ESHG - Posters: P 15 Mental Retardation


Challenges that arise from a routine MECP2 mutation testing service. 

S. Whatley 1, A. Murray 2, N. Mason 1, A. Clarke 2, D. Ravine 2;
1Department of Medical Biochemistry, University Hospital of Wales, Cardiff, UNITED KINGDOM, 2Institute of Medical Genetics, University Hospital of Wales, Cardiff, UNITED KINGDOM. 


An initial surge of demand for MECP2 mutation analysis has settled into a steady-state pattern. We have screened for mutations using bi-directional sequencing. Among 292 samples referred, results have fallen into four major categories: pathogenic nucleotide variant (74); polymorphic variant (14); variant, pathogenicity indeterminate (13); with the remainder being wildtype. Among the wildtype results, we are aware of 13 cases where the clinical diagnosis is Rett syndrome and another 10 in whom the diagnosis is strongly suspected. When an indeterminate nucleotide variant is detected, follow-up analysis is recommended. This may include evaluation of parental samples, RNA and X chromosome inactivation (XCI) studies. Three cases from the indeterminate group have been selected to reveal the complexities that can occur, which have ranged from assessing novel missense and silent nucleotide variants that have not been reported previously among more than 1400 published mutations; evaluating non-coding variants that are highly conserved; and interpreting cDNA findings in the face of skewed XCI. It is evident that laboratories that offer a comprehensive mutation testing service for MECP2 and other genes on the X chromosome must be sufficiently resourced to meet the full range of challenges that can arise.



Rett syndrome in females and in a male 

N. Misovicova 1, J. Krsiakova 1, R. Rosipal 2, J. Zeman 3, P. Martasek 2;
1Martin Faculty Hospital, Martin, SLOVAKIA, 2Medical Faculty, Charles University, Praque, CZECH REPUBLIC, 3Medical Faculty, Charles University, Praque, CZECH REPUBLIC. 


Rett syndrome is an neurodevelopmental disorder characterised by autism, dementia, ataxia and loss of purposeful hand use. The exclusive involvement of females was explained by X- linked dominant inheritance with lethality in the hemizygous males. The responsible gene has been identified in the Xq28 region and encodes the methyl-CpG binding protein 2 (MeCP2). A non fatal Rett syndrome in male has been published in the year 2000. The authors confirmed the somatic mutation in MeCP2 gene.
We have a small cohort of 8 girls with Rett syndrome. The mutation analysis revealed different mutations in MeCP2 gene ( R294X, K135E, T158M, 1069delAGC). Recently we examined a 23 year male with classical features of Rett syndrome. The normal perinatal period was followed by a period of regression, loss of acquired skills, ataxia, gait disturbance and deterioration of the brain functions. The numerical aberration of the X chromosome was excluded, the man has a normal karyotype 46,XY. We expected that the mosaic mutation using the methods based on heteroduplex analysis and sequencing will be detected.



MECP2 mutations in males; Report of a case with Prader-Willi-like phenotype 

T. Kleefstra, H. G. IJntema, A. R. Oudakker, H. van Bokhoven, B. B. A. de Vries, B. C. J. Hamel;
UMC St Radboud, Nijmegen, NETHERLANDS. 


Heterozygous mutations in the X-linked MECP2 gene have first been described in Rett syndrome (RS), a progressive neurologic developmental disorder, occurring almost exclusively in females. Diagnostic criteria for RS were established by Hagberg et al.
Recent studies indicate that MECP2 mutations are not necessarily prenathally lethal in males, but are the cause of lethal congenital encephalopathy and even of non-fatal congenital encephalopathy. Of the latter group, some patients have clinical features suggestive of the Angelman syndrome (AS), others are reported with nonspecific mental retardation (MRX) only. The clinical and molecular features of an additional male patient with a de novo MECP2 mutation are presented and compared to previous reported cases.



Refined molecular characterisation of a de novo t(5;18)(q33;q12) associated with Rett-like syndrome and autism 

C. Windpassinger 1, K. Wagner 1, S. W. Scherer 2, P. M. Kroisel 1, E. Petek 1;
1University of Graz, Graz, AUSTRIA, 2The Hospital for Sick Children, Toronto, ON, CANADA. 


Mutations in the MECP2 gene cause the severe neurodevelopmental disorder called Rett Syndrome. Children afflicted with Rett Syndrome often also exhibit autistic-like behaviours. Beside MECP2 mutations, several cases of structural abnormalities of autosomes associated with Rett Syndrome are known. Here we report on a 10-year-old boy with a reciprocal 5/18 translocation. His clinical manifestations are a combination of mild dysmorphic features and those of Rett Syndrome and autism. Due to several cases of autism in patients with 18q deletions, our primary goal was do narrow down the breakpoint region on chromosome 18. Fluorescence in situ hybridisation experiments using various region specific YAC- and BAC clones led to split signals in a 200 kb sequenced BAC clone indicating that the corresponding DNA insert is spanning the breakpoint region. Subsequent Southern blot analysis with probes derived from the spanning BAC clone was performed to isolate a junction fragment. Sequence analysis of the junction fragment will help to find candidate genes for the neuropsychiatric features seen in our patient.



Genotype / Phenotype Correlations In A Large Series Of Rett Syndrome Patients 

A. Moncla 1, J. Mancini 2, A. Kpebe 3, B. Chabrol 2, M. O. Livet 2, N. Philip 1, L. Villard 3;
1Département de Génétique Médicale, Hopital des Enfants de la Timone, Marseille, FRANCE, 2Service de Neuropédiatrie, Hopital des Enfants de la Timone, Marseille, FRANCE, 3Unité de Recherche INSERM 491, Faculté de Médecine, Marseille, FRANCE. 


