ESHG Posters 18

An sporadic case of DMD girl due to a deletion in dystrophin gene and skewed inactivation of normal X chromosome

P. Gallano¹, A. Garcia¹, A. Lasa¹, F. Martinez², M. Rodriguez¹, M. Baiget¹, M. Roig³;
¹Hospital Sant Pau, Barcelona, SPAIN, ²Hospital La Fe, Valencia, SPAIN, ³Hospital Valle Hebrón, Barcelona, SPAIN.

Duchenne muscular dystrophy (DMD) is an X-linked inherited disorder characterized by the absence of dystrophin in myofibers, with a prevalence of 1 in 3000 newborn males. Only about 8% of the heterozygote female carriers show clinical symptoms, wich can be as mild as pseudohypertrophy of the calf muscles or proximal limb weakness. In the rare cases of DMD females, cytogenetic studies have shown either the absence of one X chromosome or an X-autosome translocation. However, there are reports of rare manifesting carriers with a normal karyotype but with preferential inactivation of the normal X chromosome.
We present a young girl, an sporadic case, with a DMD phenotype. The inmonocytochemistry with anti-dystrophin antibodies (Novocastra) showed the total absence of this protein in the muscle tissue. The multiplex PCR showed a deletion of exon 3 to exon 44 (both included). The microsatellite analysis using STRs located in the deleted region (STR07A and STR44) demonstrate hemizygosity with paternal origin. The healthy brother present the same maternal haplotype. The pattern of inactivation was studied by differential methylation that exists between the active chromosome and inactive chromosome in the CpG islands of the X-linked genes, by using methylation-sensitive enzymes. The results confirmed a nearly total inactivation of the maternal chomosome.
In spite the difficulty that this kind of families, without affected boy, provides to the molecular analysis of the dystrophin gene, is important to perfoms this kind of stuies in girls with clincial symptoms of myopathy to establish the exact diagnosis of the disease.

Evidence for autosomal recessive inheritance in the infantile spinal muscular atrophy variant with congenital fractures.

W. Courtens¹, A. Johansson², B. Dachy³, F. Avni⁴, N. Telerman-Toppet³, H. Scheffer⁵;
¹Brugmann University Hospital, Brussels, BELGIUM, ²Department of Neonatology, Children’s University Hospital Queen Fabiola, Brussels, BELGIUM, ³Department of Neurology, University Hospital Brugmann, Brussels, BELGIUM, ⁴Department of Paediatric Radiology, Children’s University Hospital Queen Fabiola, Brussels, BELGIUM, ⁵Department of Medical Genetics, University of Groningen, Groningen, NETHERLANDS.

We report on a female newborn with a severe acute form of SMA, congenital bone fractures, camptodactyly of fingers and toes, bilateral hip dislocation and congenital heart defect, with early lethal outcome. DNA studies showed the absence of homozygosity for a deletion of exons 7 and 8 of the SMNt gene. A new lethal syndrome consisting of infantile spinal muscular atrophy (SMA) and multiple congenital bone fractures in 2 sibs has been suggested in 1991. Recently, another infant with a form of SMA and congenital fractures, was reported, thus validating the suggestion of a distinct and rare form of SMA associated with congenital bone fractures. Autosomal recessive inheritance was suggested in the original report, but no history of consanguinity was noted in the second. X-linked inheritance could however not be excluded since those three affected infants were male. Since our case is a female, an X-linked inheritance can be excluded. Since she was furthermore born to first cousin parents, it suggests an autosomal recessive inheritance in this rare variant of SMA type 1 with congenital fractures. We further conclude that this SMA variant, with early lethal outcome seems to have a variable clinical expression and that it is probably not linked to 5q.

H. Sibul¹, M. Hämarik²;
¹Tartu University Clinics, United Laboratories, Tartu, ESTONIA, ²Tartu University Clinics Childrens' Hospital, Tartu, ESTONIA.

The DNA testing for spinal muscular atrophy (SMA) became available in Estonia in 1997. Since then, 15 children with the clinical diagnosis of SMA have been studied. In 13 cases the diagnosis was confirmed by DNA analysis, in 2 cases the deletion was not found and the diagnosis based on clinical findings, electroneuromyography and muscle biopsy. 6 patients had type 1 (Werdnig-Hofmann), 2 had type 2 and 7 had type 3 (Kugelberg-Welander) SMA. Patients with type 2-3 disease had no complications at birth, those with type 1 SMA were asphyctic at birth, one patient was born with limb fractures. 5 out of 6 SMA type 1 patients had muscle weakness and hypotony at birth already, 1 developed those symptoms by age of 2 months. 5 patients died of respiratory insufficiency in first 6 months, one patient survived to 1.5 years. None of the SMA type 2 patients have ever walked unassisted. All SMA type 3 patients developed clinical features of the disease before the age of 3 years, 4 of them have lost the ability to walk at the age of 8-15 years.
We recommend to perform molecular testing for SMA in all children who are born in asphyxia and muscle weakness.

K. Becker¹, R. Yates²;
¹The Kennedy-Galton-Centre, Harrow, Middlesex, UNITED KINGDOM, ²Cardiology Unit, Great Ormond Street Hospital for Children, London, UNITED KINGDOM.

Winship et al. reported South African sibs with a combination of microcephaly, dilated cardiomyopathy and minor dysmorphic features in 1991. The cardiomypathy had resolved in the older child by the age of three years, and had markedly improved in the younger child on anticardiac failure therapy. The microcephaly was severe, and both children showed severe global developmental delay. The dysmorphic features were described as cupping of the outer helix of both pinnae, fifth finger clinodactyly and sandal gaps on both feet.
Kennedy et al. reported a nine year old girl with microcephaly, severe developmental delay and a dilated cardiomyopathy which had resolved at seven years of age. This patient had a sloping forehead, downslanting palpebral fissures, a narrow palate, small ears and a big sandal gap. Magnetic resonance imaging of the brain was normal.
All three children initially presented with cardiac failure, at the ages of two months, five months and neonatally respectively. There was no consanguinity in either family.
We report another case with microcephaly and a dilated cardiomyopathy, but without soft dysmorphic features, which suggests that these are more likely to represent coincidental familial traits.

M. Jelusic¹, K. Gall-Troselj², I. Jurak², K. Pavelic², H. Kniewald¹, N. Rojnic Putarek¹, I. Malcic¹;
¹University Hospital Zagreb, Zagreb, CROATIA, ²Division of molecular medicine, Ruder Boskovic Institute, Zagreb, CROATIA.

Hypertrophic cardiomiopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that in most cases familial and transmitted in a dominant fashion. More than 140 different mutations in 11 sarcomeric genes have been described to date. The most frequently affected gene codes beta-myosin heavy chain (MYH 7) (35-50%). Previous genotype-phenotype correlation studies have shown that mutations carry prognostic significance (R403Q, R719W and R719Q mutations were identified as highly malignant defects). We analysed MYH 7 in 14 patients (7 female and 7 male), members of 12 unrelated families, with HCM. The median age of patients at the time of diagnosis was 11,2 years. In 8 patients dominant inheritance was strongly suggested on the base of family history. Mutation analysis of MYH 7 was carried out for exons 8, 9, 13, 15, 16, 19, 20 and 23. Thirty-nine known mutations (9 malignant including R403Q, R719W and R719Q); 36 substitutions, 2 deletions and 1 insertion were analysed. The mutations were detected using mutation specific restriction enzyme assays and oligonucleotide sequencing. No mutation has been found in the analysed patients. The non-existence of malignant mutation amongst the analysed patients, especially those with a positive family history, it difficult to explain on the basis of published studies till 2000. The research carried out by Ackerman in 2001, has shown for the first time very low incidence of malignant mutations, less than 1%, what also confirm the results of this study.

