Azores).
No differences between normal (classes 1+2) alleles from affected individuals and controls could be shown. Distribution of the (CAG)n size showed: range 9-40 CAGs, mean 18.4±3.2, mode 17, median 17 (skewness 1.3, kurtosis 3). The 17-CAGs allele was by far the most frequent (38.0%). Intermediate alleles (27-35) represented 3.0% in the control population; 2 expanded alleles (36 and 40 repeats, 0.11%) were found. There was no evi-dence for geographical clustering of the intermediate or expanded alleles. This study showed that intermediate alleles at the HD locus are relatively frequent in the Portuguese population, which is particularly important for molecular diagnosis and genetic counselling. This will also be relevant for genetic epidemiology and evolution studies of the HD mutation.

Common trends in distribution of HLA-DQA1 and DQB1 haplotypes in patients with febrile seizures and epilepsy.

V. V. Egorov¹, S. A. Groppa²;
¹Center of Motherhood and Childhood Health Care, National Center of Family Planning and Medical Genet, Chisinau, REPUBLIC OF MOLDOVA, ²Department of Neurology, Neurosurgery and Medical Genetics, Chisinau State University of Medicine and Pharmaceutics "N. Testemitsanu", Chisinau, Republic of Moldova, Chisinau, REPUBLIC OF MOLDOVA.

Objective: The present study was designed to reveal the common trends in distribution of HLA-DQA1 and HLA-DQB1 haplotypes frequencies in patients with febrile seizures (FS) (simple and transformed to afebrile seizures (FST)) and epilepsy to determine the common traits between the pathologies.
Methods: We investigated HLA-DQA1 and HLA-DQB1 haplotypes by RFLP-analysis in the group of 68 children with FS (inclusive 12 patients with FST), 22 patients with epilepsy, and 70 individuals from control group.
Results: Frequency of HLA-DQA1 *0501 haplotype was maximal in control group (0.436), less frequent in patients with FST (0.250), in patients with FS (0.235) and in patients with epilepsy (0.023) with p < 0.001 between FS and control group, FST and epilepsy and FST and control group, and p < 0.1 between the FST and control group. Frequency of HLA-DQA1 *0201 haplotype was maximal in patients with epilepsy (0.318), less frequent in patients with FST (0.125), in patients with FS (0.081) and in control group (0.043), with p < 0.001 between epilepsy and control group. Frequency of HLA-DQB1 *0502 haplotype was maximal in control group (0.093), less frequent in patients with FS (0.088), in patients with FST (0.042) and in patients with epilepsy (0.000) with p < 0.05 between patients with epilepsy and control group.
Conclusions: The results suggests common trait in HLA-DQA1 and DQB1 haplotypes distributions in patients with FS, FST and epilepsy and, probably, have a "protective" role.

Metachromatic leukodystrophy : relations between phenotype and mutations in arylsulfatase A in adult forms.

Metachromatic leukodystrophy is due to a deficiency in arylsulfatase A which hydrolyses sulfogalactosylceramides and other sulfated glycolipids (sulfatides). In function of age, the clinical manifestations are different. The infantile form is characterized by a regression of acquired motor and later of mental activities. There are also adult forms which do not occur in the same families. Moreover, in the adult, there are two clinical variants, one in which motor signs are predominant, the other in which psychiatric symptoms dominate, although secondarily the patients become bedridden and demented. The evolution in the adult forms may be of several decades. In all those cases in the adult, the enzyme deficiency is identical as well as sulfatiduria which relates to the absence of the catabolic enzyme for sulfated glycolipids. Interestingly, it is well known from the work of V. Gieselmann (reviewed in Human Mut. 4: 233-243, 1994). that the mutations in infantile forms are different from those occurring in the adult which may explain homochrony.There seem to be specific mutations according to the motor and psychocognitive types in the adult, i.e. P426L for motor forms in a homozygote form and in the psychiatric forms a specific I179S mutation as a compound heterozygote.Studies are in progress to determine the precise clinical characteristics of the psychiatric forms and whether I179S mutation of arylsulfatase A could be a susceptibility factor of schizophrenia.

Correction of the biochemical phenotype in an X-Linked adrenoleukodystrophy mouse model by transgenic overexpression of the ALDR gene: functional redundancy at the peroxisomal membrane?

C. Camps¹, A. Pujol¹, E. Metzger¹, T. Pampols², J. Mandel¹, M. Giros²;
¹IGBMC, Illkirch, FRANCE, ²IBC, Fundacio Clinic, Barcelona, SPAIN.

X-linked adrenoleukodystrophy is a neurological disorder presenting with central or peripheral demyelination and impaired function of adrenals. X-ALD patients accumulate very long chain fatty acids (VLCFA) in plasma and tissues, mainly adrenal cortex and nervous system. The two main neurological phenotypes are the severe childhood cerebral form and the slowly progressive adult adrenomyeloneuropathy. A mouse model of the disease also accumulates VLCFAs in target organs, and has recently been shown to develop an adrenomyeloneuropathy-like phenotype (Pujol et al, Hum Mol Genet in press).
The gene mutated in the disease codes for a peroxisomal ABC transporter protein (ALDP). There is other three peroxisomal ABC transporters, ALDRP being the closest homolog (88% similarity with ALDP at the aminoacid level). A working hypothesis is that ALDRP could play a similar biochemical function. To assess whether an overexpression of ALDRP in target organs could compensate the biochemical phenotype of the ALD deficient mouse, transgenic mice overexpressing ALDRP in target organs were generated (ALDRtg), and crossed to ALD KO mice (ALD KO/ALDRtg).
We have compared the ALD KO and ALD KO/ ALDRtg have found that overexpression of the ALDRP leads to full correction of C26:0 and C24:0 fatty acids levels and of the ratios C26:0/C22:0 and C24:0/C22:0 in adrenal gland and CNS. Preliminary results indicate a correction of histopathology in adrenals. It remains to be seen whether this normalisation of the biochemical phenotype leads to an amelioration of the neurological AMN-like phenotype in mice.

New families with ataxia and hearing and visual impairment (van Bogaert-Martin syndrome) linked to 6p21-23 and refined genetic localisation

M. Gribaa¹, S. Klur¹, P. Bomont¹, R. Gershoni-Barush², B. Leheup³, M. Koenig¹;
¹igbmc, Strasbourg, FRANCE, ²Rambam Medical Center - faculty of Medicine - Department of Human Genetics, Haifa, ISRAEL, ³CHU - Service de Médecine Infantile 3 et de Génétique Clinique, Nancy, FRANCE.

The hereditary ataxias are a heterogeneous group of genetic disorders characterised by cerebellar symptoms associated with others neurological and non neurological features. Van Bogaert-Martin syndrome (MIM#271250) is defined by childhood onset autosomal recessive ataxia with optic and cochlear degeneration leading to blindness and deafness. We have previously reported that this condition is linked to a 17 cM region in 6p21-23 in an Israeli family.
We have analysed a set of patients born from consanguineous parents and for whom a diagnosis of Friedreich ataxia, ataxia with vitamin E deficiency or ataxia linked to 9q34 (with elevated alpha-foetoprotein) was excluded. Four patients, from three families were homozygous over part of the 6p21-23 interval. A sister and a brother of Turkish origin were haploidentical in 6p21-23 and homozygous over 6 consecutive markers defining an 11 cM interval. They developed ataxia and a peripheral demyelinating neuropathy before age 10, but had no hearing impairment. The sister developed retinitis pigmentosa by age 15, which was not present in the younger brother. The two other patients, of Israeli and Lebanese origin respectively, were homozygous over at least 6 consecutive markers. Albeit it cannot be formally excluded that one or both are homozygous by chance (due solely to consanguinity and not by linkage), the minimal region of homozygosity would suggest that the defective gene is located in a 7 cM interval located between markers D6S1660 and D6S265.

Further evidence that SPG3A gene mutations cause autosomal dominant hereditary spastic paraplegia

A. Magariello¹, A. Patitucci¹, A. L. Gabriele¹, F. L. Conforti¹, R. Mazzei¹, M. Caracciolo¹, G. Nicoletti¹, L. Mangone², B. Ardito³, M. Lastilla³, M. Muglia¹;
¹Institute of Neurological Sciences-CNR, Mangone (CS), ITALY, ²Institute of Neurology, School of Medicine, Catanzaro, ITALY, ³Hospital "Miulli", Acquaviva delle Fonti (BA), ITALY.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs.
In the past few years, eight spastic gait (SPG) loci have been shown to be associated with the “pure” form of AD-HSP, but only one gene responsible for the SPG4 locus has been identified.
Very recently, the gene responsible for the SPG3A locus was also identified.
The coding sequence is divided into 14 exons spanning approximately 69 Kb. The peptide encoded by SPG3A, atlastin, shows significant homology with several GTPases. Atlastin contains three conserved motifs: P-loop (74GAFRKGKS81); DxxG (146DTQG); and RD (217RD) that characterize guanylate binding/GTPase active sites.
We report a novel mutation in a large Italian pedigree from southern Italy with AD-HSP. Significant linkage to the SPG3 locus on chromosome 14 was detected with a maximum LOD score of 4.58 at D14S255.
Direct sequencing of the SPG3A gene revealed a G->A mutation at position 818 in the exon 7 of the gene. This mutation created an amino-acid change from Arg to Gln at codon 217. The variation was also confirmed with restriction enzyme Taq I, which cleaved the wild-type PCR product of 208 bp into 145 bp and 63 bp digestion fragments, but did not cleave the corresponding region of the SPG3A gene in our patients. Complete co-segregation of the heterozygous mutation with the disease was observed. This mutation was absent in 100 control chromosomes examined.
These data confirm that mutations in the SPG3A gene are causative of AD-HSP.

Linkage analysis in an italian family affected by autosomal dominant pure hereditary spastic paraplegia

V. Caputo¹, F. Brancati¹, E. Valente², E. Bertini³, C. Patrono³, F. Santorelli³, S. Salvi², A. Pizzuti¹, B. Dallapiccola¹;
¹University of Rome La Sapienza and CSS-Mendel Institute, Rome, ITALY, ²CSS-Mendel Institute, Rome, ITALY, ³Ospedale Pediatrico Bambino Gesù, Rome, ITALY.