Mutations in the human Xq28-linked MECP2 gene are responsible for 70 to 80% of Rett syndrome cases and also for atypical clinical presentations.
We are presenting the results of our studies in a serie of 64 female cases in which we looked for mutations in the MECP2 gene. These patients were divided into three groups :
- Group I (23 patients) with Rett syndrome diagnosed in our multidisciplinary consultation.
- Group II (28 patients) referred by external collaborators for a suspicion of Rett syndrome
- Group III (17 patients) from our center exhibiting a severe encephalopathy with autistic behaviour.
In the first group, 21 mutations were identified ; in the second, 8 mutations and zero in the third group. These results confirm that the vast majority of mutations in the MECP2 gene lead to a classical Rett syndrome phenotype. They also show that a large proportion of girls with a severe mental handicap are not found to be associated with a MECP2 mutation on the contrary to what was recently proposed in the litterature.
In addition, we did not find any genotype-phenotype correlations for the Rett syndrome patients taking into account the mutation type, the functional domain affected by the mutation and the X-chromosome inactivation status of these patients except for the mutation involving the C-terminal domain associated with a « forme frustre » of the disease. It is thus likely that a number of not yet identified factors are responsible for the clinical variability of this syndrome.



Fragile X syndrome : clinical and behavioral study. 

H. H. Afifi 1, A. K. Abdel-Aleem 2, O. O. Shaheen 3;
1Human Genetics Department, Clinical Genetics Section, National Research Centre, Cairo, EGYPT, 2Human Genetics Department, Molecular Genetics Section, National Research Centre, Cairo, EGYPT, 3Psychiatry Department, Faculty of Medicine, Cairo University, Cairo, EGYPT. 


The present study included 25 boys with mental retardation of unknown etiology and their mothers. Each boy manifested, at least, two of the following features: positive family history of mental retardation, long face, large ears, hyperextensible finger joints and bulbous halluces. Patients were assessed for 18 physical features and 23 behavioral features. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifies across the two KH domain regions specific for FMR1 gene. Diagnosis of patients was absolutely dependent on the detection of absent FMR1 transcriped mRNA. This approach successfully diagnosed fifteen boys (60%) from 11 families as fragile positive and ten boys (40%) from 8 families as fragile negative. Multiple regression analysis revealed 11 features (4 physical and 7 behavioral) with high predictive value for fragile X syndrome. The frequency of speech defect was significantly increased in boys with fragile X syndrome. Delayed language, numerical and time concepts, domestic activities and directive behavior were significantly delayed in those boys. Eysenk personality questionnaire, Beck depression inventory and parental attitude questionnaire were applied to all mothers and revealed increased psychoticism, neuroticism, criminality and various degrees of depression in mothers of fragile X boys. In conclusion, the combination of physical and behavioral traits is helpful in suggesting the diagnosis of fragile X syndrome. The preselection 5-criteria device proved suitable for detection of fragile X candidates, thereby improve the cost-effectiveness of fragile X molecular testing. We propose application of this device in clinical pediatric practice for early detection of fragile X syndrome in young children.



Effect of premutation in the FMR1 gene on cognitive and physical phenotype in fragile X assessed by pedigree analysis. 

D. Z. Loesch;
La Trobe University, Melbourne, Bundoora, Victoria, Australia, AUSTRALIA. 


The effect of premutation status in the FMR1 gene on cognitive and physical measures in fragile X males and females was investigated using a robust modification of the maximum likelihood estimators for pedigree data. This approach is much more powerful than standard statistics because it allows for testing model assumptions, such as concerning the effect of explanatory variables, and for reducing variability in the data by down-weighting unusual observations, and adjusting for intra-familiar variation. It also allows for estimation of heritability of complex traits. The data from extended 110 fragile X families (including 185 fragile X subjects and 110 normal relatives) were analyzed. Fragile X status was determined by the number CGG repeats in the FMR1 gene. Physical phenotype was represented by trunk, limbs, head and face measures, and neuropsychological phenotype, by the cognitive (Wechsler), and executive function measures. Evidence is presented for strong phenotypic (neuropsychological) involvement in premutation carriers, especially in males, which predominantly affects performance skills and executive cognitive functioning. The effect of premutation status on physical phenotype was less evident and concerned only some facial measures in both sexes, and the measure of joints' laxity in females. Heritability ranged from the highest (88%-90%) for physical measures, to the lowest (10%) for some executive function measures.



Should all girls learning disabled or mildly retarded be screened for FMR1 mutation? 

M. I. Tejada 1, C. Puyo 1, M. Durán 1 ,2, M. A. Lopez Ariztegui 3, M. P. Botella 4, M. Poch 5;
1Unidad de Genética. Hospital de Basurto, Bilbao, SPAIN, 2Instituto de Biología y Genética Molecular. Facultad de Medicina, Valladolid, SPAIN, 3Laboratorio de Genética. Hospital de Cruces, Barakaldo (Bizkaia), SPAIN, 4Servicio de Pediatria. Hospital Txagorritxu, Vitoria-Gasteiz, SPAIN, 5Servicio de Pediatria. Hospital San Millan, Logroño, SPAIN. 


Fragile X syndrome (FXS) is the most common inherited cause of mental retardation, with a prevalence of about 1/4.000-5.000 in males. Therefore many clinicians must deal with fragile X individuals in their daily routine, but many of them still don't recognise this syndrome. This problem is greater in women, in whom the prevalence is theoretically the same as in males, but due to the fact that at least 50% of the fully mutated women are normal, the clinical prevalence in females is in fact much lower, about 1/10.000.
Our laboratory has been working on FXS molecular testing since 1991, receiving the majority of requests for FMR1 testing from Northern Spain. Since our first description of a girl with the FRAXA full mutation with no history of mental retardation in her family (Tejada et al., 1998), we have been recommending to test women for FXS. Moreover, in cooperation with "The Fragile X Association" we have been lately developing an information program to different professionals: pediatricians, psychologists, teachers, etc.
Here we present the molecular results of the 2001-year and compared them with the 10 previous years. In this later year we have studied 38 case index females and found 3 new non-related cases of FXS (3/38= 7,89%) without affected males in their families. We also report the initial symptoms, behaviour features, physical description and mental status of these 3 girls (aged 5, 3 and 5 years respectively), to add new data on the knowledge of fragile X syndrome in women.