The possible modifier effect of mitochondrial DNA defect in familial hypertrophic cardiomyopathy causally linked to beta-MHC gene mutation.

M. Diegoli¹, E. Porcu², L. Scelsi³, A. Urrata², M. Grasso², N. Banchieri², A. Repetto³, M. Pasotti³, C. Serio³, A. Brega⁴, L. Tavazzi³, E. Arbustini²;
¹Department of Pathology - University of Pavia, Pavia, ITALY, ²Cardiovascular Pathol. and Molec. Diagn. - Res.Transplantation Lab. , IRCCS Policlinico S.Matteo, Pavia, ITALY, ³Cardiology Division, IRCCS Policlinico S.Matteo, Pavia, ITALY, ⁴Department of Genetics, University of Milan, Milan, ITALY.

Two pathological mutations, one in mitochondrial DNA (mtDNA) and one in a nuclear gene were identified in a large Italian family with hypertrophic cardiomyopathy (HCM). Patients carrying both heteroplasmic T9957C (Phe->Leu, COX III subunit) mutation of the mtDNA and Lys450Glu mutation of the beta Myosin Heavy Chain (beta-MHC) were affected by HCM and developed congestive heart failure, while patients carrying the nuclear defect, but not the mtDNA defect, did not develop heart failure. The mtDNA mutation alone was not associated to clinical phenotypes, any type. Among patients with congestive heart failure in optimal medical treatment, all those who underwent or are awaiting for hert transplantation had higher amount of mutant DNA than those who are clinically stable. The cosegregation of congestive heart failure with a heteroplasmic mtDNA defect in our large family with HCM, causally linked to a beta-MHC gene mutation, indicates that mtDNA defects are possible candidates to the role of modifiers, influencing the evolution of the disease toward heart failure.

The combination of clinical variability, anticipation and parent of origin effects creates considerable difficulties in providing accurate risk estimates for relatives at risk for myotonic dystrophy, while the recent recognition of a second locus (PROMM/DM2) is an additional factor.
Available genetic risk data from both the early and recent literature and from personal studies are reassessed and combined to give a series of estimates suitable for genetic counselling of myotonic dystrophy families. In particular it is relevant for presymptomatic testing that the risk of a clinically normal, adult first degree relative carrying the myotonic dystrophy (DM1) mutation is unlikely to exceed 10%.

Multiple consanguinity in a large Azorean family affected with Spinal Muscular Atrophy type I: Implication in genetic counselling

L. Mota-Vieira¹, L. Rodrigues², P. Melo³, C. Melo³, J. Forjaz-Sampaio³, R. dos Santos², A. Maia⁴;
¹Genetic and Molecular Pathology Unit, Hospital of Divino Espirito Santo, Azores Islands, PORTUGAL, ²Molecular Genetics Unit, Medical Genetics Institute, Oporto, PORTUGAL, ³Obstetric and Gynecological Department , Hospital of Divino Espirito Santo, Azores Islands, PORTUGAL, ⁴Pediatrics Department, Hospital of Divino Espirito Santo, Azores Islands, PORTUGAL.

In small populations, consanguinity is an important factor in the appearance of recessively transmitted hereditary diseases, like spinal muscular atrophy (SMA). This disease is classified into three types, one of which is the early infancy severe SMA type I. About 95% of the affected individuals have homozygous deletions of exons 8 and/or 7 in the SMN1 gene.
Here, we describe a multiple consanguineous kindred from the Azorean island of São Miguel, Portugal, with one child (proband) affected with SMA type I. This child, who died before 1, was the first offspring of a close consanguineous marriage (inbreeding coefficient, F= 0.0703). The ascending genealogy of the proband shows that her parents and one set of her great-grand parents were both first cousins. In addition, the extended genealogical analysis revealed another consanguineous marriage (paternal proband’s uncle and his wife) with a lower value of relationship (F= 0.0195), although relevant in terms of genetic risk to offspring. No homozygous deletion of SMN1 and NAIPt genes were found in the proband, indicating that she probably has a rare mutation. However, haplotype analysis shows homoallelism for five closely linked polymorphic markers. Considering the familiar consanguinity, the proband homoallelism suggests identity-by-descent at SMA locus. The proband’s uncle and his wife are both carriers for the same haplotype, thus each offspring has a risk of 25% to be affected with SMA type I.
In conclusion, this study shows that the extended genealogical prenatal investigation and genetic counselling are very informative in families carrying rare recessive genes. (lmv.hospdelgada@mail.telepac.pt)

Gene expression profiling analysis shows possible signal transduction pathways leading to cardiac structural changes in left ventricular hypertrophy of renal failure

C. K. Maercker¹, C. Rutenberg², E. Ritz³, G. Mall⁴, K. Amann⁵;
¹Resource Center for Genome Research, D-69120 Heidelberg, GERMANY, ²Resource Center for Genome Research, Heidelberg, GERMANY, ³Univ. Heidelberg, Dept. Internal Medicine, Heidelberg, GERMANY, ⁴Dept. Pathology, Darmstadt, GERMANY, ⁵Univ. Erlangen, Dept. Pathology, Erlangen, GERMANY.

By gene expression profiling, we have investigated signaling molecules which obviously take in a major role in re-organizing the cytoskeleton, the extracellular matrix, and the capillary density in hearts of rats with renal failure. Male Sprague-Dawley rats, which were subjected to sub-total nephrectomy (SNX), served as a model system for a gene expression profiling analysis. Poly(A)+ RNA from the hearts of SNX animals and from sham-operated rats (SHAM) as a control, was labeled and hybridized with Rat UniGene filters containing about 27.000 gene and EST sequences (Bento Soares, Univ. of Iowa). Phosphoimaging and software analysis revealed substantial changes in gene expression in SNX animals compared to SHAM: Not only integrin a1 and b1 as central players, but also genes downstream of the integrin signaling pathway, like calreticulin, rac protein kinase a, rho A and rho B have been shown to be up-regulated in SNX animals. Rho protein again, might be causal for the stimulation of the expression different enzymes of the phosphatidylinositol pathway up to cardiac dynein and actin, which also has been shown by our experiments. Therefore, the gene expression profiling experiments discussed here not only allow us to describe genes involved in activation and expansion of the non-vascular interstitial tissue in uraemic animals, like timp3, tgf-b1, osteonectin, paxillin, and laminin a1, and linker molecules like plectin, catenin, cadherin and ICAM. These experiments also make it possible to find central signaling molecules responsible for the pathomechanisms involved in cardiac structural changes upon renal failure.

Towards the identification of molecular pathways underlying ADAM 12’s role in myogenesis

B. Moghadaszadeh¹, P. Kronqvist¹, N. Kawaguchi¹, R. Albrechtsen¹, X. Xu¹, H. D. Schrøder², F. C. Nielsen³, C. Fröhlich¹, K. Iba¹, E. Engvall⁴, U. M. Wewer¹;
¹Institute of Molecular Pathology, University of Copenhagen, Copenhagen, DENMARK, ²The Department of Pathology, Odense University, Odense, DENMARK, ³The Department of Clinical Biochemistry, Copenhagen University Hospital, Copenhagen, DENMARK, ⁴The Burnham Institute, La Jolla, CA.