Hereditary spastic paraplegia (HSP) includes a heterogeneous group of neurodegenerative disorders characterised by progressive spasticity of the lower limbs. Clinically, HSP has been divided in pure and complicated forms. In pure HSP no other neurological features are present and slowly progressive spastic gait is usually associated with mild decrease of vibration sense and sphincter disturbances. The mode of inheritance may be autosomal dominant, autosomal recessive or X-linked. So far, seven loci responsible for autosomal dominant pure HSP (ADPHSP) have been mapped to chromosomes 14q (SPG3), 2p (SPG4), 15q (SPG6), 8q (SPG8), 12q (SPG10), 19q (SPG12) and 2q (SPG13). Two ADPHSP genes have been identified so far, the SPG4 gene (Spastin) and the SPG3 gene (Atlastin).
Here we report an Italian family with 10 individuals affected by ADPHSP spanning three consecutive generations. The way of inheritance is autosomal dominant, with high penetrance. The phenotype is characterised by a high incidence of urinary disturbances, mild muscle weakness and wasting, and benign course (only two patients were wheelchair-bound after 10 to 20 years of disease). Patients often complained of mild lower limbs paresthesias and diurnal fluctuations of spasticity. Mutations in the Spastin gene were excluded by direct sequencing of all 17 exons of the gene. Linkage with all known ADPHSP loci was also excluded. A genome wide search using 400 microsatellite markers covering all autosomes allowed to map a novel ADPHSP locus, termed SPG18.

Linkage analysis in an Italian family with autosomal recessive hereditary spastic paraplegia

M. Muglia¹, A. Magariello¹, A. L. Gabriele¹, R. Mazzei¹, F. L. Conforti¹, A. Patitucci¹, T. Sprovieri¹, L. Mangone², L. Morgante³, A. Epifanio³, P. La Spina³, A. Quattrone¹^,2;
¹Institute of Neurological Sciences-CNR, Mangone (CS), ITALY, ²Institute of Neurology, School of Medicine, Catanzaro, ITALY, ³Department of Neurology, University of Messina, ITALY.

Hereditary spastic paraplegia (HSP) is a heterogeneous group of disorders of the motor system, characterized clinically by slowly progressive lower extremity spasticity and weakness, in which dominant, recessive and X-linked forms have been described. While autosomal dominant HSP has been extensively studied, autosomal recessive HSP is less well known and it is considered a rare form.
Until now, five families (four Tunisian and one Algerian) with recessive HSP linked to chromosome 8p11-8q13 have been published. The HSP locus was mapped in a region of 32.2 cM flanked by the markers PLAT and D8S279.
In the current study, we report a small Italian RHSP family linked to chromosome 8. Using additional markers located between PLAT and D8S279, we were able to refine the HSP region.
Negative lod scores were obtained in the two-point linkage analysis by using STR markers on chromosome 15 and 16, whereas positive lod scores were obtained for D8S509, D8S1828, D8S285, D8S1102, D8S1723, D8S260, D8S1840 and D8S279 with a maximum lod score of 1.46 at D8S260 marker. The two affected siblings were homozygous for the markers D8S1102, D8S1723 and D8S260. According to the Genethon map, haplotype reconstruction revealed that the patients shared a common 8.2 cM region of homozygosity encompassing the D8S1102, D8S1723 and D8S260 markers. Based on recombination events in our Italian family, we could map the RHSP gene between D8S285 and D8S1840, thus refining the HSP region by approximately 20 cM, from 32.2 to 12cM.

Linkage of a new locus for autosomal recessive axonal form of Charcot-Marie-Tooth disease to chromosome 8q21.3.

C. Barhoumi¹, R. Amouri², C. Ben Hamida², M. Ben Hamida², S. Machghoul¹, M. Guediche¹, F. Hentati²;
¹Hopital Militaire Principal de Tunis, Montfleury, TUNISIA, ²National Institute of Neurology, Tunis, TUNISIA.

We report clinical and genetic linkage analysis of large Tunisian family including 13 affected patients suffering from a particular form of Charcot-Marie-Tooth (CMT) with pyramidal feature. The inheritance was autosomal recessive. The clinical phenotype was sterotyped in all patients and characterized by onset during the first decade, a progressive course and distal atrophy in four limbs associated with a mild pyramidal syndrome. Nerve biopsy performed in two patients showed severe axonal neuropathy. Genetic linkage studies allowed to exclude linkage with known loci of different forms of CMT, familial spastic paraplegia and Juvenile Amyotrophic Lateral Sclerosis. A significant lod score obtained with marker D8S286, confirming linkage to chromosome 8q21.3. The clinical syndrome observed in this family corresponds to a new genetic form of autosomal recessive CMT.

Fine mapping of disease locus and histopatholgical studies in Russian family with autosomal dominant Charcot-Marie-Tooth neuropathy type 2F

S. Ismailov¹^,2, V. Fedotov³, E. Dadali¹, V. Ivanov¹, I. Ivanova-Smolenskaya², S. Illarioshkin², A. DeSandre-Giovannoli⁴, I. Bocaccio⁴, N. Levy⁴;
¹Russian State Medical University (RSMU), Moscow, RUSSIAN FEDERATION, ²Institute for Neurology of Russian Academy of Medical Sciences, Moscow, RUSSIAN FEDERATION, ³Genetic Counselling Department of Voronezh Diagnostic Centre, Voronezh, RUSSIAN FEDERATION, ⁴Inserm U491, Faculty de Medecine de la Timone, Marseille, FRANCE.

Charcot-Marie-Tooth disease (CMT) is the most common inherited motor and sensory peripheral neuropathy. The axonal form of CMT, or CMT type 2 (CMT2), is clinically and genetically heterogeneous with several assigned autosomal dominant loci.
Last year, at the 10th International Congress of Human Genetics in Vienna, Austria, we reported of the new locus (CMT2F) for autosomal dominant Charcot-Marie-Tooth type 2 neuropathy that maps within 15 cM region on chromosome 7q11-q21 in an extended Russian family.
By this moment we performed fine mapping for CMT2F locus using set of short tandem repeat (STR) markers located inside of the previously defined region and flanking markers.
Using these additional markers we got narrowing CMT2F locus.
Haplotype analysis with the new additional markers demonstrates that the disease gene maps at chromosome 7q11-q21 within reduced to 8 cM region.
Presently we are also performing histopatholgical examination of peripheral nerves in affected patients from CMT2F family and these results will be ready by the ECHG 2002 conference and can be presented at the conference too.

Axonal Autosomal Recessive forms of Charcot Marie Tooth disease: genes (GDAP1 and LMNA)frequencies and spectrum of their mutations.

H. Azzedine¹, N. Birouk², G. Durosier³, M. A. M. Salih⁴, D. Ente¹, D. Mahmoudi⁵, A. N. Masmoudi⁵, A. Vandenberghe⁶, M. Tazir⁷, D. Grid⁸, G. Stevanin¹, A. Brice¹, E. Le Guern¹;
¹INSERM U289 Hopital de la Salpetriere, Paris, FRANCE, ²Laboratoire de Neurogenetique Hopital des Specialites, Rabat, MOROCCO, ³Centre Hospitalo-Universitaire, Port-au-Prince, HAITI, ⁴Department of Paediatrics (39) College of Medecine&KKUH King Saud University, Riyadh, SAUDI ARABIA, ⁵Service de Neurologie, Hopital de Bab el Oued, Algiers, ALGERIA, ⁶Laboratoire de Neurogenetique, Hopital de l'Antiquaille, Lyon, FRANCE, ⁷Service de Neurologie, Hopital Mustapha, Algiers, ALGERIA, ⁸AFM, Hopital Salpetriere, Paris, FRANCE.

Charcot Marie Tooth Disease (CMT) is a pathologically and genetically heterogeneous group of hereditary motor and sensory neuropathies characterised by slowly progressive weakness and atrophy, primarily in peroneal and distal leg muscles. Two major types have been distinguished, in which the neuropathy is either demyelinating or neuronal. Electrophysiological studies on median motor conduction velocity (MNCV) have confirmed the distinction between the demyelinating and axonal forms of the disease. Several loci and various modes of inheritance were described: autosomal dominant, X linked and autosomal recessive (ARCMT). More than 30 loci and 10 genes are implicated. Only 3 loci were reported on the axonal form of ARCMT: 1q21, 8q13 and 19q13.
36 consanguineous families with axonal ARCMT originating from the Mediterranean basin were selected and screened by linkage analysis and homozygocity mapping to these loci. Nine families were linked to the 1q21 locus (LMNA gene), 10 to the 8q13 locus (GDPAD1 gene) and only one family was linked to 19q13. The GDAP1- and LMNA-linked forms were equally frequent, representing 25% and 28% of our series, respectively. The 19q13 locus is the rarest one (3%). Therefore, ~44% remains unlinked to any known axonal ARCMT locus. Furthermore, 5 families with demyelinating ARCMT were found to be linked to the 8q13 locus. The mutation screening in the axonal and demyelinating families is ongoing and the results will be presented at the meeting.

Clinical and genetic studies of a large pedigree with rolandic epilepsy and speech dyspraxia co-inherited as a dominant trait

G. Rudolf¹, P. Roll², S. Jamali², R. Carcangiu¹, M. P. Valenti¹, S. Finck¹, C. Seegmuller¹, A. de Saint Martin³, C. Marescaux¹, A. Lemainque⁴, M. N. Metz-Lutz¹, M. G. Lathrop⁴, E. Hirsch¹, P. Szepetowski²;
¹Neurology Unit, Strasbourg, FRANCE, ²INSERM U491, Marseille, FRANCE, ³Paediatric Unit, Strasbourg, FRANCE, ⁴CNG, Evry, FRANCE.

The complex relationship between epilepsy and language impairment is well known but poorly understood. Rolandic epilepsies (RE) account for about one-sixth of all childhood epilepsies. Language processing deserves particular attention in RE because discharges involve the perisylvian language areas. Consequently, several studies have shown that language dysfunction may be associated with RE. One family with nocturnal oro-facio-brachial seizures and centro-temporal epileptiform discharges associated with oral and speech dyspraxia and cognitive impairment had been described (Scheffer et al., 1995).
A new family was identified (Rudolf et al., manuscript in preparation) in which the same syndrome is inherited as a dominant trait with full penetrance. Epilepsy is of the rolandic type and language dysfunction is of the same type as in the former family. Video-EEG in the youngest patient showed asynchrone biphasic spikes predominant over the centro-temporal regions increased by sleep. The five subjects who performed neuropsychological testing showed a mental delay with significantly lower verbal performances. The main verbal deficits involved speech articulation and auditory verbal memory span.
The family is composed of eleven affected individuals, of whom ten are alive. Blood samples from all ten affected patients as well as from four unaffected members were collected. Genome screen was initiated with markers spaced regularly across the genome. Statistical studies are being performed assuming a dominant mode of inheritance with full penetrance and preliminary linkage data will be presented.

The -463G/A myeloperoxidase promoter polymorphism is associated with reduced risk for late-onset sporadic Alzheimer’s disease.