Fmr-1 full mutation in leukocytes of a female not affected by X-Fragile syndrome. 

P. Battaglia, E. De Fanti, R. Grimau Merino, L. Giardina, R. Schiavon;
Legnago Hospital, Legnago, ITALY. 


A 35 years old woman was evaluated for FMR-1 mutation because her children (a male and a female) were found to be FMR-1 full mutation (CGG > 200 repeats). The laboratory tests performed to investigate the X fragile syndrome were: 1) chromosomal analysis 2) PCR of blood sample 3) Southern Blot method after treating DNA from leukocytes with Eag and EcoRI restriction enzymes. Results: 1) normal 46, XX caryotipe in blood nucleated cells without fragile sites detected, 2) a single faint band of PCR product following FMR-1 gene amplification, 3) the absence of 2.8 Kb band corresponding to the unmethylated FMR-1 allele, accompanied by a smear in correspondence of the methylated allele. To interpret these findings we hypotize that in blood cells of this women the ratio between FMR-1 full mutation and FMR-1 normal allele might be in favour of the abnormal allele. This could be supported by the not clear result on PCR. The peculiarity of this young woman is the absence of mental retardation and other classical abnormality typical of the X fragile syndrome, so that we can suppose a rare form of FMR-1 abnormality involving at least her blood leucocytes. Further investigations about other forms of tissue mosaicism are in progress.



Genetic-Diagnostic Survey of 3570 Children with Mental Retardation 

A. Yuksel 1, H. Kayserili 2, T. Tukel 2, S. Basaran 2, M. Yuksel Apak 2;
1Genetics and Teratology Research Center, Istanbul University, Istanbul, TURKEY, 2Division of Medical Genetic, Institute of Child Health, Istanbul University, Istanbul, TURKEY. 


This study was conducted on 3570 subjects who were referred to our outpatient clinic from July 1, 1985 to December 31.1998 for evaluation of mental retardation (MR) Medical records and photographic documentation were reviewed in all. History, physical and neurological examinations, chromosomal analysis, molecular studies, skeletal surveys; cranial imaging studies, electroencephalography and metabolic screening test were performed as indicated.
Patients were classified into two groups as patients who have isolated MR only, and MR with multiple congenital abnormalities (MKA+MR).
MR group (n: 582) consisted of known monogenic syndromes (n: 90), enzyme deficiencies (n: 112), patients of unknown etiology with MR (n: 380),
MKA+MR group (n: 2988) consisted of chromosome abnormalities (n: 1384)., recognizable syndromes (n: 55), known monogenic disease (203), structural central nervous system abnormalities (n: 540) and unclassified patient with MKA+MR (n: 806)
Out of 582 patients with MR and 2988 patients with MKA+MR etiology were noticed into 202(34.7 %) and2182 (72.02 %) respectively.
The results were compared with the similar studies in literature.



High occurrence of Brachydactyly-Mental Retardation syndrome among mentally retarded subjects in Italy 

A. Ragusa 1, O. Galesi 1, M. A. Lo Giudice 1, M. Fichera 1, E. Borgione 1, T. Mattina 2, M. Pierluigi 3, F. Faravelli 3, F. Dagna Bricarelli 3, D. Greco 1, P. Failla 1, C. Romano 1;
1Oasi Institute, Troina, ITALY, 2Clinica Pediatrica, Catania, ITALY, 3Laboratorio di Genetica Umana, E.O. Galliera, Genova, ITALY. 


Brachydactyly-Mental Retardation syndrome (BDMRS), MIM 600430, also defined as Albright Hereditary Osteodystrophy-like (AHO3) syndrome, is associated to distal deletion on 2q37 region. This association was first described independently by Phelan et al and Wilson et al (1995).
Results MultiFISH based screening for subtelomeric chromosome defects (Cytocell) was performed on 250 mentally retarded patients. The observed frequency of subtle chromosomal abnormalities was 6% (15/250). Among the fifteen subjects carrying chromosomal rearrangements six showed 2q37 de novo deletions (2.4%) associated to BDMRS. Since the probe included in the kit still detects 2q37 telomeric polymorphism (Macina et al, 1994; Knight et al, 1997), for all patients the 2q37 deletion was mapped by microsatellites and FISH analysis using genomic probes (PAC and BAC) encompassing the chromosomal region. Parental origin was determined by microsatellites analysis. The occurrence of the length polymorphism at 2q37 was also found in 17 subjects (7%). In two of them the polymorphism was present in compound heterozygosity with the BDMRS-associated deletion.
Conclusion Advancement of FISH strategies allowed to screen large series of mentally retarded patients and consequently improve the frequency definition of some recurrent chromosomal aberrations escaping from conventional karyotype. Among previously reported patients (Phelan et al and Wilson et al, 1995) with 2q37 deletion, only one of nine showed a normal karyotype. The group of patients presented here carried submicroscopic deletions allowing the evidence that BDMRS is an emerging clinical grouping. The presence of chromosomal polymorphisms is under investigation to verify a genetic predisposition to 2q37 rearrangements.



X-linked Lissencephaly with Absent Corpus Callosum and Ambiguous Genitalia (XLAG). Clinical, MRI and Neuro-Pathological Findings. 

D. Bonneau 1, A. Toutain 2, P. Saugier-Veber 3, A. Gélot 1;
1Génétique Médicale, Angers, FRANCE, 2Génétique Médicale, Tours, FRANCE, 3Génétique Médicale, Rouen, FRANCE. 