The ADAMs (A Disintegrin And Metlloprotease) are a recently described family of cell membrane anchored glycoproteins. During mouse development ADAM 12 is expressed in several tissues including muscle. Postnatally, the expression of ADAM 12 in the muscle ceases and only reappears transiently during muscle regeneration.
To study the role of ADAM 12 in vivo, we generated transgenic mice that overexpress ADAM 12 in the muscle beyond embryonic life. These mice showed accelerated regeneration following acute injury. Interestingly, when ADAM 12 transgenic mice were paired with dystrophin-deficient mdx mice, the enhanced expression of ADAM 12 resulted in a decrease of muscle cell necrosis and inflammation and a more than 50% reduction in serum creatine kinase.
In order to begin to decipher the molecular mechanism of ADAM 12’s action in muscle, we examined on DNA microarray chips the expression of 12500 genes in ADAM 12 transgenic mice and their littermate controls. Three independent experiments gave similar results; in particular, compared to their littermate controls, the transgenic mice showed an increase in the expression of myogenin, myosin, troponin and acetylcholine receptor which are implicated in muscle development and structure. Besides, P21 cycline-dependent kinase inhibitor, which is implicated in cell survival showed an increased expression in transgenic mice. The analysis of mdx and ADAM 12/mdx microarray experiments are underway.

Muscle specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cell cultures

A. Buj-Bello¹, D. Furling², H. Tronchère³, J. Laporte¹, B. Payrastre³, G. Butler-Browne², J. Mandel¹;
¹IGBMC, CNRS/INSERM/ULP, Illkirch, CU. Strasbourg, FRANCE, ²Faculté de Médecine Pitié-Salpêtrière, CNRS UMR 7000, Paris, FRANCE, ³Institut Fédératif de Recherche en Immunologie Cellulaire et Moléculaire, Toulouse, FRANCE.

The myotubularin-related 1 (MTMR1) gene belongs to a highly-conserved family of phosphatases, which includes hMTM1, mutated in X-linked myotubular myopathy, a severe congenital disorder that affects skeletal muscle, and hMTMR2, mutated in Charcot-Marie-Tooth type 4B, a recessive demyelinating neuropathy with a specific Schwann cell pathology. We and others recently showed that the MTM1 gene product, myotubularin, is a potent phosphatidylinositol 3-phosphate (PI(3)P) phosphatase. We now demonstrate that this function is conserved amonsgt other members of the family, in particular MTMR2 and MTMR1 proteins. Whereas no mutations in the hMTMR1 gene have been associated with a human disorder so far, this gene, that arose from an ancient hMTM1 duplication and is adjacent to it in Xq28, may share some biological functions with MTM1, as the corresponding proteins are 57% identical. We investigated whether MTMR1 could play a role in myogenesis by analysing its expression pattern during muscle differentiation both in vitro and in vivo. We have identified 3 novel coding exons in the MTMR1 intron 2 that are alternatively spliced, giving rise to at least four mRNA isoforms. One of the transcripts is muscle-specific. We analysed MTMR1 alternative splicing in muscle cells derived from patients with congenital myotonic dystrophy (cDM1), a disease with RNA splicing disturbances. We have found a reduction of muscle-specific isoform levels and the appearance of an aberrant MTMR1 transcript in cDM1 myotubes in culture. Our results suggest that MTMR1 plays a role in muscle formation and represents a novel target for aberrant pre-mRNA splicing in myotonic dystrophy.

The neuron-specific RNA binding proteins CELF3 is a component of the DMPK mRNA-associated ribonucleoprotein complex: implications for myotonic dystrophy

M. D'Apolito¹, M. Bozzali², A. Caruso³, S. Quattrone⁴, A. Grifa¹, T. Dottorini², M. Gennarelli⁵, A. Pizzuti², B. Dallapiccola², A. Quattrone¹;
¹IRCCS CSS, S. Giovanni Rotondo, ITALY, ²Istituto CSS Mendel, Roma, ITALY, ³Dipartimento di Fisiologia Umana e Farmacologia, Univ. "La Sapienza", Roma, ITALY, ⁴Dipartimento di Anatomia, Istologia e Medicina Legale, Univ. di Firenze, Firenze, ITALY, ⁵IRCCS Fatebenefratelli, Brescia, ITALY.

Cognitive impairment is a common finding in congenital myotonic dystrophy type 1 (DM1), being also associated with late onset DM1. The relative independency from the muscular deficits of this DM1 psychiatric feature could be explained on the basis of a molecular perturbation selective of the CNS, which is at the moment completely unknown. A CTG microsatellite expansion in the 3’ UTR of the DMPK mRNA is the cause of DM1, and a substantial body of evidence is indicating that the majority of the clinical features of DM1 are consequent to a still undefined perturbation in the cellular ribonucleoprotein infrastructure, due to an aberrant interaction of RNA binding proteins with the expanded DMPK mRNA. Here we further characterize a family of six human RNA binding proteins, ortholog of the Drosophila Bruno translational repressor, which are component of the DMPK mRNA-associated ribonucleoproteins. Two of these proteins, CELF3 and CELF5, display a strictly neuron-specific pattern of expression. CELF3 appears to be extremely well conserved in evolution and selectively expressed in certain regions of the mouse adult brain, while its developmental expression pattern in the mouse is indicative of a role in brain formation. Therefore, a perturbation of localization or activity of CELF3 could be involved in the mental deficiencies suffered by DM1 subjects.

Functional Consequence of Two Novel Dominant Mutations in the Muscle Chloride Channel Gene CLCN1 Causing Thomsen’s Syndrome

M. Dunø¹, E. Colling-Jørgensen², M. Schwartz¹, J. Vissing²;
¹Dept. of Clinical Genetics, 4062, University Hospital Copenhagen, DENMARK, ²Dept. of Neurology, 2082, University Hospital Copenhagen, DENMARK.

Autosomal dominant myotonia congenita - Thomsen’s disease - and autosomal recessive myotonia congenita - Becker’s disease - are rare mostly nondystrophic disorders both due to mutations in the CLCN1 gene encoding the muscle chloride channel 1. In an attempt to categorize Danish patients with myotonia congenita genetically, we sequenced all 23 exons of the CLCN1 gene in ten selected patients and identified four novel mutations. Two missense mutations (E193K, M128V) were found in dominant myotonia whereas one missense (T328I) and one nonsense (nt2517DCT) mutation were found in recessive myotonia. Apart from the novel mutations we also found the previously described mutations; P480L, G285E, F307S and ntD1437-1450. Surprisingly, the recurrent R894X mutation was found in four pedigrees segregation both in a dominant and a recessive fashion, and the F307S mutation, which has priviously thought to be strictly dominant, was found together with the nt2517DCT nonsense mutation, suggesting recessive behavior in this family. Thus, the relation between genotype and phenotype is not straightforward in myotonia congenita. In order to shed light on the genotype - phenotype relation we examined the electrophysiological features of the patients caring the two novel dominant mutations and unexpectedly, found no decrease of the decrement. The functionality of the two mutations was further characterized by whole-cell patch-clamp.

I. Novakovic¹, S. Apostolski², S. Todorovic³, L. Lukovic¹, V. Bunjevacki¹, D. Bojic⁴, L. Mestroni⁵, J. Milasin⁶;
¹Institute of Biology and Hum. Genetics, School of Medicine, Belgrade, YUGOSLAVIA, ²Institute of Neurology, KCS, Belgrade, YUGOSLAVIA, ³Clinic for Pediatric Neurology and Psychiatry, Belgrade, YUGOSLAVIA, ⁴Institute of Cardiovascular Diseases "Dedinje", Belgrade, YUGOSLAVIA, ⁵ICGEB, Trieste, ITALY, ⁶Institute of Biology and Hum. Gentics, School of Stomatology, Belgrade, YUGOSLAVIA.