I. Manna, V. Andreoli, A. La Russa, G. La Porta, G. Nicoletti, P. Serra, R. Cittadella;
CNR Institute of Neurological Science, Cosenza, ITALY.

Myeloperoxidase (MPO) is a myeloid-specific enzyme that catalyses the reaction of chloride and hydrogen peroxide to yield hypochlorus acid, a strong ossidant, and its reactive by-products have been linked to DNA-strand breakage. MPO is present at high levels in circulating neutrophils and monocytes but is not detectable in microglia in normal brain tissue . However, MPO presence has been demonstraded in microglia associated with Alzheimer’s disease (AD) plaques, suggesting that MPO gene expression may play a role in neurodegenerative diseases involving macrophage-microglia. One portion of the gene thought to be involved in regulation of MPO expression is the proximal 5’ flanking region. The polymorphic G/A nucleotide base shift, 463 bases upstream from the transcription start site, negates the binding region for the general transcription factor SP1, and results in reduced gene expression, which would imply lower susceptibility to Alzheimer’s risk.
In this work, we present a case-control study to test this hypothesis. We have examined this polymorphism in a sample of 162 characterized AD cases compared with 158 cognitively normal subjects from the same geographic background. MPO genotypes were examined by PCR-RFLP. The allele frequency for MPO -463A was found to be 22.5% for cases and 34.2% for controls (OR=0.560, 95% CI=0.40-0.8, p=0.0011). These results
suggest that MPO -463 A allele reduces the risk of the Alzheimer ‘s disease.

S. Barlati¹, C. Izzi¹, A. Barbon¹, R. Kretz², T. Sander²;
¹University, Brescia, ITALY, ²University, Berlin, GERMANY.

Hereditary factors play a major role in the aetiology of juvenile absence epilepsy (JAE). Sander et al. (1997) reported an allelic association of JAE with the nine-copy allele of a tetranucleotide repeat polymorphism in the third intron of the kainate-selective GluR5 receptor gene (GRIK1) and supportive evidence for linkage of IGE to GRIK1 in families of JAE probands. These findings suggest that a major genetic determinant of GRIK1 confers susceptibility to JAE. We have sequenced the coding regions and regulatory sequences of the GRIK1 gene in 8 JAE patients who carry the nine-repeat allele of the GRIK1 tetranucleotide repeat polymorphism to detect a putative functional GRIK1 mutation that is in linkage disequilibrium with the nine-repeat allele. Seven of them were derived from families showing positive evidence for linkage to GRIK1. Our mutation analysis of coding regions and splice junctions revealed only two silent polymorphisms (A522C and C1173T) out of the five SNPs present in public databases and no mutations affecting protein structure. No significant differences were found in the allele frequencies of the detected polymorphisms between the JAE patients and controls. High levels of sequence conservation were also found in the promoter, in the 5’ and both the 3’ untranslated regions and in the RNA secondary structure involved in the editing reaction. These results indicate that mutations in the coding sequences, in the intron-exon boundaries and in the main regulatory and editing regions of the GRIK1 are not commonly involved in the aetiology of JAE.

P. Giannakopoulos¹, F. Herrmann¹, G. Gold¹, C. Bouras¹, R. Mulligan¹, G. Duriaux-Sail², A. Michon¹, S. E. Antonarakis², J. L. Blouin²;
¹Department of Psychiatry and Geriatrics, University Hospitals, Geneva, SWITZERLAND, ²Medical Genetics, University Hospitals and School of Medicine, Geneva, SWITZERLAND.

Alzheimer's disease (AD) is characterized neuropathologically by neurofibrillary tangles and senile plaques in brain. A key component of plaques is Aß, a 40-42 residue polypeptide derived from Aß-precursor-protein (APP) through proteolytic cleavage catalyzed by ß and g-secretases. ß-secretase is the rate-limiting enzyme in this process, which represents an alternate to normal a-secretase cleavage. Sequence variation in genes BACE1 (chromosome11q23.3) and BACE2 (chromosome 21q22.3), which encode two closely related proteases that appear to act as the AD ß-secretase, may represent a strong genetic risk factor for AD. In order to address this issue, we analysed the frequencies of 2 SNPs in BACE1 (V262, dbSNP rs#638405) and BACE2 (chr.21-cSNP database #hc21s00169, dbSNP rs#12149) respectively in a community-based sample of 96 individuals with late-onset AD followed in geriatric and psychiatric clinics (mean age = 79.9; SD 9.3 ; 45% men) and 170 controls randomly selected among residents of the same community who participated in an epidemiological study of dementia and underwent extensive mental status testing (mean age = 74.7 ; SD 7.4 ; 48% men). Genotype and allele distribution in both study groups were compared using Fisher's exact test and did not demonstrate any association between AD and BACE1 or BACE2. These data do not support BACE1 or BACE2 involvement in genetic risk of late onset AD in agreement with the recently published studies (Nowotny et al., 2001, Murphy et al., 2001).

Absence of association between multiple sclerosis and the -463 promoter region polymorphism of the human myeloperoxidase gene.

R. Cittadella¹, I. Manna¹, V. Andreoli¹, A. La Russa¹, G. La Porta¹, P. Serra¹, P. Valentino², F. Ruscica², L. Mangone², D. Messina¹, A. Quattrone²^,1;
¹CNR Institute of Neurological Sciences, Cosenza, ITALY, ²Institute of Neurology, University Magna Graecia, Catanzaro, ITALY.

Multiple Sclerosis (MS), the common autoimmune demyelinating disease of young adults, is a chronic inflammatory disease of the central nervous system (CNS) characterized by primary demyelination with relative axonal sparing. Although the pathogenesis of MS is not fully understood, the role of genetic factors is firmly established. Several association studies of single genes have illustrated that genetic factors contribute to the increased risk to develop MS . Myeloperoxidase (MPO) catalyses a reaction between chloride and hydrogen peroxide to generate hypochlorous acid and other reactive compounds that have been linked to DNA damage. Reactive oxygen species generated by macrophages have been implicated in inflammatory or neurodegenerative disorders. MPO is expressed in macrophages and microglia, which play a key role in the demyelination of nerve axons in Multiple Sclerosis (MS). A G-to-A substitution polymorphism in the promoter region of the MPO gene has been suggested in vitro studies to decrease gene transcription. We tested the association of this polymorphism with Multiple Sclerosis in a population-based case-control study of 131 cases and 163 controls from the same geographic background. We did not found an association with gender, age at onset, susceptibility to or the course of MS, according to the specific genotypes of the polymorphism of the MPO gene.

Association between some candidate regions on the chromosome 6p21 and Multiple Sclerosis: a family based study in population of the Central Sardinia, Italy

I. Prokopenko¹, L. Bernardinelli¹, L. Foco¹, C. Montomoli¹, L. Musu², M. Piras², A. Ticca², P. Holmans³, B. Murgia²;
¹University of Pavia, Pavia, ITALY, ²St.Francesco Hospital, Nuoro, ITALY, ³MRC Biostatistics Unit. Institute of Public Health., Cambridge, UNITED KINGDOM.

Multiple Sclerosis (MS) is an immune-mediated disease with the complex interplay between genetic and environmental factors noted in its aetiology.
Population of Sardinia is an isolated genetically homogeneous, distinct and different from other European populations that originates from a limited number of Caucasian ancestors, it has high degree of consanguinity, low migration rate, high prevalence of MS cases (157/100000 inhabitants) and high proportion of MS cases in the young ages.
6p21 region covering major histocompartibility complex (MHC) has been identified as promising in previous genome screenings.
A haplotype-based strategy of TDT mapping was adopted. The aim of this stage of the study was to evaluate the association in the 6p21 region with MS in Sardinian population.
Blood samples were obtained from 120 recruited MS trios (nuclear MS families from the MS register, Nuoro province, Central Sardinia). Mapping in some candidate regions was performed to search for the susceptibility genes. In this analysis 7 microsatellite markers (D6S2222, D6S276, STR-MICA, MICB, TNFalfa, D6S273, DQCARII) covering of about 4 cM in the MHC region (6p21) were chosen to study candidate regions identified before and according to the prior hypothesis involving TNFalfa and MICB genes.
To test for the allelic association, we performed the classical TDT test using TRANSMIT programme (D.Clayton) that allows for incomplete data.
Analysis of linkage disequilibrium and results on allelic and haplotype association between microsatellites in study and MS will be presented. Hypothesis about the role of some small regions of MHC region in the disease aetiology to be discussed.

Linkage disequilibrium analysis indicates a multiple sclerosis susceptibility effect in vicinity to the protachykinin-1 gene (TAC1) on chromosome 7q21-22.

A. Goris¹, I. Alloza², P. Fiten¹, S. Heggarty³, E. Cocco⁴, S. A. Hawkins⁵, C. A. Graham³, M. G. Marrosu⁴, G. Opdenakker¹, K. Vandenbroeck²;
¹Rega Institute, K.U.Leuven, Leuven, BELGIUM, ²Cytokine Biology and Genetics Programme, School of Pharmacy, QUB, Belfast, UNITED KINGDOM, ³Department of Medical Genetics, QUB, Belfast, UNITED KINGDOM, ⁴Department of Neuroscience, University of Cagliari, Cagliari, ITALY, ⁵Department of Neurology, Royal Victoria Hospital, Belfast, UNITED KINGDOM.

Chromosome 7q21-22, and in particular the region surrounding D7S554, emerged from the recent American genome screen in multiple sclerosis (MS) as the most promising region genome-wide for harboring a disease susceptibility gene. Also in the Canadian genome screen linkage was found with this chromosome region. We tested association between D7S554 and MS in 217 Sardinian trio MS families by the transmission disequilibrium test (TDT), and in a Northern Irish case-control study comprising 542 individuals. In both populations we found evidence for significant allelic association (Pc = 0.04 and Pc = 0.0002, respectively). In a second stage, we analysed 5 microsatellite markers in a 4 megabase interval on chromosome 7q21-22 in the same set of Sardinian families. Parental transmission of a single allele of one of these markers, i.e. D7S3126, was significantly distorted (Pc = 0.008). D7S554 and D7S3126 are located at distances of respectively 40 and 81 kb 5‘ from the startcodon of the protachykinin-1 gene (TAC1), and occur in strong linkage disequilibrium (P < 10^-7). Our study indicates that the previous finding of linkage with D7S554 refers possibly to the presence of an MS susceptibility effect in vicinity to TAC1. In addition, a second independent association was uncovered between a microsatellite polymorphism in the plasminogen activator inhibitor-1 gene, i.e. D7S477, and MS. Overall, the analysis presented here may contribute to the increasingly refined genomic map of MS, and underscores the requirement for a further high-resolution screening of chromosome 7q21-22.