X-linked Lissencephaly with absent corpus callosum and ambiguous Genitalia (XLAG) is a newly recognized syndrome responsible for a severe neurological disorder of neonatal onset in boys. Based upon the observations of three new cases, we confirm the phenotype in affected boys, we describe additional MRI findings, we report the neuropathological data and, we show that carrier females may exhibit neurological and MRI abnormalities. In affected boys, consistent clinical features of XLAG are intractable epilepsy of neonatal onset, severe hypotonia, poor responsiveness, genital abnormalities and early death. On MRI, a gyration defect consisting of anterior pachygyria and posterior agyria with a moderately thickened brain cortex, dysplastic basal ganglia and complete agenesis of the corpus callosum are consistently found. Neuropathological examination of the brain shows a tri-layered cortex containing exclusively pyramidal neurons, a neuronal migration defect, a disorganization of the basal ganglia and a gliotic and spongy white matter. Finally, females related to affected boys may have mental retardation and epilepsy, and often display agenesis of the corpus callosum. These findings expand the phenotype of XLAG, may help in the detection of carrier females in affected families and give arguments for a semidominant X-linked mode of inheritance.



Long Term Follow-up Of A Girl With Oro-facio-digital Syndrome Type I Due To A Mutation In The Ofdi Gene 

C. Stoll 1, P. Sauvage 2;
1Hôpital de Hautepierre, Strasbourg, FRANCE, 2Hôpital de Hautepierre, Chirurgie Infantile, Strasbourg, FRANCE. 


In 1954 Papillon-Léage and Psaume described a dominant, X-linked condition which they named oro-facio-digital (OFD). This condition was split in at least 9 syndromes, the more common being OFDI. We report a girl with OFDI syndrome followed-up during 24 years.
The parents were unaffected. An older brother had hydrocephaly, and mental retardation. A younger sister is unaffected.
The proband was examined for the first time when she was 3 months old for median cleft lip and cleft palate, lingual frenula and hypoplasia of the maxillary and the mandible. She had clinodactyly of the 5th fingers, shortening of fingers and toes 3, 4 and 5 and syndactyly. She was operated on several times.
At 19 years of age renal insufficiency appeared. Renal transplantation was performed.
A mutation, an insertion of a G leading to a frameshift in the OFDI gene encoding a protein containing coiled-coil a-helical domains was identified in the patient who was asking for prenatal diagnosis when she was 24 years old.
Associated malformations of the OFDI syndrome are cerebral, and renal. There was no developmental delay and no cerebral malformations in this patient. There were multicystic kidneys with renal insufficiency leading to renal transplantation. The results were good.
In conclusion a girl with OFD type I syndrome was followed up during 24 years. A renal transplantation was performed when she was 20 years old. A frameshift mutation of the OFDI gene was identified.



X-linked mental retardation with cerebellar hypoplasia 

N. M. Philip 1, L. Villard 2, C. Raybaud 3, B. Chabrol 4;
1Département de Génétique Humaine, Hôpital d'Enfants de la Timone, Marseille, FRANCE, 2INSERM U491, Marseille, FRANCE, 3Service de Neuroradiologie, Hôpital d'Enfants de la Timone, Marseille, FRANCE, 4Service de Neuropédiatrie, Hôpital d'Enfants de la Timone, Marseille, FRANCE. 


We report a three genration family including 4 boys aged 3 to 17 years presenting with moderate mental retardation, abnormal facial appearance and brain anomalies. Mean age at walking was 30 months. Language was severely delayed and reading was not acquired in the eldest ones. All had tall stature above 2SD. Facial appearance was similar with macrocephaly, deeply-set eyes and and prominent chin. Brain MRI showed the same findings in all affected boys: supratentorially, mild to large dilatation of the ventricles was noted without cortical dysplasia. On of the boys had vetriculo-peritoneal shunt because of marked dilatation of the third and lateral ventricles. The most prominent abnormalities were seen in the posterior fossa: the cerebellar vermis was hypoplastic with various degrees of Dandy-Walker complex. The two obligate carrier females demontrated mild mental retardation and tall stature. Brain MRI showed diffuse cerbral atrophy in both and a slight supratentorial expansion of the cisterna magna through a posterior hiatus of the tentorium in one.
We compared the phenotype of these patients with the one described in previously reported families with X-linked mental retardation with cerebellar abnormalities. In most of these reports, the clinical picture was characterized by progressive cerebellar atrophy and clinical ataxia, which is very different from the pure mental retardation with congenital cerebellar hypoplasia seen in our patients.
Assuming X-linked inheritance, linkage analysis using 32 highly polymorphic parkers evenly distributed along the X chromosome was performed. There was no recombination between markers DXS1039 (Xp11.23) and DXS1047 (Xq25).



Clinical investigation and candidate gene screen of families mapped to the Partington syndrome region in Xp22.1. 

S. G. M. Frints 1, J. Fryns 2, M. Borghgraef 2, K. Hollanders 1, P. Marynen 1, G. Froyen 1;
1Human Genome Laboratory, Flanders Interuniversity Institute for Biotechnology, Department of Human Genetics, Leuven, BELGIUM, 2Department of Clinical Genetics, University Hospital Leuven, Department of Human Genetics, Leuven, BELGIUM. 


Partington et al. (1988) described a three-generation family (MRXS1, MIM *309510, PRTS) with a syndromic form of X-linked mental retardation (XLMR). The clinical features in 10 affected males included mild to moderate mental retardation, dystonic movements of the hands, dysarthria and seizures. Through linkage analysis a maximum LOD-score of 3.1 was obtained at marker DXS989 with flanking markers DXS365 and DXS28 (Xp22.1). The PRTS region comprises about 10 megabase and includes 9 novel transcripts and 13 known genes that are expressed in brain and muscle. The PRTS is a rare and specific clinical entity. So far, no patients other than the original family have been described. We present two brothers with PRTS and three males of a two-generation family with non-specific XLMR (MRX36), who after clinical reinvestigation show PRTS features. Neurological features in the affected males include moderate mental retardation, dysarthria, facial muscle weakness, severe dysdiadochokinesis, slow dystonic movements and mild spasticity of the hands without ataxia or spasticity of the legs. The symptoms are non-progressive, extra-pyramidal and without cerebellar involvement. We try to further delineate the PRTS phenotype and based on recent findings of Dr. J. Gecz and Dr J. Chelly, we will present data on a candidate gene screen performed on the two brothers with PRTS, MRX36, a family with West syndrome and on a selected group of MR male-patients.