In-frame deletions of distal part of the dystrophin gene are generally associated with classic Becker muscular dystrophy (BMD). Skeletal myopathy has benign course with later presentation and slower progression, but cardiac disorders could show clinical diversity. In this study we analyzed correlation between gene defect and clinical phenotype in a group of BMD patients with dystrophin gene deletions encompassing exons 45-60. Dystrophin gene deletions were detected by standard multiplex PCR method based on simultaneous amplification of deletion prone exons. Clinical evaluation included neurological and detail cardiological examination. The mean time of onset of disease in our patients was 14.4 y. and skeletal myopathy had relatively slow progression rate, so none of them was in severe stage or wheelchair bound. All of the patients were without symptoms of heart failure, but we detected diffrent forms of cardiological disorders, ranged from benign ECG changes to moderate heart function impairment (EF=40%). Cardiac disorders were in correlation with patient age and, to a lesser extent, with muscle dystrophy severity. Herat dysfunction was associated with different types of gene deletions. For example, moderate systolic function impairment had one patient with single exon 45 deletion (age 19 y.) and another patient with deletion of exons 45-47 (age 31 y.).

Deletion patterns of dystrophin gene and carrier analysis in Hungarian families with Duchenne/Becker muscular dystrophies

H. Piko¹, I. Nagy², A. Herczegfalvi³, A. Herczegfalvi³, E. Endreffy⁴, V. Karcagi²;
¹National Center for Public Health, Budapest, HUNGARY, ²National Center for Public Health, National Institut of Enviromental Health, Budapest, HUNGARY, ³Bethesda Children's Hospital, Budapest, HUNGARY, ⁴University of Szeged, Faculty of Medicine, Pediatric clinic, Szeged, HUNGARY.

Duchenne muscular dystrophy is an X-linked progressive muscular disorder with an incidence of 1 per 3500 live-born males. Patients become wheelchair-bound at the age of 18-25 years. Becker muscular dystrophy is a less severe allelic form of the disease with an incidence of 1 per 30 000 live-born males.
Deletion pattern analysis of the dystrophin gene was performed in 49 Hungarian patients with Duchenne/Becker muscular dystrophy. The detection of deletions was performed by multiplex PCR technique that enables the simultaneous screening of 18 exons of the dystrophin gene. In 29 cases (59% of total patients), deletions were detected in the most commonly affected exons. With respect to the proximal-distal distribution of the deletions, 82% of the patients had deletions at the 3’ end of the gene, 18% of the deletions affected only the 5’ end. Distribution pattern in the dystrophin gene deletions showed similarity to that observed in various Western European populations.
If deletions were detected in the index patient, identification of female carriers in the affected family was carried out by radioactive Southern blot hybridization using special cDNA probes, a new method in Hungary introduced by our laboratory. In the 15 families examined so far, 46% of female relatives proved to be carriers of the DMD/BMD gene deletions. The cDNA analysis also enables determination of the exact localization and the full size of the deletion in patients. Therefore, the analysis was also performed in 38 patients and additional exon deletions of the dystrophin gene were detected.

j. Knezevic¹, K. Gall-Troselj², J. Pavelic¹;
¹"Rudjer Boskovic" Institute, Zagreb, CROATIA, ²"Rudjer Boskovic" Institute,, Zagreb, CROATIA.

Duchenne muscular dystrophy and Becker muscular dystrophy are X-linked recessive neuromuscular diseases caused by mutations in the gene coding for the 427-kD cytoskeletal protein dystrophin. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene are responsible for about 65% of DMD or milder, BMD cases. Within the dystrophin gene, these deletions tend to cluster in hot spot. Frameshift deletions result in DMD (with no functional dystrophin protein produced), while deletions that maintain the reading frame produce the BMD phenotype (partially functional dystrophin present). Approximately two-thirds mothers of affected males with known deletions are asymptomatic carrier of DMD and about 30% percent of affected males represent de novo mutations. Current methods used in carrier testing are directed to multiplex PCR and quantitative analysis of products. However these methods are difficult to perform and interpretation can be subjective. In our study we try to develop an effective and exact assay of carrier testing through cDNA. Illegitimate transcription has made possible the analysis of dystrophin mRNA from peripheral blood lymphocytes. Thanks to the fact that deletions are clustered in hot spots we have designed two sets of primers which span the regions of interest. In a case of female carrier two bands should be recognised; one from normal allel and second related to DMD allel. Here we would like to show preliminary data, which in our opinion will be of great benefit in diagnostic laboratory procedure. We believe that in this way any subjective interpretation will be overcome.

Loss of the chloride channel in DM1 skeletal muscle due to misregulated alternative splicing: a likely cause of myotonia

N. Charlet-B., R. S. Savkur, G. Singh, A. V. Philips, E. A. Grice, T. A. Cooper;
Department of Pathology, Baylor College of Medicine, Houston, TX.

Myotonic dystrophy type 1 (DM1) is the most common form of adult onset muscular dystrophy (1 in 8500 individuals). It is a dominantly inherited disorder caused by a CTG trinucleotide expansion in the 3' untranslated region of the DMPK gene. Nuclear accumulation CUG)n RNA from the expanded allele is proposed to be pathogenic in DM1 by disrupting the function of the splicing regulator, CUG-binding protein (CUG-BP). A predominant feature of DM1 is myotonia, manifested as delayed skeletal muscle relaxation after voluntary contraction. In humans or animal models myotonia can be due to loss of the muscle-specific chloride channel (ClC-1). Here we demonstrate by western blot and RNase protection analysis loss of ClC-1 mRNA and protein in DM1 skeletal muscle. The likely cause is nonsense mediated decay, as premature stop codons are incorporated in the ClC-1 mRNA by aberrant alternative splicing of intron 2 and exons 6b and 7a. We were able to reproduce the DM1 aberrant splicing pattern in normal cells by coexpressing CUG-BP with a ClC-1 intron 2 minigene. We conclude that aberrant regulation of alternative splicing leads to a predominant pathological feature of DM1. We predict that other targets of CUG-BP are misregulated in DM1 patients causing other symptoms of the disease

A 12-year experience in molecular diagnosis of Duchenne and Becker muscular dystrophies: a comprehensive strategy for mutation detection allows to detect the molecular defect in 90% of the DMD/BMD patients.

S. Tuffery-Giraud¹, S. Chambert¹, C. Saquet¹, C. Coubes², F. Rivier³, B. Echenne³, M. Claustres¹;
¹Laboratoire de Génétique Moléculaire, CHU, Montpellier, FRANCE, ²Consultation de Génétique Médicale, CHU, Montpellier, FRANCE, ³Service de Neuropédiatrie, CHU, Montpellier, FRANCE.