Susceptibility and disease progression in Portuguese patients with multiple sclerosis - study of the role of APOE and SCA2 loci

M. Santos¹, M. C. Costa¹, M. E. Rio², M. J. Sá², M. Monteiro², A. Valença³, A. Sá⁴, J. Dinis⁵, J. Figueiredo⁶, L. Bigotte de Almeida⁷, A. Valongueiro⁵, I. Coelho⁸, M. T. Matamá¹, J. Pinto-Basto¹^,9, J. Sequeiros¹^,10, P. Maciel¹^,10;
¹UnIGENe-IBMC, Porto, PORTUGAL, ²Hosp. S. João, Porto, PORTUGAL, ³Hosp. Força Aérea, Lisboa, PORTUGAL, ⁴Hosp. Dist. Sto André, Leiria, PORTUGAL, ⁵Hosp. Dist. Sta Luzia, Viana do Castelo, PORTUGAL, ⁶Hosp. S. Marcos, Braga, PORTUGAL, ⁷Hosp. Garcia de Orta, Almada, PORTUGAL, ⁸Hosp. Dist. Sta Oliveira, Guimarães, PORTUGAL, ⁹Igm, Porto, PORTUGAL, ¹⁰Icbas, Porto, PORTUGAL.

Multiple sclerosis (MS) a is a major demyelinating disease with a highly variable clinical presentation, following a progressive or relapsing-remitting course that affects around 1:500 European young adults. Genetic factors are thought to play a role in susceptibility to MS and its progression.
In order to determine the influence of the APOE and SCA2 loci on susceptibility to multiple sclerosis and their correlation with disease severity, we studied 243 Portuguese patients, who were matched by sex, age and region of origin to 192 healthy controls. Both parents of 92 patients were also studied. We did not detect any significant difference when APOE and SCA2 allele frequencies of cases and controls were compared (McNemar's test), or when we compared cases with primary progressive versus other forms of the disease (Fisher's exact test). Disequilibrium of transmission was tested for both loci in 92 trios, and we did not observe segregation distortion for any allele at both loci.
To test the influence of the APOE epsilon 4 and SCA2 22 CAG alleles in the severity of the disease, we compared the age of onset, severity and progression rate between the groups with and without those alleles. We did not observe an association of the epsilon 4 or the 22 CAG alleles with severity or rate of progression of MS in our population.
Given the importance of gene-environment interactions in MS, it is possible that different genetic factors are relevant to its pathogenesis in different populations.

No evidence of association between alpha 2 macroglobulin gene and Parkinson's disease in a case- control sample

G. Annesi¹, A. A. Pasqua², P. Spadafora², E. V. De Marco², D. Civitelli², S. Carrideo², I. C. Cirò Candiano², F. Annesi², P. Serra², L. Mangone³, M. Zappia³, G. Nicoletti²;
¹Insitute of Neurological Sciences National Research Council, Mangone (Cs), ITALY, ²Institute of Neurological Sciences, National Research Council, Mangone (CS), ITALY, ³Institute of Neurology, University Magna Graecia, Catanzaro, ITALY.

Parkinson's disease is one of the most frequent neurodegenerative disorders. The pathological hallmarks of PD are: a) the presence of Lewy bodies (cytoplasmatic eosinophilic hyaline inclusions) in all affected brain-stem regions, especially the dorsal motor nucleus of the vagus; and b) a massive loss of dopaminergic neurons in the pars compacta of the substantia nigra.
Lewy body contains a variety of costituents, and its antigenic determinants can be divided into four groups: structural elements of Lewy bodies during their formation. The mechanism of Lewy-body formation, the importance of Lewy body to the pathogenesis of Parkinson's disease, and its role in the neurodegenerative process remain unknown. Alpha-2 macroglobulin (A2M) is a component of Lewy bodies; two alpha2 macroglobulin polymorphisms were described: a five nucletide deletion at the 5' splice site of exon 18 and a valine (Val) to isoleucine (Ile) exchange in aminoacid position 1000 near the thiolester active site. We present a study of these polymorphisms in a case-control sample consisting of 158 PD patients to verify if a potential functional alteration of this protein would be a risk factor for PD. No significant difference regarding allelic and genotypic distribution was found between cases and controls between early and late onset PD patients. These data suggest that these polymorphisms do not represent risk factors for PD and do not modulate age at onset of PD.

Mammalian, yeast, bacterial and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy

Y. Bao, D. C. Rubinsztein;
Cambridge Institute for Medical Research, Cambridge, UNITED KINGDOM.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is characterized pathologically by intranuclear inclusions in skeletal muscles and is caused by the expansion of a 10 alanine stretch to 12-17 alanines in the intranuclear poly(A) binding protein 2 (PABP2). While PABP2 is a major component of the inclusions in OPMD, the pathogenic mechanisms causing disease are unknown. Here we show that polyalanine expansions in PABP2 cause increased numbers of inclusions and enhance death in COS-7 cells. We observed similar increases of protein aggregation and cell death with nuclear-targeted green fluorescent protein (GFP) linked to longer vs. shorter polyalanine stretches. Intranuclear aggregates in our OPMD cell model were associated with HSP40 (HDJ-1) and HSP70. Human HDJ-1, yeast hsp104, a bacterially-derived GroEL minichaperone and the chemical chaperone DMSO reduced both aggregation and cell death in our OPMD model without affecting levels of PABP2 and similar trends were seen with GFP with long polyalanine stretches. Thus, polyalanine expansion mutations in different protein contexts cause proteins to misfold/aggregate and kill cells. The situation in OPMD appears to have many parallels with polyglutamine diseases, raising the possibility that misfolded, aggregate-prone proteins may perturb similar pathways, irrespective of the nature of the mutation or protein context.

D. Helmlinger, G. Yvert, K. Merienne, Y. Trottier, L. Ben-Haïem, C. Weber, J. Mandel, D. Devys;
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, FRANCE.

Nine neurodegenerative disorders are caused by expansions of CAG repeats within the coding region of the disease-causing genes, encoding polyglutamine tracts in the corresponding proteins. Among these disorders, spino-cerebellar ataxia type 7 (SCA7) is the only one affecting the retina.
To investigate the mechanisms of expanded ataxin-7 neurotoxicity, we generated transgenic mice overexpressing full-length human ataxin-7 (containing 10 or 90 glutamines) in rod photoreceptors, by using the rhodopsin promoter. Expression of the mutant protein triggers a clear, easily quantifiable, retinal dysfunction associated with neurodegeneration. Intranuclear inclusions (NIs) form by accumulation of an N-terminal cleaved fragment of mutant ataxin-7, are ubiquitinated and recruit chaperones (HDJ2, HSP70) and proteasomal subunits.
Because retina is suitable for molecular analysis and for testing therapeutic strategies, we initiated two different studies using this model.
First, we addressed in vivo normal ataxin-7 fate in presence of the mutant form, by crossbreeding SCA7-Q10 and SCA7-Q90 mice. Interestingly, photoreceptors expressing both transgenes lose rapidly, progressively and completely normal ataxin-7 protein, as shown by a complete disappearance of its cytoplasmic staining, whereas aggregation still occurs. Whether mutant ataxin-7 or any polyglutamine containing proteins induce a transcriptional repression or a specific proteolytic response is now investigated.
Second, we wanted to determine whether inducing chaperone activity could allow significant protection against polyglutamine-induced retinal toxicity in our SCA7 model. Mice overexpressing either HDJ2 or HSP70 under the same rhodopsin promoter are currently characterized. Preliminary results on mice expressing mutant ataxin-7 and HDJ2 suggest a specific partial reduction of histological abnormalities.

Ataxin-7, the gene product involved in Spinocerebellar ataxia 7, interacts with Sprouty-1 and 2: the Cbl-associated protein connection.

A. S. Lebre¹, J. Takahashi², H. Fujigasaki¹, C. Duyckaerts², J. H. Camonis³, A. Brice¹;
¹INSERM U289, Paris, FRANCE, ²Laboratoire de Neuropathologie, Hôpital de la Salpêtrière, Paris, FRANCE, ³INSERM U528, Paris, FRANCE.

Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by a polyglutamine expansion in ataxin-7. The molecular basis of SCA7 disease and the normal function of ataxin-7 remain unknown. Since SCA7 patients have retinal degeneration, we used a two-hybrid approach to screen a human retina cDNA library for ataxin-7 binding proteins and have isolated Sprouty-1. Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. To date, four mammalian Sprouty genes have been identified. Human Sprouty-1 was the most frequently cDNA isolated in this screening. By Northern blot, we observed that Sprouty-1 is expressed in brain and peripheral tissues. As Sprouty-2 is also expressed in brain, the subcellular localizations of Sprouty-1 and 2 were examined by immunohistochemistry in mouse brain: both are expressed in the Purkinje cells affected in SCA7. The interaction between ataxin-7 and Sprouty-1 and 2 were confirmed by GST pull-down. Sprouty associates directly with c-Cbl, a known down-regulator of receptor tyrosine kinase signaling. A short sequence in the N-terminus of the Sprouty proteins was found to bind directly to the RING finger domain of c-Cbl. Interestingly, we previously identified a variant of another Cbl-associated protein, CAP, as an ataxin-7 binding protein which colocalized with mutated ataxin-7 in neuronal intranuclear inclusions of SCA7 patients. More investigations are underway to confirm and further characterize the role of Cbl and Cbl-associated proteins in the normal function of ataxin-7 and their involvment in the pathophysiology of SCA7.

Processing of Huntington’s Disease protein and formation of intracellular aggregates.

Y. Trottier¹, A. Lunkes², K. S. Lindenberg³, L. Ben-Haiem¹, D. Devys¹, G. B. Landwehrmeyer³, J. L. Mandel¹;
¹IGBMC, Illkirch-Strasbourg, FRANCE, ²European Molecular Biology Organization, Heidelberg, GERMANY, ³Dept Neurology, University of Ulm, Ulm, GERMANY.