X-linked lissencephaly with absent corpus callosum and ambiguous genitalia (XLAG) : a new family with severe expression in a girl 

L. Burglen 1, I. Desguerre 2, D. Rodriguez 3, G. Ponsot 2;
1Unit of Medical Genetics, Department of Neuropediatrics, Hôpital Trousseau, Paris, FRANCE, 2Department of Neuropediatrics, Hôpital Saint Vincent de Paul, Paris, FRANCE, 3Department of Neuropediatrics, Hôpital Trousseau, Paris, FRANCE. 


X-linked lissencephaly with absent corpus callosum and ambiguous genitalia (XLAG) is one of the distinct malformation syndrome associated with lissencephaly. Only 9 male patients were reported since the first description by Berry-Kravis in 1994. All were severely affected with intractable epilepsy from birth, profound mental retardation, temperature instability, growth retardation and ambiguous genitalia. Neuroradiologic findings were lissencephaly with a posterior-to anterior gradient, intermediate increase in cortical thickness, corpus callosum agenesis. Recently, Bonneau et al. described female carriers phenotype: all carriers (5/5) had partial or complete corpus callosum agenesis with no or mild mental retardation. None had severe mental retardation nor seizures. We report a new family. The propositus, a male, presented at birth with the classical type of the disease and died at day 10. His sister had seizures at day 2. At 3 years, she had profound mental retardation and severe epilepsy. RMI showed corpus callosum agenesis, posterior agyria-pachygyria and abnormal white matter. Karyotype was normal 46 XX. No mutation was detected in XLIS gene. X-inactivation study is on progress. The mother had normal intelligence but RMI showed partial corpus callosum agenesis. This observation confirm the frequency of corpus callosum agenesis in XLAG carriers. In contrast, a severe phenotype was never observed in female carriers and skewed X-inactivation is supposed to be responsible for severe expression in this girl.



Report of 2 new FRAXE families detected by FRAXA and FRAXE post-natal screening 

M. Doco-Fenzy 1, P. Cornillet-Lefebvre 2, P. Helms 3, E. Ulrich 1, J. Couchot 4, M. Mozelle 5, M. Quillerou 5, D. Gaillard 1;
1Department of Genetics, CHRU, Reims, FRANCE, 2Department of Haematology, CHRU, Reims, FRANCE, 3Department of Pediatrics, CHRU, Reims, FRANCE, 4Department of Pediatrics A, CH, Charleville, FRANCE, 5Department of Pediatrics, CH, Troyes, FRANCE. 


FRAXA is one of the most common cause of mental retardation for boys, FRAXE is rarely reported. From 1997 to 2001, 857 families were screened for FRAXA and FRAXE amplification. The study concerned 581 boys and 276 girls showing non-explained developmental delay. CGG and GCC repeat amplifications have been studied for respectively FMR1 and FMR2 genes.. Through this screening 11 new FRAXA families and 2 new FRAXE families were detected using respectively Pergolizzi and Knight’s modified PCR protocole (Cell 1993) and chemiluminescence for agarose gel revelation.
The first FRAXE proposant was an 8 years old boy affected by mental retardation, stuttering and frontal headache, constipation, cryptorchidy and corpus callosus agenesis.He had no facial dysmorphism but strabismus. WISC-R showed: 54 (verbal : 66, performance : 51) , he was not efficient in mathematics.
PCR revealed an abnormal amplification over 350 GCC repeats for him, 115 repeats for his mother and 71 repeats for his grand-mother.
The second FRAXE proposant was a 14 years old boy, with long face and nose, a mouth finely drawn, small ears, photosensible seizures, and headache. He was very anxious, and had difficulties in mathematics. PCR revealed an abnormal amplification over 320 GCC repeats for him, 203 repeats for his healthy sister, 121 for his mother, and 50 repeats for his grand-mother. four other men and women had amplification over 83 repeats in this family. A prenatal diagnosis was realised for his sister and only the sexe status of the female foetus was announced .



Coffin-Lowry's syndrome: About a familial case 

F. Maazoul, L. Ben Jemaa, R. Mrad, M. Chaabouni, M. Ksontini, H. Chaabouni;
Congenital and Hereditary Service, EPS Charles Nicolle, Tunis, TUNISIA. 


Coffin-Lowry's syndrome is individualized in 1975 by Temtamy from 2 descriptions princeps of Coffin and Lowry. It is an X-linked semi- dominant inherited disorder, mental delay is deeper at the boy's, the gene is localized and identified on the short arm of the X chromosome, in Xp22-2, it codes for a protein kinase RSK2. The incidence of this syndrome is to be about 1 per 50-100 000 males/ year.
We report the observation of a mother and its daughter presenting a mental retardation, an obesity, a facial dysmorphy, a digital abnormalities and a deformations of the skeleton, evoking Coffin-Lowry's syndrome .The investigates genetic find similar cases in the family. We begin in this work a comparative study with the data of the literature, we discuss the differential diagnosis, and we shall insist on the interest of the molecular study in a purpose of antenatal diagnosis.



Prospective Evaluation Of Audio-Visual Function In Down Syndrome Infants 

A. Ozkilic, B. Eroglu Kesim, A. Deviren, S. Hacihanefioglu, A. Yuksel;
Genetic and Teratology Research Center, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY. 


There is limited knowledge of early development of visual and auditory function in Down syndrome.
In this study audio-visual function of 35 Down syndrome infants (0-6 months old) were analyzed and followed prospectively 3-6 months later.
In assessment of auditory function, other than the 7 infants with chronic otitis media, brain-stem auditory evoked potential (BAEP) testing was applied to the remaining 28 infants. Compared with age-matched 62 normal children the major anomalies were significantly elevated response threshold (%39 hearing loss) and poorly differentiated wave I. Latency of wave V and I-III, III-V and I-V interpeak intervals were shorter but were within the 2 standart deviation. As a developmental change, follow-up testing showed that interpeak intervals continued to shorten with age.
In assessment of visual function, 35 Down syndrome infants (0-6 months old) were evaluated with visual examination and flash visual evoked potentials (FVEP) testing. Compared with age-matched 36 normal infants, latency of N2 and P2 were longer (p< 0.05) and amplitude of N2 and P2 were smaller in Down syndrome patients (p< 0.05).
The results of this study reveal that in Down syndrome the development of peripheral hearing and vision is delayed and also abnormal. BAEP and VEP testing are proposed to assess the development and detecting the problems of hearing and vision in Down syndrome infants. These tests should be repeated between periods of 6-12 months even if the results of initial tests are normal.