Since 1989, 258 families have been referred to our laboratory for molecular diagnosis of Duchenne (DMD) or Becker (BMD) muscular dystrophies. We have developed a hierarchical mutation screening strategy for mutation identification in the dystrophin gene (Xp21) including the following steps (1) multiplex PCR to detect large intragenic deletions (2) RT-PCR coupled to the protein truncation test (PTT) to scan for rare deletions, duplications, and point mutations, and (3) sequencing, PCR/restriction, DHPLC, or gene dosage analysis (LightCycler, Roche Diagnostics) to confirm point mutations and test for gene dosage alterations at the genomic level. This strategy allows to detect the molecular defect in 90% of the investigated patients. As a result, the families are currently benefiting from accurate carrier-status assessment. Up to now, a total of 71 mutations have been found by the RT-PCR/PTT procedure consisting in 6 exon deletions, 6 duplications, and 59 point mutations (26 nonsense, 17 splice mutations and 16 frameshift). The effects of nucleotide alterations in splice sites were precisely determined by examination of muscle transcripts, and accurate genotype/phenotype correlation was delivered to the clinicians. Further investigations are required to identify the cause of DMD or BMD in the remaining 10% patients in whom the mutation is not identified yet. An alternative mutation scanning method, the Base Excision Sequence Scanning (BESS), is currently being tested in those patients.
Support : Association Francaise contre les myopathies (AFM).

Insertion of mid-intron cryptic exons in dystrophin mRNA: a novel mechanism of dystrophinopathy

F. Leturcq, C. Beroud, A. Carrie, C. Beldjord, N. Deburgrave, S. Llense, N. Carelle, J. C. Kaplan, D. Recan;
Institut Cochin de Génétique Moléculaire, Paris, FRANCE.

We describe two cases of Becker Muscular Dystrophy with an aberrant dystrophin transcript containing an unknown sequence precisely intercalated between two intact exons (89 nt between exons 60 and 61 in patient #1; 90 nt between exons 9 and 10 in patient #2). Both insertions introduce a premature stop codon into the transcript. An in silico survey of the now available entire DMD gene sequence showed that these inserts are present in the mid-part of intron 60 (95 kb) and intron 9 (52 kb) respectively, both being flanked by cryptic splice sites. By sequencing each putative cryptic exon in the two patients we found a single substitution ( G->T in patient #1; C->T in patient #2), converting a weak donor splice site into a perfect one, corroborating the assumption that the inserted sequences were cryptic exons activated by a point mutation.
Both patients exhibited a BMD phenotype, consistent with the coexistence of the aberrant transcript with a normally spliced transcript and a weak normal sized dystrophin. Patient #1 was severely mentally retarded.
The activation of cryptic exons by a point mutation is not a novel mechanism, but to our knowledge it has not been reported so far in the recently deciphered gigantic DMD gene introns. This mechanism seems to be unfrequent since we found only 2 such cases in our collection of 720 DMD/BMD patients with a documented mutation. We emphasize that this type of mutation, now explorable, cannot be directly detected at the genomic level without prior transcript analysis.

Sequencing Of The 79 Exons Of The Dystrophin Gene In Duchenne And Becker Muscular Dystrophies: Identification Of 45 Point Mutations.

L. Michel-Calemard¹, E. Ollagnon², M. P. Cordier³, F. Prieur⁴, N. Streichenberger⁵, L. Féasson⁶, H. Plauchu⁷, P. Guibaud⁸, Y. Morel¹;
¹Laboratoire de Biochimie Endocrinienne et Moléculaire, Hôpital Debrousse, Lyon, FRANCE, ²Service de Génétique, Hôpital de la Croix-Rousse, Lyon, FRANCE, ³Service de Génétique, Hôpital E. Herriot, Lyon, FRANCE, ⁴Service de Génétique, Hôpital Nord, St-Etienne, FRANCE, ⁵Laboratoire d'Anatomie-Pathologique, Hôpital Neurologique, Lyon, FRANCE, ⁶Service d'Explorations Fonctionnelles Musculaires, Hôpital St Jean Bonnefonds, St-Etienne, FRANCE, ⁷Service de Génétique, Hotel-Dieu, Lyon, FRANCE, ⁸Service de Pédiatrie et Génétique, Hôpital Debrousse, Lyon, FRANCE.

In 1996, we took over molecular diagnosis of dystrophinopathies from the Rhône-Alpes region of France, previously performed in two distinct laboratories. 1816 DNA from 326 families are progressively reanalyzed. In 161 studied families, 81 deletions were identified by multiplex-PCR 18 exons (50%).
In the absence of deletion, our strategy to explore the gene depends on the feasibility of a muscle biopsy. When muscular tissue is available, sequencing of cDNA is used to seek for mutations. 5 point mutations, 1 deletion and 1 duplication were identified this way.
If the biopsy is impossible, strategic choice becomes delicate. As the gene is large, sequencing of all exons doesn't seem the appropriate one at first. It is though the approach we chose for different reasons: 1) we have in our laboratory an old version of sequencer which is not very suitable for screening techniques, 2) the patients being hemizygotes, one sequence is sufficient to explore an exon. A little more than 2 gels are necessary to sequence a patient's all exons, 3) samples were usually collected long ago and the patients are not approachable for biopsy.
Some exons are co-amplified, reducing the number of PCRs to perform (69 vs 79).
77 patients were sequenced: 69 on genomic DNA, 8 on cDNA, allowing 73% detection: 5 deletions outside the hot-spots, 1 duplication and 50 point mutations (20 non-sense, 16 splicing, 14 frameshift mutations). Among the 21 patients without mutation, only 7 were completely sequenced. They probably carry undetected duplications or intronic mutations.

S. Nasti¹, C. Autore², S. C. Barillà², G. D’Amati³, D. Pistilli³, F. Sironi⁴, G. Ghigliotti¹, C. Brunelli¹, R. Casadonte⁵, P. Spirito⁶, G. Cuda⁵, D. A. Coviello⁴;
¹Dipartimento di Medicina Interna, Università di Genova, Genova, ITALY, ²Dipartimento di Scienze Cardiovascolari e Respiratorie, Università la Sapienza Roma, Roma, ITALY, ³Dipartimento di Medicina Sperimentale e Patologia, Università la Sapienza Roma, Roma, ITALY, ⁴Laboratorio di Genetica Medica, Dipartimento di Medicina di Laboratorio, Istituti Clinici di Perfezionamento, Milano, ITALY, ⁵Dipartimento di Medicina Sperimentale Clinica, Università Magna Graecia, Catanzaro, ITALY, ⁶Divisione di Cardiologia, Ospedali Galliera, Genova, ITALY.

Many genetic conditions are considered a single disease. However, molecular analysis often revealed a wide genetic heterogeneity. Recently, new classifications based on the molecular defect, rather than clinical presentation, have been proposed.
Familial Hypertrophic Cardiomyopathy (FHC) is transmitted as autosomal dominant trait with a prevalence of about 1/500. The disease is characterised by a hypertrophied and non-dilated left ventricle. The clinical course of the disease is heterogeneous: some patients remain asymptomatic, others die suddenly. Mutations causing disease in ten cardiac contractile proteins have been identified in FHC patients. Recently mutations on a non sarcomeric protein gene have also been identified as responsible of FHC. Genotype-phenotype correlation is crucial to the understanding of the natural history of FHC and possibly to separate heterogeneous clinical presentations into different diseases. Genetic definition of FHC may also have to be reconsidered including the clinical interpretation of possible recessive mutations, double heterozygous mutations, and mutations on two genes in the same subject.
We believe that it is crucial to perform the molecular characterisation of patients on several loci. Due to the large number of genes responsible for this disease, we have started a pilot study to organise an Italian laboratory diagnostic network, and we look forward to join other European laboratories working in the same field.
Our activity has focused on search of mutations in MYH7, MYBPC3, TPM1 and TNNT2 genes using the DHPLC technology and automated sequencing. A total of 26 different mutations have been identified. Specific cases will be reported in our presentation.