Huntington’s Disease (HD), an inherited neurodegenerative disorder, is caused by an expansion of a polyglutamine stretch localized in the amino-terminal region of huntingtin, a cytoplasmic protein of 350 kDa. The polyglutamine expansion, which modifies the antigenicity of huntingtin and promotes its aggregation, confers a gain of “toxic” property by a yet unkown mechanism. Animal and cellular model systems for HD suggest that processing of mutant huntingtin is a key event in the pathogenesis of HD, as mutant huntingtin breakdown products, which aggregate in cytoplasm and nucleus, are more harmful to neurons than the full length form. Studies on the proteolysis of huntingtin could reveal potentiel targets for therapeutic interventions by uncovering proteolytic sites. In this study, we focus on the characterization of the molecular nature of the inclusions and the proteolytic activities generating huntingtin fragments, which aggregte in neurons. We report that cytoplasmic and nuclear aggregates detected in HD brain and in a neuronal cell model of HD are made up of huntingtin breakdown products differing in size. The shorter of these fragments is the major huntingtin product that form aggregates in the nucleus, and is released by cleavage in a 10 aminoacid domain. Deletion and aminoacid replacement of the proposed cleavage domain either abolishes or diminishes cleavage. We also provide evidence that these huntingtin fragments are generated by proteases, which act in concert with the proteasome to ensure the normal turn over of huntingtin.

A. Sulek¹, D. Hoffman-Zacharska¹, E. Zdzienicka¹, J. Empel², J. Zaremba¹;
¹Institute of Psychiatry and Neurology, Warsaw, POLAND, ²Warsaw Uniwersity, Warsaw, POLAND.

Autosomal dominant ataxias are a group of neurodegenerative disorders characterized by progressive cerebellar ataxia of gait and limbs, dysarthria and other neurological signs. Several genes have been linked to different types of spinocerebellar ataxias (SCAs). Six types of SCA (SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA) are caused by an expanded CAG repeats in coding region of corresponding genes. The frequency of particular types of SCAs present an ethnical diversity.
In 2001, a new form of spinocerebellar ataxia - SCA17, with expansion of CAG repeats in coding region of TBP gene was identified in a few Japanese families. Besides typical cerebellar ataxia symptoms, patients with SCA17 presented extrapiramidal signs and intellectual deterioration.
Analysis of CAG polymorphism in TBP gene was performed in Polish control group consisted of 100 unrelated, healthy individuals with no neurological signs. The non-pathogenic repeat number in our group has ranged from 32 to 45 CAG.
Molecular analysis among 50 Polish patients with other types of SCA excluded, revealed one familial case with expansion of CAG in TBP gene. The proband was a 38 year-old woman with ataxia, bradykinesia, parkinsonism, dystonia and dementia. Her sister presented similar features and both had cognitive disturbances. Analysis of expanded alleles showed 55 CAG repeats in TBP gene in both sisters, with existing CAA interruptions.
Up to date, SCA1 and SCA2 were the only types of SCA we identified among a large group of Polish patients. These results suggest that SCA17 may be another, rare form of spinocerebellar ataxia in Poland.

A. Maat-Kievit¹^,2, M. Losekoot², K. Zwinderman³, M. Vegter-van der Vlis², R. Belfroid², G. J. van Ommen², R. Roos⁴;
¹Dept of Clinical Genetics, Erasmus University Medical Center, Rotterdam, NETHERLANDS, ²Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, NETHERLANDS, ³Dept of Medical Statistics, Leiden University Medical Center, Leiden, NETHERLANDS, ⁴Dept of Neurology, Leiden University Medical Center, Leiden, NETHERLANDS.

Huntington disease (HD) is an autosomal dominant, progressive neuropsychiatric disorder with chorea, dementia and changes in personality, mood and -behavior. The disease is incurable and leads to death usually within 17 years after onset (range: 2 to 45 years). The age at onset ranges from 2 to 80 years, with a mean between 46.0 and 48.9 years and the number of repeats, in the causal CAG repeat expansion, is inversely related to the age at onset and accounts for 50-77% of the variation in age at onset.
We analysed a Dutch cohort of 755 individuals retrospectively to assess the probability of onset for any given CAG repeat.
The repeat size is the major determinant of age at onset, with a correlation of -0.74, stronger (-0.84) in paternal than in maternal inheritance (-0.64), consistent with increased repeat expansion and stronger anticipation in the paternal line. The age at onset within families was more similar, than could be explained by the resemblance of the repeat size of persons in the same family. We hypothesised that if environmental factors were principally responsible for this familiar aggregation, one would expect a greater correlation for sibs than for parents and children. Our observations suggest that genetic factors may play a greater role in the onset of HD than a shared environment. Finally we discuss several explanations for the fact that the Dutch median age at onset for all expanded repeat sizes studied is significantly later -about 10 years- than a Canadian study.

Genetic heterogeneity of Huntington's Disease : screening of Huntington’s disease-like 1 and 2 loci

G. Stevanin¹, A. S. Lebre¹, C. Jeannequin¹, C. Julien¹, A. Camuzat¹, R. Sahloul², C. San³, R. L. Margolis⁴, C. Dode³, A. Durr¹^,5, A. Brice¹^,5;
¹INSERM U289, Paris, FRANCE, ²-, Colombes, FRANCE, ³Groupe Hospitalier Cochin-Port Royal, Paris, FRANCE, ⁴John Hopkins University School of Medicine, Baltimore, MD, ⁵Fédération de Neurologie et Département de Génétique, Cytogénétique et Embryologie, Hôpital de la Salpêtrière, Paris, FRANCE.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting mainly from the loss of neurons in the striatum. Patients suffer from progressive and unremitting chorea, rigidity and cognitive impairment. A CAG repeat expansion in the IT15 gene on chromosome 4 accounts for 93% of typical HD cases in our series. Recently, 2 new mutations were reported in HD-like (HDL) patients : a 192 nucleotide insertion in the PRPN gene encoding the prion protein (HDL1) and a CTG/CAG repeat expansion in the gene encoding Junctophilin-3 (HDL2). We analyzed 74 HD patients without expansions in the IT15 and DRPLA genes for these 2 mutations. The 192 nucleotide insertion in the PRNP gene was not detected in our series. The CAG/CTG repeat at the HDL2 locus was polymorphic in the controls and ranged from 8 to 28 units with a 62% heterozygosity rate. Only one Moroccan HD patient carried 50 uninterrupted CTG/CAG repeats at the HDL2 locus. This patient, a 44 year old woman with no know family history, presents with a 2 years duration of mild choreic movements of the face and extremities and sub-cortical dementia. Enlargement of the anterior horn of the lateral ventricles was observed on cerebral MRI suggesting atrophy of the head of the caudate nucleus. Cortical atrophy was marked. In conclusion, CTG/CAG repeat expansions at the HDL2 locus account for 0.2% of our series of HDL patients while the 192 nucleotide insertion in the PRNP gene was not found. Further genetic heterogeneity is then expected.

Polyalanine accumulation and toxicity in a CAG tract disorder, Machado-Joseph Disease.

A. Toulouse, C. Gaspar, D. Rochefort, J. Roussel, G. A. Rouleau;
McGill University Health Centre Research Institute, Montreal, PQ, CANADA.

Machado-Joseph disease (MJD) is part of a group of neurodegenerative disorders caused by the expansion of a polyglutamine-coding CAG repeat. A pathological hallmark of these disorders is the presence of intranuclear inclusions (INIs) which are aggregates of insoluble proteins including the expanded polyglutamine protein. The presence of such INIs in patient material and cellular models has lead to a cellular toxicity model. Similar INIs are found in oculo-pharyngeal muscular dystrophy (OPMD), which is caused by the short expansion of an alanine-encoding GCG repeat. The similarities between OPMD and the CAG tract disorders INIs led us to hypothesize that frameshifting events leading to polyalanine accumulation could contribute to the toxicity observed in CAG tract disorders. We previously demonstrated that such events occur in patient material and in a cellular model of MJD. Here we propose a frameshifting mechanism for CAG tracts involving RNA structure formation, similar to that previously implicated in retroviral organisms. We also investigate the role of polyalanine accumulation in the cellular toxicity observed in MJD.

L. Veneziano¹, S. Guida², E. Mantuano¹, L. D'Urbano², S. Pagnutti³, A. Tottene³, T. Fellin³, L. Verriello⁴, K. A. Stauderman⁵, M. E. Williams⁵, D. Pietrobon³, C. Jodice², M. Frontali⁶;
¹Istituto di Neurobiologia e Medicina Molecolare, C.N.R., Roma, ITALY, ²Dipartimento di Biologia, Università di Tor Vergata, Roma, ITALY, ³Dipartimento di Scienze Biomediche, Università di Padova, Padova, ITALY, ⁴Cattedra di Neurologia, Università di Udine, Udine, ITALY, ⁵SIBIA Neurosciences, La Jolla, CA, ⁶Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, ITALY.

CACNA1A gene encodes the a1A subunit of P/Q Ca2+channels and its mutations are responsible for 3 disorders: Familial Hemiplegic Migraine (FHM), preferentially associated with missense mutations, Episodic Ataxia type 2 (EA2) and Spinicerebellar ataxia type 6, thought to be due, respectively, to premature stop mutations and to small expansions of a CAG repeat.
A mutation screening, by SSCP and DHPLC, of the CACNA1A gene, still ongoing, was performed on 26 ataxia patients from different families. So far 4 patients with a mutation of CACNA1A gene were identified. Two families showed a CAGn expansion, and 2 a previously undescribed point mutation: 1 missense and 1 deletion in frame. In addition single nucleotide substitutions have been detected in introns or in exons, without aminoacid change, possibly affecting exon splicing enhancers sites. No mutations leading to premature stop were so far identified. The clinical picture was that of EA2 or of SCA6, showing once more the wide overlap between the 2 disorders. The functional analysis of some of the above EA2 point mutations favors the hypothesis of a loss of channel function. Overall the present and literature data suggest that ataxia-causing point mutations, different from those leading to truncated protein, are relatively frequent and appear to be preferentially located in the III protein domain, while missense mutations causing FHM appear to be widely spread in all protein domains.
Telethon Grant E847 to MF, Grant of Ministry of Health to MF and LM

Ataxin 7: A Nuclear Protein Involved In Caspase cleavage-mediated Apoptotic Cell Death

L. G. Gouw¹, S. S. Propp², L. M. Ellerby²;
¹University of Utah, Salt Lake City, UT, ²Buck Institute, Novato, CA.

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine disorder characterized by specific degeneration of cerebellar, brainstem and retinal neurons. Although they share little sequence homology, proteins implicated in other trinucleotide (CAG)n repeat disorders have similar attributes beyond their characteristic polyglutamine tract and neurodegenerative pathology. These attributes include a nuclear aggregation phenotype associated with apoptotic cell death and roles as caspase substrates. In this study we present evidence that ataxin-7, the product of the SCA7 gene, has a predominantly nuclear distribution in HEK 293 cells that does not depend upon on isoform, polyglutamine tract length or the presence or absence of a putative nuclear localization sequence. However, the morphology of nuclear aggregation appears to depend upon polyglutamine length. Ataxin-7 is implicated in apoptotic cytotoxicity by qualitative as well as quantitative parameters . Consistent with its nuclear localization and apoptotic propensity, ataxin-7 acts as a substrate for an activated caspase. Ataxin-7’s susceptibility to caspase action was eliminated by mutation of specific residues. The data support a model whereby one of ataxin-7’s roles in the nucleus is as a modulator

Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington’s disease.