Cognitive and Behavioral Phenotype of Children with Some Common Dysmorphic Syndromes 

S. A. Shopova 1, E. G. Simeonov 2, G. Shalamanov 3;
1Section of Clinical Genetics, Department of Pediatrics, Medical University, Sofia, BULGARIA, 2Section of Clinical Genetics, Department of Pediatrics,Medical University, Sofia, BULGARIA, 3Technical University, Sofia, BULGARIA. 


Testing the cognitive capacity is a relatively new approach when examining patients having genetic diseases. These diseases often tend to be diagnostic problems due to their wide variety. That renders the precise estimation of the cognitive and behavioral phenotype a great importance.
Aim: to study cognitive and behavioral characteristics of patients with dysmorphic syndromes most often met in clinical practice (Down, Frigali X, Prader-Willi, Williams-Beuren, Noonan and Cornelia de Lange) and to define the specific cognitive and behavioral phenotypes.
Results and discussion:The different dismorphic syndromes manifest cognitive capacity deviations of different severety. IQ level in an individual patient changes with age in different ways. In our experience patients with Down, FRAX-A and CdeL syndromes show an intellectual decline with age, as other authors have observed, while patients having PWS, WBS and NS manifest an unequivocal tendency to imrove their cognitive skills. The most maladaptive behavior is shown by patients with CdeL and FRAX-A. The relationship between intellectual functioning and adaptive skills is in inverse proportion - for example for CdeL syndrome there are low IQ values and a large number of psychopathological behavioral symptoms hampering adaptability, while for NS there are comparatively high IQ values and negligible behavioral deviations.
Conclusions: The patients with some dismorphic syndromes manifest more or less specific psychological profiles of cognitive and behavioral characteristics. These profiles may be successfully used both in support of diagnostic process and as a basis for adequate medical and psychological intervention and counselling of parents.



Asperger syndrome and severe language impairment in two siblings, as the type and countertype of an unbalanced cryptic familial 22qter rearrangement. 

P. De Mas 1, N. Chassaing 1, C. Pienkowski 2, S. Julia 1, M. Vincent 1, G. Bourrouillou 1, P. Calvas 1, E. Bieth 1;
1Service de Génétique Médicale, Hôpital Purpan, Toulouse, FRANCE, 2Service d'Endocrinologie et Génétique Médicale, Hôpital des Enfants, Toulouse, FRANCE. 


Numerous cytogenetical and molecular evidences suggest that terminal 22q region is involved in language development. Indeed, many 22qter cryptic deletions were recently reported from patients harbouring a mild mental retardation, absent or few dysmorphic traits, frequent behavioural problems and severe speech impairment as a constant feature. We report clinical and molecular data from a family where a terminal 22q deletion was identified. The proband, a 8 years old girl suffered of a global development delay with a patent discrepancy between development of speech and of the other areas. This deletion was inherited from a paternal balanced translocation between the terminal end of a 22q arm and the p arm satellites of a chromosome 14. The karyotype of her father was 46,XY,ish t(14;22)(p11.2;q13.33)(D22S1726+,ARSA+;D22S55+,D22S1726-,ARSA-). Interestingly a 22qter partial trisomy was identified on a proband’s brother affected with Asperger’s syndrome, including a very precious language, rich vocabulary and pedantic speaking style. So, one or more genes localised in this critical terminal 22q region might be implicated in language development through gene dosage effect.



Peculiar facies and mental retardation in mother and daughter: a possible new syndrome? 

E. Lapi, A. Cecconi, E. Andreucci, M. L. Giovannucci Uzielli;
University of Florence, Firenze, ITALY. 


The Authors describe a couple mother-daughter (35 and 11 yrs old respectively), whose phenotype is characterized by a peculiar facies, minor anomalies of the limbs and mental retardation in a medium range. Both of them have broad nasal bridge, upslanting palpebral fissures, high cheekbones, bulbous nasal tip, anteverted nares, long philtrum, thin and inverted V-shaped upper lip and full lower lip: they appear to be of South East Asian origin rather than Caucasian (which they are, also because of their olive complexion). They have a rough voice; hands with short digits(<3 rd centile), partial skin syndactyly and clinodactyly of the 5 th; stocky feet, with a large big toe. The mother’s final stature is154cm, quite below her genetic target. The daughter is growing in the 10 th centile (Tanner B1,P1 ). Behavioural alterations have been noticed: attention deficit, hyperactivity, anxiety in both and depressive tendency in the mother. A brain MRI does not show anomalies in the daughter, while the mother’s left cerebral hemisphere appears underdeveloped, with a “simplified“ cortical design. EEGs are alterated with no correlated clinical signs. A good quality, 450 G bands karyotype appears normal in both our propositae; fragile X is negative; the research of subtelomeric rearrangements is in progress.
Using a computerized dysmorphology database, no condition had a combination of such peculiar facies, minor anomalies of the limbs and mental retardation. To the best of our knowledge it looks like we are in front of a recognizable and transmissible syndromic pattern, possibly unique.



Angelman syndrome: a genetic and clinical survey of 243 patients. 