Various forms of worldwide quadriceps sparing myopathy are caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase gene

I. Eisenberg¹, G. Grabov-Nardini², H. Hochner², T. Potikha², V. Askanas³, T. Bertorini⁴, W. Bradley⁵, G. Karpati⁶, L. Merlini⁷, M. Sadeh⁸, Z. Argov², S. Mitrani-Rosenbaum²;
¹The Hebrew University- Hadassah Medical School, Jerusalem, ISRAEL, ²The Hebrew University-Hadassah Medical School, Jerusalem, ISRAEL, ³University of Southern California, Los Angeles, CA, ⁴The University of Tennessee, Memphis, TN, ⁵University of Miami School of Medicine, Miami, FL, ⁶Montreal Neurological Institute, Montreal, PQ, CANADA, ⁷Istituto Ortopedico Rizzoli, Bologna, ITALY, ⁸Wolfson Hospital, Holon, ISRAEL.

Hereditary Inclusion Body Myopathy (HIBM) (MIM600737) is a unique group of neuromuscular disorders characterized by adult onset, slowly progressive distal and proximal weakness and a typical muscle pathology including rimmed vacuoles and filamentous inclusions. The autosomal recessive prototype form described in Jews of Persian descent and later of other Middle Eastern origins (Iraq, Afghanistan, Kurdistan, Uzbekistan, Egypt) affects mainly leg muscles but with an unusual distribution that spares the quadriceps. We have identified the gene encoding for UDP-N-acetylglucosamine2-epimerase/N-acetylmannosamine kinase (GNE), at chromosome 9p12, as the disease causing gene in this community, where a single homozygous missense mutation (Met712Thr) has been found. Further study of the involvement of GNE in HIBM affected families from various ethnic origins identified ten novel mutations: an homozygous missense mutation in a consanguineous family from Mexico and distinct compound heterozygotes in HIBM-quadriceps sparing non Jewish families from Germany, The Bahamas, Italy, Georgia (USA), and in a large family from East India. Furthermore, interestingly, the GNE "Persian mutation" was also found in HIBM atypical patients with unusual muscle weakness distribution (quadriceps involvement, unusual proximal leg involvement, mild facial weakness) and with unusual occurrence of inflammation, known to appear only in the sporadic form of inclusion body myositis (IBM).
The identification of GNE as the responsible gene for HIBM allows not only the re-evaluation of the phenotypic and genotypic scope of multiple worldwide recessive HIBM forms, but also its involvement in the sporadic form of the disease which is the most common myopathy in individuals over age fifty.

Familial and sporadic forms of central core disease are associated with mutations in the C-terminal domain of the skeletal muscle ryanodine receptor

J. Lunardi¹, N. Romero², N. Monnier³, P. Landrieu⁴, Y. Nivoche⁵, M. Fardeau²;
¹Université Joseph Fourier, Grenoble, FRANCE, ²U523-Institut de Myologie, Paris, FRANCE, ³CHU, Grenoble, FRANCE, ⁴CHU-Bicêtre, Le Kremlin-Bicêtre, FRANCE, ⁵Hôpital R. Debré, Paris, FRANCE.

Central core disease (CCD) is an autosomal dominant congenital myopathy. Diagnosis is based on the presence of cores in skeletal muscles. CCD has been linked to the gene encoding the ryanodine receptor (RYR1) and is considered as an allelic disease of Malignant Hyperthermia Susceptibility. However, the report of a recessive form of transmission together with a variable clinical presentation has raised the question of the genetic heterogeneity of the disease. Analyzing a panel of 34 families exclusively recruited on the basis of both clinically and morphologically expressed CCD, 12 different mutations of the C terminal domain of RYR1 have been identified in 16 unrelated families. Morphological analysis of the patients' muscles showed different aspects of cores, all of them being associated with mutations in the C terminal region of RYR1. Furthermore, we characterized the presence of neomutations in the RyR1 gene in four families. This indicates that neomutations into the RyR1 gene are not a rare event and must be taken in account for genetic studies of families that present with congenital myopathies type "Central Core Disease". Three mutations led to the deletion in frame of amino acids. This is the first report of amino-acid deletions in RYR1 associated with CCD. According to a 4- transmembrane domains model, the mutations concentrated mostly in the myoplasmic and luminal loops linking respectively transmembrane domains T1 and T2 or T3 and T4 of RYR1.

Results of mutation analysis in candidate genes for Emery-Dreifuss muscular dystrophy

M. S. Wehnert¹, C. Wasner², G. Bonne³, A. van der Kooi⁴, D. Recan⁵, D. Toniolo⁶;
¹Institute of Human Genetics, D-17487 Greifswlad, GERMANY, ²Institute of Human Genetics, Greifswald, GERMANY, ³INSERM UR523, Paris, FRANCE, ⁴Department of Neurology, Amsterdam, NETHERLANDS, ⁵Hopital Cochin, Paris, FRANCE, ⁶IGBE, Pavia, ITALY.

So far two disease genes, STA and LMNA, have been associated to Emery-Dreifuss muscular dystrophy (EDMD). Screening of patients with EDMD revealed, that mutations in these two genes together account for only 45 % of the cases. Obviously, further genes are likely to be involved in EDMD. Unfortunately, most patients are sporadic cases and families for a positional cloning approach are rare. Thus, we started a candidate gene approach. Emerin encoded by STA and lamin A/C encoded by LMNA are components of the inner nuclear membrane and the nuclear lamina. Thus it seems very likely, that other genes encoding functionally related proteins can cause this disease. Additionally, we considered such genes as candidates, which are specifically expressed in heart and skeletal muscle, that represent the mainly affected tissues in EDMD. Thus, 118 EDMD patients, that were excluded to have mutations in STA and LMNA, were included in the candidate gene mutational analysis. Using genomic DNA of the patients, the exons including the intron/exon boundaries and the promoter regions of candidate genes were amplified by PCR and screened by heteroduplex analysis combined with direct sequencing. Currently, 12 genes (BAF, DDX16, FLNC, LAP1, LAP2, LBR, LMNB1, LMNB2, MAN1, NRM, PSME3, SMPX) are under investigation. So far no disease causing mutation has been found. However, 17 intragenic SNPs have been identified including one in the translated region of FLNC leading to an amino acid exchange. Further upcoming genes related to the nuclear membrane and lamina have to be involved to complete the study.

First description of mild phenotypes of Ullrich congenital muscular dystrophy caused by mutations in COL6A3.

P. Guicheney¹, E. Demir¹, V. Allamand¹, P. Sabatelli², B. Echenne³, H. Topaloglu⁴, L. Merlini⁵;
¹INSERM U523, Paris, FRANCE, ²CNR, Bologna, ITALY, ³Hôpital Saint Eloi, Montpellier, FRANCE, ⁴Hacettepe Children's Hospital, Ankara, TURKEY, ⁵IOR, Bologna, ITALY.

Ullrich Congenital Muscular Dystrophy (UCMD) is an autosomal recessive disorder characterized by generalized muscular weakness, contractures of multiple joints and distal hyperextensibility. Homozygous mutations of COL6A2 on chromosome 21q22 have recently been shown to cause UCMD. We performed a genome-wide screening with microsatellite markers in a consanguineous family with three UCMD affected sibs. Linkage of the disease to chromosome 2q37 was found in this family and others. Analysis of COL6A3, which encodes the alpha3 chain of collagen VI, led to the identification of a homozygous mutation in three families.
A nonsense mutation, R2342X, caused absence of collagen VI in muscle and fibroblasts and a severe phenotype, as described in UCMD patients. A splice site mutation (6930+5A>G), leading to the skipping of an exon, caused a partial reduction of collagen VI in muscle biopsy and an intermediate phenotype. A nonsense mutation R465X was associated with only a limited reduction of collagen type VI around patient muscle fibers. This was due to nonsense mediated exon skipping and could explain the mild phenotype of the patient who was ambulant at the age of 18 years and showed an unusual combination of hyperlaxity and finger contractures.
Mutations in COL6A3 are described in UCMD for the first time, and illustrate the wide spectrum of phenotypes which can be caused by collagen VI deficiency.