A. Sittler¹^,2, R. Lurz², G. Lueder², J. Priller³, H. Lehrach², M. Hayer-Hartl⁴, U. Hartl⁴, E. E. Wanker²;
¹IGBMC, CU Strasbourg, Illkirch, FRANCE, ²Max Planck Institute of Molecular Genetics, Berlin, GERMANY, ³Charité, Humboldt-University, Berlin, GERMANY, ⁴Max Planck Institute of Biochemistry, Munich, GERMANY.

Huntington’s disease (HD) is a progressive neurodegenerative disorder with no effective treatment. The formation of neuronal inclusions with aggregated huntingtin protein is associated with the progressive neuropathology in HD. Our previous work suggested that inhibition of huntingtin protein aggregation in patients by small molecules could be a promising therapeutic strategy (Heiser et al, 2000, Proc. Natl. Acad. Sci. USA, 97, 6739-6744). Geldanamycin is a benzoquinone ansamycin that binds to the heat shock protein Hsp90. Geldanamycin disrupts a complex consisting of Hsp90 and the heat shock transcription factor HSF1 and triggers the activation of a heat shock response in mammalian cells. In this study, we show by using a filter retardation assay and immunofluorescence microscopy that treatment of mammalian cells with geldanamycin at nanomolar concentrations induces the expression of Hsp40, Hsp70 and Hsp90 and inhibits HD exon 1 protein aggregation in a dose-dependent manner. Similar results were obtained by overexpression of Hsp70 and Hsp40 in a separate cell culture model of HD. This is the first demonstration that huntingtin protein aggregation in cells can be suppressed by chemical compounds activating a specific heat shock response. These findings may provide the basis for the development of a novel pharmacotherapy for HD and related glutamine repeat disorders.

I. Silveira¹, T. Matamá¹, S. Martins¹, L. Guimarães¹^,2, J. Sequeiros¹^,2;
¹UnIGENe, IBMC, Porto, PORTUGAL, ²Icbas, Porto, PORTUGAL.

The spinocerebellar ataxias (SCAs) are neurodegenerative disorders clinically and genetically very heterogeneous. Dentatorubropallidoluysian atrophy (DRPLA) is a type of SCA, characterized by gait ataxia, myoclonic epilepsy and dementia, due to a (CAG)n expansion on chromosome 12p13. DRPLA is prevalent in Japan, but several families of non-Japanese ancestry have been identified. Expanded DRPLA alleles of Asian and Caucasian ancestry share a common haplotype, which is associated with longer repeats commonly found in Asians. Associations between prevalence of dominantly inherited SCAs and frequency of large normal alleles have indicated that these may contribute to the generation of expanded alleles. We identified five families with DRPLA in Portugal. Interestingly, no association between the frequency of DRPLA and the frequency of large normal alleles was found in the Portuguese population. To identify the origin of the expanded DRPLA alleles in Portuguese we studied two previously reported intragenic polymorphisms in introns 1 (A1010G; system A) and 3 (T1865C; system B). Polymorphisms were detected by PCR-SSCP or/and sequencing. We found that all expanded DRPLA alleles in Portuguese share the same polymorphism in intron 3, which is common to that found in Asian expanded chromosomes. We are presently assessing the phase of expanded alleles with system A. Our preliminary results, thus, seem to indicate that expanded DRPLA alleles in Portuguese share a common haplotype.

Familial essential tremor is not associated with SCA 12 mutation in southern Italy

F. Annesi¹, G. Nicoletti², S. Carrideo², A. A. Pasqua², P. Spadafora², I. C. Cirò Candiano², D. Civitelli², L. Mangone³, M. Zappia³, G. Annesi²;
¹Insitute of Neurological Sciences National Research Council, Mangone (Cs), ITALY, ²Institute of Neurological Sciences, National Research Council, Mangone (CS), ITALY, ³Institute of Neurology, University Magna Graecia, Catanzaro, ITALY.

Autosomal dominant cerebellar ataxias (ADCA) are a heterogeneous group of hereditary neurodegenerative disorders characterized primarily by progressive cerebellar ataxia and classified clinically in various subgroups. Recently, Holmes et al. identified in a large American pedigree of German descent, a novel form of ADCA termed spinocerebellar ataxia 12 (SCA 12). SCA 12 was mapped on chromosome 5q31-33. Affected subjects had expansions containg 66 to 78 CAG repeats in the 5'-untranslated region of the gene encoding PPP2R2B, a brain-specific regulatory subunit of protein phosphatase PP2A, whereas controls had 7 to 28 repeats. SCA 12 is a slowly progressive ADCA that differs from other SCAs becouse it typically begins with tremor of head and arms, often diagnosed as essential tremor (ET). We conducted a study to screen individuals who presented with familial ET for SCA 12 mutation. Thirty index cases (12 men and 18 women, from families) with familial ET underwent to DNA analysis for the SCA 12 mutation. As controls we enrolled 58 (23 men and 35 women) healthy subjects. All cases and controls were from the same ethnic and socio-economic background (Southern Italy). The range of allele size was 9 to 21 in both patients and controls; the most frequent allele was the 10 repeat allele (44.8% among controls, 43.3% among patients) no patient with ET presented a repeat larger then 19. In the current study, no patient with familial postural and tremor of head or arms harboured the SCA 12 mutation confirming that ET and SCA 12 are distinct diseases.

A. R. Noorian¹, P. Pasalar¹, B. Moghimi¹, A. Jannati¹, A. Soltanzadeh¹, T. Krefft², R. Crook³, H. Najmabadi⁴, J. Hardy³^,5;
¹Tehran University of Medical Sciences, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Department of Neurology, Mayo Clinic, Jacksonville, Florida, FL, ³Department of Neuroscience, Mayo Clinic, Jacksonville, FL, ⁴Genetics Research Center the Social Welfare and Rehabilitation University., Tehran, IRAN (ISLAMIC REPUBLIC OF), ⁵Laboratory of Neurogenetics, NIA/NIH, Building 10, 6C, Bethesda, MD.

Despite the fact that APP mutations were the first described cause of Alzheimer`s Disease relatively few pathogenic mutations have been described. Here we describe the occurrence of a novel APP mutation (T714A) in an Iranian family with documented affected individuals in 3 generations. The phenotype in this family is similar to that of many of the other families with APP mutations, with an onset age In the mid 50.

Mutations in the parkin gene in patients from Russia is associated with parkinsonism

O. Miloserdova¹, S. Illarioshkin², I. A. Ivanova-Smolenskaya², S. Selestova¹, P. Slominsky¹, S. A. Limborska¹;
¹Institute of Molecular Genetics, Moscow, RUSSIAN FEDERATION, ²Institute of Neurology, Moscow, RUSSIAN FEDERATION.

Until now only two genes have been associated with PD coding for the proteins a-synuclein and parkin. Exonic deletion of the parkin gene and parkin mutations have been assocoated with autosomal recessive juvenile parkinsonism (with onset before age 40). We studied parkin gene mutations in 55 patients from Moscow. 1, 2, and 7 exones were examined by SSCP analysis. We found an aberrant band pattern in exon 1 in 3 patients, in exon 2 in 1 patient and in exon 7 in 1 patient. Daughter of patient with alteration in exon 7 has the same pattern.
Subjects were screened for mutations in the PARK2 gene with use of a semiquantitative PCR assay that simultaneously amplified several exons too. We found alterations of gene dosage in 5 of 55 Moscow’s patients with PD. Mutations included heterozygous deletion of exons 4, 10 and 12, homozygous deletion of exon 5.
Among the 55 patients with early-onset Parkinson disease, was founding 10 (18%) PARK2 mutations. At present time our workgroup continue searching mutations in other exons.

Parkin mutations are frequent in patients with isolated early-onset parkinsonism

M. Periquet¹, M. Latouche¹, E. Lohmann¹, N. Rawal¹, G. De Michele², S. Ricard³, H. Teive⁴, A. Ouvrard-Hernandez⁵, D. Nicholl⁶, N. W. Wood⁷, S. Raskin⁸, J. Deleuze⁹, Y. Agid¹, A. Dürr¹, A. Brice¹;
¹Inserm U289, Paris, FRANCE, ²Universita Federico II, Naples, ITALY, ³Aventis-Pharma, Vitry, FRANCE, ⁴Hospital de Clinicas, Curibita, BRAZIL, ⁵CHU de Grenobles, Grenobles, FRANCE, ⁶Queen Elizabeth Hospital, Birmingham, UNITED KINGDOM, ⁷Queen Square, London, UNITED KINGDOM, ⁸Laboratorio Gentika, Curibita, BRAZIL, ⁹Aventis-Pharma, Paris, FRANCE.

Parkin gene mutations have been reported to be a major cause of early onset parkinsonism in families with autosomal recessive inheritance and in isolated juvenile-onset parkinsonism (age at onset <20 years). However, the precise frequency of parkin mutations in isolated cases is not known. In order to evaluate more accurately the frequency of parkin mutations in patients with isolated early onset parkinsonism, we studied 146 patients of various geographical origin with an age at onset <45 years. All were screened for mutations in the parkin gene with the use of semi-quantitative PCR combined with the sequencing of the entire coding region. We identified parkin mutations in 20 patients including 4 new exons rearrangements and a new missense mutation. Taken together with our previous study (Lücking et al. 2000), these data show that parkin mutations account for at least 15% (38/246) of early-onset cases without family history, a proportion significantly decreasing when age at onset increases. There were no clinical group differences between parkin cases and other patients with early onset parkinsonism. However, a single case presenting with cerebellar ataxia several years before typical parkinsonism allows to extend the spectrum of parkin disease.

Mutations in the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) are associated with axonal or demyelinating recessive Charcot-Marie-Tooth disease (CMT).