D. Giardino 1, M. T. Bonati 1, F. Cogliati 1, G. Gottardi 1, F. Cavalleri 1, F. Natacci 2, A. Selicorni 3, P. Bonanni 4, A. Romeo 5, S. Russo 1, L. Larizza 6 ,1;
1Istituto Auxologico Italiano, Milan, ITALY, 2Istituto Sacra Famiglia, Cesano Boscone, ITALY, 3II- Clinica pediatrica, Università di Milano, Milan, ITALY, 4(5) D.U.NPI, IRCCS Stella Maris,, Calambrone ( Pisa), ITALY, 5C.R.E.I.- Osp. Fatebenefratelli, Milan, ITALY, 6Dipartimento di Biologia e Genetica, Università di Milano, Milan, ITALY. 


Angelman syndrome (AS) is a complex neurodevelopmental disorder with a difficult clinical diagnosis and a heterogeneous genetic basis. More than 70% of patients carry a 15q microdeletion detectable by FISH. Up to 5% of patients have maternal UPD, identifiable by segregation analysis of chromosome 15 microsatellites. Up to 5-10% of cases are due to an abnormal imprinting process. M-PCR test reveals all genetic defects except mutations in the UBE3A/E6-AP gene, which are responsible for the remaining 20% of cases. We report on 243 patients with a clinical diagnosis of AS analyzed by one or more of the following techniques: FISH, M-PCR, chromosome 15 haplotyping, UBE3A mutation screening. In order to define the recurrence risk in IC patients, microdeletions were tested by Southern-blot analysis and sequencing of the minimal overlapping region of AS deletion. Fifty-five cases negative just by FISH or M-PCR were excluded by further analyses because of insufficient diagnostic criteria at clinical re-evaluation. A laboratory test confirmed the diagnosis in 66 out of the remaining 188 patients, with 42 deletions, 18 (including 4 familial cases) mutations of UBE3A/E6-AP gene, 5 defects of the imprinting process and one maternal UPD. We think that the low percentage of positive cases is likely due to scarce diagnostic criteria leading to misdiagnosis: work on a check list of clinical signs which can predict AS-specific genetic lesions is in progress. We also performed comparison of the clinical manifestations of our confirmed AS patients across the different subgroups aiming at assessing a putative phenotype-genotype correlation.



sleep and its disturbances in Angelman syndrome 

H. de Leersnyder;
Hopital Necker, Paris, FRANCE. 


Angelman syndrome (AS) is a severe neurogenetic disorder, characterised by severe mental retardation, ataxia, seizures, absence of speech and characteristic facial features. We have studied sleep behavior of 62 AS patients aged 2 to 20 years through sleep questionnaires filled by the parents. Bedtime was between 8 and 10.30 p.m. , awakenings between 6 and 9 am, these results were similar to control group (30 healthy children aged 2 to 20 years), 53% of the children woke-up 1 to 3 times each night, and need parent’s attendance to go back asleep. These sleep disturbances have a major impact on parents and family members, who report tiredness, depression, becoming sleep-deprived themselves. After the age of 3 years, children had no naps during the day , and do not objective fatigue. Adding anti-epileptic medication, 42% of the patients take different medications for sleep. Patients had maternal deletion 15q11-q13 (84%), paternal disomy (10%), imprinting defects (1%) or mutations of the UBE3A gene (4%). There was no phenotype-genotype correlation for sleep disturbances in AS. The mechanism of sleep disorders in AS is complex, insomnia may be related to abnormalities in brain development which may include abnormal function of the circadian system and seizures play a part in night awakenings. Psychological impact of lack of language could find its expression during the night, pointing to anxiety. Therapeutic management of sleep disorder in AS remains a necessity for the physician and parents.



Cellular mosaicism of maternal 15q11 imprinting in an atypical Angelman syndrome without obesity 

B. LeHeup 1, C. Philippe 1, K. Buiting 2, M. Juif-Elmerich 3, P. Jonveaux 1;
1Departement de Genetique Medicale, CHU de Nancy, Vandoeuvre les Nancy, FRANCE, 2Institut fur Humangenetik, Universitatklinikum, Esseb, GERMANY, 3Service de Pediatrie, Hopital Ste-Croix, Metz, FRANCE. 


Atypical forms of Angelman syndrome with obesity, muscular hypotonia and ability to speak have been reported in patients with methylation pattern of the 15q11 region which supports the hypothesis of cellular mosaicism for a maternal imprinting (Eur J Hum Genet 1999, 7, 638) . We herewith report a new case with the same pattern of the 15q11 methylation, which extends further the clinical pattern of such anomalies.
The girl was born from healthy parents aged 37 years at conception and she is the second child. Pregnancy was uneventful. Length and birthweigth were normal. She walked unaided at 18 months. First words were said at 12 months. Language did not develop thereafter. At 3 years 6 months her weight and height were at the mean for the age. Her head circumference was at -1 sds for age. She exhibited mainly hyperactivity and was joyful. Cytogenetic analysis was normal. Because of the behavior characteristics the methylation pattern of the 15q11 SNRPN was evaluated. The maternal band was found fainted on three different samples (SNRPN exon alpha, Southern blot, XbaI and NotI double digestion). Methylation-specific PCR at the SRPN locus confirmed a faint maternal band. Quantitative Southern blot analysis of the critical IC region (AS-SRO) showed a normal dosage. Therefore IC deletion is unlikely. Sporadic imprinting defect and cellular mosaicism are likely explanations for such a pattern. The present observation without obesity further extends the clinical delineation of this anomaly.
Ackownledgement: We thank the help of B Horsthemke, Institut fur Humangenetik, Universitatklinikum, Essen



A 17p11.2 deletion associated with a mild Smith-Magenis phenotype and GH deficiency. 

P. Maraschio 1, G. L. Marseglia 2, C. Danesino 1, E. Spadoni 1, P. Colapietro 3, L. Tiepolo 1;
1University of Pavia - Dipartimento di Patologia Umana ed Ereditaria, Pavia, ITALY, 2Clinica Pediatrica - IRCCS San Matteo, Pavia, ITALY, 3Dipartimento di Biologia e Genetica per le Scienze Mediche - University of Milano, Milano, ITALY. 