D. Coprean¹, M. Popescu², M. Militaru¹, B. Bosca¹;
¹"Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, ROMANIA, ²"Horea Radu" Center of Neuromuscular Pathology, Valcele, ROMANIA.

Facioscapulohumeral muscular dystrophy (FSHMD) is characterized by a considerable variability in terms of the severity of symptoms, onset age and changes at muscular level. FSHMD can be clinically distinguished from the other progressive muscular dystrophies by: changes in the face appearance (tapir lip) and scapulohumeral girdle, as well as slow evolution. FSHMD is a myopathy with autosomal dominant inheritance and incomplete gene penetrance. The locus of FSHMD gene maps to 4q35-3 ter. The studies were performed on 180 FSHMD cases. The analysis of the pedigrees of the patients investigated confirms the autosomal dominant mode of inheritance. The analysis of FSHMD onset age in patients from the same family (ancestry and descent) shows the presence of the anticipation phenomenon (earlier age at onset in successive generations). Incomplete FSHMD gene penetrance is demonstrated by the intrafamilial variability of the severity and evolution of the disease: from almost asymptomatic patients to wheelchair dependent patients. Our study found a slight prevalence of the disease in the male sex (52.68%) compared to the female sex (47.32%). Epidemiological studies have found a higher FSHMD incidence in Brasov, Constanta, Ilfov, Prahova, Salaj and Sibiu districts, which can be explained by the effect of the founder couple and genetic drift. The study of the incidence and clinical genetic aspects of this form of myopathy which represents 9% of all PMD types and generates serious socio-economic problems (being a disabling disease) is motivated by the necessity of offering efficient genetic counseling to FSHMD patients and their families.

Molecular and Clinical Studies of Facioscapulohumeral Muscular Dystrophy (FSHMD) in Greece

H. Fryssira, K. Kekou, C. Sofocleous, S. Youroukos, A. Manta, A. Mavrou, C. Metaxotou;
Medical Genetics, University of Athens Medical School, "Aghia Sophia" Childrens Hospital, Athens, GREECE.

FSHMD is a myopathy transmitted by autosomal dominant inheritance.The genetic locus has been mapped to the 4q35 subtelomeric region. The telomeric probe p13E-11 has been shown to detect EcoRI polymorphic fragments shorter than 35kb and an EcoRI-BlnI (or AvrII) digestion is used to avoid the interference of small EcoRI polymorphic fragments of 10qter
origin. We studied 45 Greek families, 59 affected and 21 unaffected individuals at risk of inheriting or transmitting the FSHMD shorter frgments. Restriction analysis of genomic DNA using EcoRI and EcoRI/AvrII enzymes, followed by pulse-field or conventional gel electrophoresis and non radioactive hybridization with p13E-11 probe, were performed. The results revealed an EcoRI/AvrII fragment, ranging between 7.5 and 34kb, in 32 families (74%), comprising 19 familial and 13 isolated cases. In all, except one, FSHMD familial cases the same size fragment segregated in the family. In two isolated cases, the presence of three shorter fragments, complicated the interpretation of Southern blot analysis. An overall correlation has been found, between fragment size, age of onset and disease severity, indicating that patients with EcoRI/AvrII fragment smaller than 20kb are more severely affected than patients with larger fragments. The application of double digestion, identifies FSHMD alleles even in pre-symptomatic cases, facilitates clinical prognosis and allows genetic counselling of the disease.

Mutations in the Selenoprotein N gene (SEPN1) cause congenital muscular dystrophy with early rigidity of the spine and restrictive respiratory syndrome

N. Petit¹, B. Moghadaszadeh¹, C. Hu¹, A. Lescure², S. Quijano³, B. Estournet³, L. Merlini⁴, F. Muntoni⁵, H. Topaloglu⁶, A. Krol², U. Wewer⁷, P. Guicheney¹;
¹INSERM 523, Institut de Myologie, GH Pitié-Salpêtrière, Paris, FRANCE, ²IBMC du CNRS, UPR 9002, Strasbourg, FRANCE, ³Hôpital Raymond Poincaré, Paris, FRANCE, ⁴IOR, Bologna, ITALY, ⁵Hammersmith Hospital, London, UNITED KINGDOM, ⁶Hacettepe Children's Hospital, Ankara, TURKEY, ⁷University of Copenhagen, Copenhagen, DENMARK.

Rigid Spine Muscular Dystrophy (RSMD) is a rare autosomal recessive neuromuscular disorder characterized by early rigidity of the spine, axial and proximal muscle weakness associated with a dystrophic pattern of patient muscle biopsies, limb-joint contractures, and restrictive respiratory insufficiency requiring nocturnal ventilation. We recently reported the refinement of the RSMD1 locus on 1p35-36 to a 1cM region by linkage disequilibrium and the identification of mutations in SEPN1, the gene encoding a recently described selenoprotein of unknown function, selenoprotein N. Selenoproteins have in common to contain selenium as selenocysteine. One of the unique features in the incorporation of selenocysteine is the use of a UGA codon, which normally serves as a termination signal and needs a mRNA stemloop structure located in the 3’ unstranslated region and specific translation factors to be recognized as the codon for selenocysteine insertion. Fourteen different mutations including frameshift, missense, nonsense mutations in the coding sequence and a splice-site mutation, have been identified in SEPN1. Previous Northern blot experiments showed an ubiquitous expression of SEPN1. Polyclonal antibodies were developed in order to perform additional studies at the protein level. Biochemical studies with these antibodies allowed the detection of a 70 kDa band corresponding to the full-length protein present in control fibroblasts or myoblasts. However, this band could not be detected in total proteins extracted from patients cells bearing a homozygous frameshift mutation. The cellular localization of the selenoprotein N is currently underway and might help to better understand the role of this protein in skeletal muscle.

Conversion analysis between SMN 1 and SMN 2 genes in patients with a spinal muscular atrophy from North-West region of Russia.

A. Glotov, S. Mogilina, A. Kiselev, T. Ivaschenko;
Institute of Obstetrics & Gynecology, St.Petersburg, RUSSIAN FEDERATION.

Spinal muscular atrophy (SMA) is the second most common fatal autosomal recessive disease with the frequency 1: 10 000 newborn. The results of deletion and conversion analysis in 53 SMA families from North-West region of Russia are reported. Homozygous deletions of SMN 1 gene were identified in 96% of our patients. 21% of our SMA patients had the absence of exon 7 SMN1 gene but retention of exon 8. This complex rearrangements might result either from extensive deletion (up to exon 8) or from gene-conversion event giving rise to the origin of “chimeric” gene. The latter has been registered in 13% of SMA patients. All our conversion cases could be attributed to the formation of “chimeric” genes consisting of 5' area of SMN 2 gene (exon 7) and 3' area of SMN 1 gene (exon 8). Thus two types of conversion chromosomes were identified: with the deletion of exon 7 of SMN 2 gene as result of “chimeric” gene formation (1) and without such a deletion (2).