E. Nelis¹, S. Erdem², M. C. Belpaire-Dethiou³, A. Cuesta⁴, L. Pedrola⁴, C. Verellen⁵, P. De Jonghe¹^,6, F. Palau⁴^,7, P. Y. K. Van den Bergh³, E. Tan², H. Topaloglu⁸, V. Timmerman¹;
¹Molecular Genetics Department, Flanders Interuniversity Institute of Biotechnology (VIB), Born-Bunge Foundation (BBS), University of Antwerp (UIA), Antwerpen, BELGIUM, ²Department of Neurology and Neuromuscular Diseases Research Laboratory, Hacettepe University, Ankara, TURKEY, ³Centre de Référence Neuromusculaire, Cliniques universitaires St-Luc, Université catholique de Louvain (UCL), Brussels, BELGIUM, ⁴Laboratory of Genetics and Molecular Medicine, Instituto de Biomedicina, CSIC, Valencia, SPAIN, ⁵Unité de Génétique Médicale, Université catholique de Louvain (UCL), Brussels, BELGIUM, ⁶Department of Neurology, University Hospital Antwerp (UZA), Antwerpen, BELGIUM, ⁷Department of Genetics, Universitat de Valencia, Burjassot, SPAIN, ⁸Department of Pediatric Neurology, Hacettepe Children's Hospital, Ankara, TURKEY.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. The disease can be demyelinating as well as axonal and is inherited as an autosomal dominant, autosomal recessive or X-linked trait. Mutations in the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) were recently shown to be responsible for recessive CMT4A (chromosome 8q21.1). GDAP1, comprising an ORF of 1077 nucleotides, encodes a protein of 358 amino acids containing a glutathione S-transferase (GST) domain. GST enzymes are known to function in antioxidant pathways and in detoxification. Therefore, GDAP1 might play a role in protecting myelin membranes against free radical-mediated damage, to which it is susceptible. We screened the GDAP1 gene for the presence of mutations in 7 recessive CMT families compatible with linkage to CMT4A. A homozygous nonsense mutation in exon 5 of GDAP1 (c.581C>G, S194X) was found in a consanguineous Moroccan family. Clinical, electrophysiological and neuropathological examination of the patients showed an axonal CMT phenotype. A homozygous 1 bp deletion in exon 6 (c.786delG, G262fsX284) of GDAP1 was identified in two probably related Turkish families. Electrophysiological as well as neuropathological data support a demyelinating CMT phenotype. In a Turkish consanguineous family a homozygous missense mutation (c.844C>T, R282C) in exon 6 of GDAP1 was detected. Electrophysiological examination of the patient revealed an axonal CMT phenotype. In conclusion, we detected 3 distinct homozygous GDAP1 mutations (a nonsense, a frameshift and a missense mutation) in families with recessive CMT. The CMT phenotype associated with GDAP1 mutations can be axonal as well as demyelinating.

Charcot-Marie-Tooth disease type 1B (CMT1B): GFP is a versatile tool to visualize in vivo effects of Myelin Protein Zero (P0) mutations.

A. B. Ekici¹, S. Oezbey¹, E. Nelis², C. Van Broeckhoven², M. Schachner³, B. W. Rautenstrauss¹;
¹Institute of Human Genetics, Erlangen, GERMANY, ²Laboratory of Neurogenetics, University of Antwerp, Antwerp, BELGIUM, ³Centre for Molecular Neurobiology, University of Hamburg, Hamburg, GERMANY.

P0 is a well characterized cell adhesion molecule playing a crucial role during myelination in the peripheral nervous system. GFP is a helpful tool to observe cellular events in vivo and is frequently used for this purpose in various cells or even organisms. P0 mutations cause Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas-Syndrome (DSS) and congenital hypomyelination (CH), diseases of widely varying phenotypes, even if the same position is substituted by different amino acids. We used two insect cell lines, S2 and HighFive, in order to develop an adhesion test system to determine in vivo effects of P0 mutations. We analyzed 3 pathogenic mutations for their effect on adhesion capability in a S2 cell system and found a direct correlation of adhesion capability and severity of the disease phenotype. Subsequently we constructed fusion proteins with GFP for P0wt and the Ala221fs mutation to visualize the effect in vivo both on the intracellular localization and on the changes in the adhesion capability. The GFP-P0wt fusion revealed the expected adhesion capability and moreover the membrane insertion of the fusion protein was clearly visible using a fluorescent microscope. The Ala221fs mutation is expressed, however, no membrane insertion takes place. This may explaine the lack of its adhesion capability. Hence, this system is suitable to predict the severity of the phenotype based on expression of in vitro mutated P0 and to visualize the effect of mutations in vivo.

G. Zanni¹^,2, V. des Portes¹, K. Poirier¹, S. Illarioshkin³, E. Bertini², J. Chelly¹;
¹ICGM Cochin Port-Royal INSERM U129, Paris, FRANCE, ²Bambin Gesu' Childrens' Hospital Dept of Molecular Medicine, Rome, ITALY, ³Department of Neurogenetics, Russian Academy of Medical Sciences, Moscow, RUSSIAN FEDERATION.

X-linked congenital cerebellar atrophy (XCA) is a rare genetic disorder characterized by severe hypotonia at birth, delayed motor development, dysarthria, slow eye movements and nonprogressive ataxia with normal/borderline intelligence. Global cerebellar hypoplasia is generally evident after age 2.
The XCA locus was assigned to a large genetic interval of 54cM on Xp11.21-Xq24, flanked by markers DXS991 and DXS1001 (Illarioshkin et al 1996)
In two additional XCA families an exclusion mapping strategy was adopted,
reducing the critical interval to two subregions: Xp11.21-q21.33 and Xq23-q24 flanked by markers DXS991-DXS990 and DSX424-DXS1001 respectively (Bertini et al 2000).
We have been initially focusing on the Xp11.21-Xq13 subregion. Genes or transcripts identified by bioinformatic tools, have been screened by RT-PCR to test their expression profile in the cerebellum and other tissues. Out of 77 genes, 59 have been found positive for cerebellum expression. Candidate genes have been selected and tested for mutation screening by DGGE, DHPLC or direct sequencing,according to the following criteria:
1)genes or their homologs strongly expressed during cerebellar development:EPLG2, LMO6
2)genes in which mutation or disruption resulted in cerebellar defects in either human pathology or animal models:CXCR3, NDUFA1
3) Xq deletion associated with cerebellar hypoplasia.(AR and OPHN1 genes are deleted resulting in a contiguous gene syndrome with mental retardation and androgen insensitivity) The involvement of OPHN1 gene in XCA was also excluded.
Additional XCA families are being recruited to further narrow down the critical interval.
The project is funded by Italian Telethon (492/B)

A novel PLP mutation in X-linked hereditary spastic paraplegia (SPG2) further broadens the extent of heterogeneity at this locus.

C. K. Hand, M. Dubé, M. Shevell, G. A. Rouleau;
McGill University Health Centre Research Institute, Montreal, PQ, CANADA.

Hereditary Spastic paraplegias (HSP) are a large group of genetically heterogeneous neurodegenerative disorders. The disease is characterized by progressive lower limb spasticity. Three X linked HSP loci have been identified and mutations in the proteolipid protein (PLP) gene have been identified in families linked to the uncomplicated SPG2 locus on Xq22. The PLP gene encodes 2 myelin proteins in the CNS, PLP and DM20 by alternate splicing.
Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder which is also caused by PLP mutations. PMD normally manifests in infancy or early childhood with nystagmus and cognitive impairment with progression to severe spasticity and ataxia. The most common mutation in PMD is a duplication of the entire PLP gene, resulting in a mild phenotype, while point mutations often result in a more severe disease.
Here we present findings in a large French Canadian family with a complicated X-linked recessive HSP linked to the SPG2 locus. The proband has been wheelchair-bound since the age of 3 years and at 11 years old he presents a severe spastic paraplegia, mild upper limb spasticity and mild developmental delay. Mutation analysis of the PLP gene identified a segregating mutation in the initiation codon. This G to A mutation is expected to result in complete absence of PLP. Mutations in this codon have been identified in PMD patients with mild forms of the disease, but not in HSP, thus extending the spectrum of mutations found in these allelic disorders and indicates further clinical heterogeneity than previously thought.

Refined SPG11 locus in autosomal recessive hereditary spastic paraplegia with thin corpus callosum

G. Montagna¹, E. M. Valente², E. Bertini¹, F. Brancati², F. M. Santorelli¹^,3, B. Dallapiccola²^,4, C. Casali³;
¹Bambino Gesù Hospital, Rome, ITALY, ²CSS-Mendel, Rome, ITALY, ³Inst.of Neurology, La Sapienza University, Rome, ITALY, ⁴Inst. of Genetics-La Sapienza University, Rome, ITALY.

Objective: To study nine Italian families with Hereditary Spastic Paraplegia (HSP) and thin corpus callosum (TCC).
Background: HSP is a genetically heterogeneous group of upper motor neuron syndromes and has been divided into pure and complicated forms. Both forms are classified by inheritance: autosomal dominant (AD), recessive (AR), X-linked. Complicated HSP is mainly recessive and five loci have been mapped. A locus on chromosome 15q13-15 (SPG11) has been identified in Italian and American pedigrees and in Japanese AR-HSP+TCC families.
Methods: We studied a total of 39 individuals from nine non-consanguineous Italian families with AR-HSP+TCC. Genetic linkage analyses were carried out with polymorphic DNA markers of 15q13-15 using an ABI 3100 Sequencer. Pairwise LOD scores were obtained using the FASTLINK version of the MLINK program. Multipoint LOD scores were generated by use of SIMWALK2.
Results: Linkage with the SPG11 region could neither be confirmed nor excluded with certainty in five families. Three families showed evidence for linkage to the SPG11 locus. Analysis of recombination events combined with data from published pedigrees narrowed the SPG11 locus to a 17.7 cM region. The upper extent of the region is between markers D15S1007 and D15S971, while the lower extent of the region is between markers D15S659 and D15S978. In one family, linkage to SPG11 could be excluded, supporting genetic heterogeneity.
Conclusions: Our analyses suggested that AR-HSP+TCC is more frequent than previously believed in Italy, narrowed the SPG11 locus, and corroborated genetic heterogeneity in AR-HSP.
Supported by the Italian Ministry of Health and MIUR

Autosomal recessive ataxias: a new gene – aprataxin – responsible for ataxia-ocular apraxia 1, and a new locus on 9q34

M. C. Moreira¹^,2, S. Klur¹, C. Barbot²^,3, N. Tachi⁴, P. Bomont¹, M. Watanabe⁵, M. Shoji⁵, J. M. Warter⁶, P. Aubourg⁷, A. Dürr⁸, A. H. Németh⁹, R. Amouri¹⁰, F. Hentati¹⁰, A. Alurkar¹¹, D. Divekar¹¹, P. Mendonça¹², J. Sequeiros², P. Coutinho²^,13, M. Koenig¹;
¹IGBMC - CNRS, INSERM, ULP, Strasbourg / Illkirch, FRANCE, ²UnIGENe - IBMC, ICBAS Univ. Porto, Porto, PORTUGAL, ³Hospital de Crianças Maria Pia, Porto, PORTUGAL, ⁴School of Health Sciences, Sapporo Medical University, Sapporo, JAPAN, ⁵Department of Neurology, Gunma University School of Medicine, Maebashi, JAPAN, ⁶Service de Neurologie, Hôpital Civil, Strasbourg, FRANCE, ⁷Hôpital Saint Vincent de Paul, Pédiatrie C, Paris, FRANCE, ⁸INSERM U289, Département de Génétique, Cytogénétique et Embryologie, et Fédération de Neurologie, Hôpital de la Salpêtrière, Paris, FRANCE, ⁹Wellcome Trust Centre for Human Genetics and Department of Clinical Genetics, The Churchill Hospital, Oxford, UNITED KINGDOM, ¹⁰Institut National de Neurologie, Tunis, TUNISIA, ¹¹Pune Institute of Neurology, Pune, INDIA, ¹²Department of Hematology, Hospital do Divino Espirito Santo, S. Miguel, Açores, PORTUGAL, ¹³Hospital Säo Sebastiäo, Sta. Maria da Feira, PORTUGAL.