V. Cusin¹, O. Clermont², D. Chantereau¹, B. Gérard³, E. Bingen², J. Elion¹;
¹Service de Biochimie Génétique, Hopital R. Debré, Paris, FRANCE, ²Laboratoire de Génétique bactérienne (EA 3105), Hôpital R. Debré, Paris, FRANCE, ³Service de Biochimie hormonal, metabolique et génétique, Hôpital Bichat, Paris, FRANCE.

Spinal muscular atrophy (SMA) is linked to 5q13 locus in 95% patients. Among them, 98% show homozygous deletion of SMN1 gene, while about 2% of them show heterozygous deletion of SMN1 associating a mutation on the only SMN1 copy they present, and thus required a specific approach to reach diagnosis.
Here we show the results of SMN1 analysis in this specific group of SMA patients among those referred for molecular diagnosis to our laboratory. Genotype was established in all patients with clinical course and electromyography consistent with spinal muscular atrophy diagnosis and not showing homozygous SMN1 deletion. Detection of patients carrying a heterozygous SMN1 deletion was performed according to the fluorescent quantitative assay described by Gerard et al. (2000). Three patients carrying this genotype were identified and screened for SMN1 mutation with a standardized method associating single strand conformational polymorphism analysis and long range PCR to demonstrate that the detected mutations are indeed localized on SMN1 not SMN2, his homologous gene. Using this strategy, we successfully identified the causative mutations in all 3 studied patients. Two novel mutations were described in exon 3 and the third one was the previously described Y272C in exon 6.

I. Shagina, E. Dadali, A. Polyakov;
Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION.

Spinal muscular atrophy is a common often lethal neurodegenerative disorder with three major clinical phenotypes (SMA type I, II, III). The disease is caused by deletion, conversion or point mutations in the telomeric survival motor neuron gene (SMNt). In our study, we present the molecular-genetic analysis of 372 patients from 369 Russian SMA families. Homozygous deletions of either one exon 7 or both exons 7 and 8 of SMNt have been demonstrated in 96,2% of our patients (99%, 96% and 90% for SMA I, II and III respectively). The absence of SMNt exon 7 but retention of exon 8 were revealed in 59 (16 %) SMA patients. “Chimeric” (SMNc-SMNt) gene was found in 9% of SMA I, in 16% of SMA II and in 15% of SMA III patients. SMNt/SMNc ratio was analyzed by densitometry analysis (Gel Doc 2000/ Bio-RAD) of 7 and 8 exons PCR products digested by Eco RV and Bse NI respectively. Twenty families were referred for prenatal diagnostics without accessible material from affected child. Parents in sixteen families were heterozygous carriers of SMNt deletion. Prenatal diagnostics has been performed in 118 SMA families by means of deletion analysis of SMNt gene and polymorphic DNA-markers (D5S435, D5S557 and D5S629). We identified 31 affected, 58 carrier and 29 normal individuals among observed fetuses.

B. H. Trülzsch, K. Davies, M. J. A. Wood;
Dept of Human Anatomy and Genetics, Oxford, UNITED KINGDOM.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disease caused by loss of functional survival motor neuron gene (SMN) product. SMA ultimately leads to progressive loss of motor neuron function and muscular atrophy. Although the SMN gene is ubiquitously expressed, the cause for selective motor neuron loss is unknown. Study of the disease has been hampered by the fact that the condition is embryonal lethal for mice, and other currently available transgenic mice models are not viable for long periods of time. Goal of this study is to develop a cell culture system in which the SMN gene expression can be varied using catalytic nucleic acids. Catalytic nucleic acids are short sequences of RNA (ribozymes) or DNA (DNAzymes) capable of sequence specific cleavage of a target mRNA, thus downregulating gene expression. We designed three ribozymes and three DNAzymes targeted against the murine Smn RNA sequence. All ribozymes and DNAzymes effectively cleaved the full length Smn RNA in a sequence specific manner, while inactive versions of the molecules had no effect. Cleavage of target RNA was observed at magnesium concentrations as low as 2 mM, which corresponds to the intracellular Magnesium concentration of mammalian cells. Cleavage increased in a time and concentration dependent manner. These results indicate that catalytic nucleic acids effectively cleave Smn target RNA in a cellular environment and thus have great potential for interference with Smn gene expression in cells and in vivo.

Hyperacetylating agents activate SMN2 gene expression in fibroblast cultures from spinal muscular atrophy (SMA) patients

C. Andreassi, F. D. Tiziano, C. Angelozzi, T. Vitali, E. De Vincenzi, G. Neri, C. Brahe;
Università Cattolica del Sacro Cuore, Rome, ITALY.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder caused by homozygous loss of the Survival of Motor Neuron (SMN1) gene. SMN1 is located in a large inverted duplicated region on 5q13 where an almost identical copy, SMN2, is also present. The two genes differ only for a silent mutation in the reading frame, resulting in the majority of SMN2 transcripts lacking exon 7. All patients retain at least one, more often two to four, copies of SMN2. Milder phenotypes are generally associated with higher gene copy number and higher levels of protein, although patients with different phenotype can carry the same number of SMN2 copies. These data suggest that SMN2 genes are functionally different and one possible mechanism responsible of such differences could be epigenetic modifications, like DNA methylation and/or histone acetylation. Thus, upregulation of SMN2 by chromatin remodelling agents can be a potential target for a therapeutic approach to SMA.
We have currently undertaken a study to evaluate the effect of hyperacetylating agents, such as sodium butyrate and derivatives, on SMN2 expression in primary fibroblast cultures from patients with different disease severity. Preliminary results show that the treatment significantly increases the production of full length SMN2 transcripts and the number of gems (the nuclear structures where the SMN protein concentrates). These encouraging results suggest that hyperacetylating agents can be beneficial in SMA treatment protocols.

S. S. Shishkin¹, N. I. Shakhovskaya², L. F. Skosobzeva², S. B. Artemieva², I. N. Krakhmaleva¹, A. A. Khodunova¹, N. L. Gerasimova³;
¹Research Centre for Medical Genetics RAMS, Moscow, RUSSIAN FEDERATION, ²Children’s Psycho-Neurological Hospital of Moscow Region, Moscow, RUSSIAN FEDERATION, ³Interregional Association of assistance to people suffering neuromuscular diseases, Moscow, RUSSIAN FEDERATION.

Two approaches in therapy for muscular dystrophies were developed as a part of program for the long-term support for families with hereditary neuromuscular disorders (HNMD) in which included clinical trials of medicaments treatment (prednisolone, cyclosporine) and myoblast transplantation for DMD gene correction; the creation of computer database of Russian families with HNMD; the use of common diagnostic criteria with DNA-analysis et cetera.
94 DMD/BMD and 10 LGMD patients-volunteers participated in double-blind controlled prednisolone trial during 1 year with alternate-day schedule (0.5 mg/kg/day in treatment day). In 80% cases were obtained some beneficial effects and in all cases were absent the manifested side-effects. 17 DMD/BMD patients obtained this treatment 3-6 years and maintained relatively good conditions with low progression of muscular weakness.
For the other approach it was developed special technique of preparation of human myoblast cultures. 5 DMD patients-volunteers participated in clinical trial of myoblast transplantation by protocol “single muscle treatment”. Every recipient received cyclosporine two weeks before transplantation and one month after transplantation. 50-90 millions of myoblast cells was transplanted into m. tibialis anterior of one leg. 6 months after transplantation in biopsy specimens were revealed the presence of donor’s DNA (in three cases) and the expression of dystrophin (in two cases). Dystrophin and donor’s DNA were absent in the sham-injected muscles.