The newly recognized ataxia-ocular apraxia 1 is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy. However, it does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes aprataxin, a new and ubiquitously expressed protein, comprising three domains that share distant homology with the amino-terminal domain of polynucleotide 5'-kinase 3'-phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. The results suggest that aprataxin is a nuclear protein with a role in DNA repair, reminiscent of the function of the protein defective in ataxia-telangiectasia, but would cause a phenotype restricted to neurological signs when mutant.
A second AOA locus - on 9q34 - was initially identified in a Japanese and a Pakistanese family. We have since then identified families from Portugal, France, Algeria and Turkey. In most cases, we found an association with elevated serum alfa-fetoprotein, adolescent age of onset, and ocular apraxia defined by hypometric saccades. The critical interval was reduced to a 3 cM region.

Infantile Onset Progressive Ascending Spastic Paralysis is Linkes to ALS2/Alsin Gene on 2q33-35

E. Eymard-Pierre¹, E. Bertini², G. Lesca³, M. di Capua², R. Cusmai², V. Leuzzi⁴, A. Ponsone⁵, O. Boespflug-Tanguy¹^,6;
¹INSERM U384, Clermont-Ferrand, FRANCE, ²Dept of Pediatric Neurology, Bambino Gesu Children's Hospital, Rome, ITALY, ³Service de Génétique, Université de Lyon, Lyon, FRANCE, ⁴La Sapienza, Roma, ITALY, ⁵Ospedale Infantile Regina Margarita, Torino, ITALY, ⁶Fédération Génétique Auvergne, CHU, Clermont-Ferrand, FRANCE.

We report on 15 patients from 10 families (4 familial and 6 sporadic forms) who presented severe progressive ascending spastic paralysis. They were considered normal at birth and spastic paraplegia initiated during the two first years of life. Weakness and spasticity extended to upper limbs around the age of 7-8 years. The patients were all wheel-chair bound by the age of 10 years and during the second decade the disease progressed to tetraplegia, anarthria, dysphagia and slow eye movements. Muscle Biopsy and EMG were normal, PEM showed abolition of corticospinal response en contrast with normal SEP. Genotyping analysis excluded linkage to previously reported loci for dominant as well as and recessive Hereditary Spastic Paraplegia and established linkage to ALS2 locus on chromosome 2q33-35 with a lod score of 6.66, raising the possibility that this infantile onset ascending spinobulbar paralysis is allelic to the condition previously reported as juvenile amiotrophic lateral sclerosis (ALS2). Therefore, we tested as gene candidate the ALS/Alsin gene recently reported mutated in 3 families consanguineous Arabic families with ALS2 and 1 consanguineous Saudi family with a childhood-onset primary lateral sclerosis (PLS). Alsin mutations were found in 4 families: 3 deletions and 1 splice site mutation leading to truncated Alsin protein. In the six remaining families absence ALS2/Alsin gene mutation suggest an heterogeneity in infantile onset progressive ascending spastic paralysis.

De Novo mutations in the sodium channel gene SCN1A cause severe myoclonic epilepsy of infancy

L. R. F. Claes¹, J. Del-Favero¹, B. Ceulemans², L. Lagae³, C. Van Broeckhoven¹, P. De Jonghe²;
¹University of Antwerp, Antwerp, BELGIUM, ²University Hospital Antwerp, Antwerp, BELGIUM, ³University Hospital Gasthuisberg, Leuven, BELGIUM.

Severe myoclonic epilepsy of infancy (SMEI) is a rare disorder occurring in patients without a family history of a similar disorder. Early manifestations of the disease are tonic, clonic and tonic-clonic seizures occurring within the first year of life. These seizures are often prolonged, generalized and associated with fever. Later in life, SMEI patients suffer from afebrile seizures, including myoclonic, tonic-clonic, absences, simple and complex partial seizures. Early psychomotor and speech development is normal, but in the second year of life developmental stagnation occurs. Patients often become ataxic and speech development is delayed. In general, SMEI is very resistant to all anti-epileptic drugs. A mild type of epilepsy associated with febrile and occasionally afebrile seizures in adulthood is generalized epilepsy with febrile seizures plus (GEFS+). Missense mutations in the gene coding for a neuronal voltage-gated sodium channel a-subunit (SCN1A) were identified in families with GEFS+. Since both GEFS+ and SMEI show fever-associated seizures we screened 13 unrelated SMEI patients for mutations in SCN1A. Mutation analysis was performed using DHPLC and DNA sequencing. We identified a single heterozygous mutation in each patient: 4 frameshift, 2 nonsense, 2 splice donor and 5 missense mutations. Using a multi-allelic marker we genotyped the patients and the parents to confirm paternity. Sequencing demonstrated that all mutations are de novo mutations. Pyrosequencing was used to confirm that none of the mutations occurred in 184 control chromosomes.

Disruption of the serine threonine kinase 9 gene (STK9) as the cause of X-linked infantile spasms

V. M. Kalscheuer¹, T. Jiong¹, G. Hollway², E. Schwinger³, M. Hoeltzenbein¹, H. Eyre², N. Tommerup⁴, H. H. Ropers¹, J. Gecz²;
¹Max-Planck-Institute for Molecular Genetics, Berlin, GERMANY, ²Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, and Department of Paediatrics (J.G.) University of Adelaide, Adelaide, AUSTRALIA, ³Institute for Human Genetics, Universitätsklinikum Lübeck, Lübeck, GERMANY, ⁴Wilhelm Johannsen Centre for Functional Genome Research, IMBG, The Panum Institute, University of Copenhagen, Copenhagen, DENMARK.

X-linked infantile spasms (ISSX) or West-syndrome (WS) are characterised by onset of generalized seizures between 3 and 7 months of age, hypsarrhythmia on the electroencephalogram (EEG) and mental retardation (MR). In the pursuit of the ISSX/WS gene we have acquired two patients with balanced translocations 46,X,t(X;6)(p22.3;q14) and 46,X,t(X;7)(p22.3;p15) and clinical features of severe ISSX/WS. Both patients had early onset of seizures at age 2-3 month, and developmental arrest with profound mental retardation. Cloning of the X-chromosome breakpoints revealed the serine threonine kinase 9 gene (STK9, Montini et al. Genomics 51:247-433, 1998) to be interrupted by these rearrangements. Mutation screening of the 21 exons of the STK9 gene in three ISSX families was negative. In the meantime the ISSX/WS gene interval was refined on one Canadian family to ~7cM region in Xp21.3-Xp22.1 (Bruyere et al. Clin Genet 55:173-181, 1999); importantly, excluding the STK9 gene. More recently, the ISSX gene (MIM # 308350) has been identified from this interval (J. Gecz, unpublished). Based on the identical phenotype of our two patients with balanced X-autosome translocations we suggest, that there are at least two genes for ISSX on the human X-chromosome. We propose, that lack of a functional Stk9 protein is responsible for a severe form of West-syndrome.

S. Baulac¹, I. An-Gourfinkel², G. Huberfeld¹, M. Baulac², A. Brice¹, R. Bruzzone³, E. Le Guern¹;
¹INSERM U289, Pitié-Salpetriere hospital, FRANCE, ²Epilepsy center, Pitié-Salpetriere hospital, FRANCE, ³NSNR, Institut Pasteur, FRANCE.

GABAergic neurotransmission is known to be involved in epilepsy since many decades. We report a K289M mutation in the GABAA receptor gamma2 subunit gene (GABRG2) that segregated in a family with a phenotype closely related to Generalized Epilepsy with Febrile Seizures Plus (GEFS+), an autosomal dominant disorder associating febrile seizures and generalized epilepsy that had so far been linked only to mutations in sodium channel genes. The K289M mutation affects a highly conserved residue located in the extracellular loop between transmembrane segments M2 and M3. Functional analysis in Xenopus oocytes confirmed the genetic evidence of the implication of this mutation. The K289M mutation caused a dramatic decrease in the amplitude of GABA-activated currents compared to wild-type receptor. This study provides the first genetic evidence that a GABAA receptor is directly involved in a human idiopathic epilepsy.

Molecular characterisation of a 11q14.3 microdeletion associated with leukodystrophy of unknown cause.

B. Arveiler¹, C. Goizet¹, I. Coupry¹, L. Taine², C. Rooryck¹, D. Lacombe²;
¹Université Victor Segalen Bordeaux 2, Bordeaux, FRANCE, ²CHU de Bordeaux, Bordeaux, FRANCE.

We have previously described a de novo 11q14.3 microdeletion in a boy with leukodystrophy of unknown cause and oculocutaneous albinism. The detection of this chromosomal rearrangement revealed a new chromosomal region susceptible to bear a gene involved in the pathogenesis of leukodystrophy. We have molecularly characterized this microdeletion in order to identify the causative gene. Eighteen polymorphic microsatellite markers mapped in 11q14.3 were selected to test the patient for hemizygosity. The maternal alleles of two of them, D11S1780 and D11S1367, were deleted, while eleven were not and five were uninformative. The genetic size of the defined region is 2.4 cM. We have constructed a contig of BACs encompassing the entire region of interest that allowed us to obtain a physical map of this region. Only one gap of unknown size persists among this BAC contig. The DNA of several BACs from this contig was extracted and used as molecular probes for FISH analysis on patient's chromosomes. The size of the deleted region was reduced to 2 Mb. Sequence annotation of this region was performed in order to identify candidate genes implicated in leukodystrophy determinism. We are now testing the potential implication of these candidate genes both in our patient and in a group of children with leukodystrophy of unknown cause. Identification of the causative gene will be useful for elucidation of the molecular bases of leukodystrophies, the cause of which remains unknown in 30% of cases.