A 7-years old girl was referred for chromosome analysis because of recurrent infections and microcephaly. High resolution banding revealed an interstitial deletion in 17p11.2. The deletion was confirmed by FISH with the SMS probe (Oncor).
The girl presents with few facial dysmorphic features: anteverted ears, full upper lip with a "tented" appearance and mild micrognathia. Overall facial "gestalt" does not resemble the Smith-Magenis Syndrome (SMS). Thyroid function is in the normal range, while GH secretion was reduced upon stimulation. Her height, at 3rd percentile until 5.5 years, has improved to the 10th percentile after starting replacement therapy. She is microcephalic (below the 3rd percentile) and has short hands. Immunity function is normal apart from a IgA value at the lower limit of normal range. The girl had occasional absence-like episodes and repeated EEGs showed non-specific alterations. Psychomotor development evaluation revealed a borderline I.Q. She was diagnosed with ADHD (Attention Deficit Hyperactivity Disorder); she has temper tantrums, occasional stereotypic behaviours and mild sleep disturbances, while she never attempted to self-injurious behaviours, typical of SMS. FISH, aimed at defining deletion extent and boundaries, was carried out with YACs 795c9 and 828b9 mapping in tel cen order within the SMS deletion interval. Both signals were given by y795c9, while a strongly diminished signal was detected on one17p by means of y828b9, pointing out that the deletion is more centromerical than the typical SMS one. Further FISH experiments are in progress in order to address the genotype-phenotype correlation on such peculiar 17p microdeletion case.



Results of subtelomeric screening in 52 families with unspecific mental retardation. 

Z. Helias-Rodzewicz 1 ,2, E. Bocian 1, P. Stankiewicz 1, E. Obersztyn 1, E. Kostyk 3, K. Jakubow-Durska 1, A. Kutkowska-Kazmierczak 1, T. Mazurczak 1;
1Dept. Medical Genetics, Institute of Mother and Child, Warsaw, POLAND, 2Postgraduate School of Molecular Medicine, Warsaw, POLAND, 3Univeristy Children Hospital, Cracow, POLAND. 


Recently, it has been shown that subtelomeric chromosome rearrangements may be a common cause of idiopathic mental retardation (MR). Such aberrations were found in ~7 % of patients with severe or moderate MR and dysmorphic features. However, further studies are needed to elucidate their prevalence and to establish more cost-effective selection criteria for subtelomeric test. We report results of screening studies in 52 families with idiopathic MR. Apart from MR, the main inclusion criteria were clinical features suggestive of a chromosomal aberration. Fluorescence in situ hybridisation with Chromoprobe Multiprobe T System (Cytocell) was used. In five cases subtle rearrangements were found. Verification of the karyotype at a higher resolution level has revealed that two rearrangements were half cryptic and in two cases both products of identified translocation could be seen. In one of them it was not a subtelomeric rearrangement but it could not be seen in the routine karyotype because of similar banding pattern and the size of involved regions. The prevalence of subtelomeric abnormalities was 4/52 (7,7%). All identified aberrations were familial in the origin and no abnormalities were shown in patients with mild MR. Our results confirm previous findings, indicating the important role of subtelomeric rearrangements in the aetiology of MR. They also emphasise the preferential occurrence of such abnormalities in patients with severe MR and positive family history for intellectual disability. Moreover, they also show that higher banding resolution should be used in the routine karyotyping in this group of patients.



14q32.2 subtelomeric deletion in a child with severe congenital anomalies and a cryptic t(Y;14)(p11.3;q32.2). 

M. Poscente 1, R. Rinaldi 2, E. Buffone 3, B. Grammatico 2, L. Florean 4, M. Assumma 3, P. Grammatico 4 ,2;
1Medical Genetics, University, Rome, ITALY, 2Medical Genetics, San Camillo-Forlanini Hospital, Rome, ITALY, 3Neonatal Intensive Therapy, S. Camillo-Forlanini Hospital,, Rome, ITALY, 4Medical Genetics, University "La Sapienza", Rome, ITALY. 


We describe a newborn with a de novo subtelomeric deletion of the terminal portion of the long arm of chromosome 14 due to a t(Y;14)(p12.3;q32.2).
The child was born at 36 weeks of gestation from healthy non-consanguineous parents. Fetal ultrasound examination at 28 weeks showed intrauterine growth retardation and anhydramnios. At birth the child demonstrated: weight 1160 g (<3th centile), length 40 cm (<3th centile), head circumference 28,5 cm (<10th centile), short nose with broad nasal root, low set dysmorphic ears, hands fingers anomalies, male external genitalia, undescendent left testis, imperforate anus and interventricular defect; umbilical cord was mono-arterial. The child died on day 19th .
Cytogenetic analysis using GTG tecnique revealed a 45,X karyotype. Because of the presence of external male genitalia, a FISH analysis with a SRY specific BAC probe (PHU 14) was performed, showing the presence of fluorescent signal in 14q telomeric region .
Further FISH analysis carried out with YAC and BAC probes specific for 14q32.3-qter region revealed a subtelomeric deletion with a breakpoint beyond 116 cM.
Father’s karyotype with GTG and FISH analysis with PHU 14 probe was normal.
At least ten patients with a 14q terminal deletion have been reported with some common clinical features: an exact characterization of the deleted segment in our case can contribute to the delineation of a distinct 14qter deletion syndrome.



Subtelomeric Chromosome Abnormalities in Patients with Developmental Disorder 

K. Rapakko, A. Mustonen, T. Lopponen, H. Kokkonen, J. Leisti;
Department of Clinical Genetics, Oulu University Hospital/University of Oulu, Oulu, FINLAND. 


Subtle chromosomal abnormalities have been reported to occur in 7.4% of patients with moderate to severe mental retardation as identified with a multiprobe telomere FISH protocol (Knight et al. 1999). We have studied 69 patients with mental retardation or developmental delay, and non-specific dysmorphic features, who had a normal 550 band G-banded karyotype. FISH testing was performed using the Cytocell Multiprobe-T system. Clinically significant abnormalities were found in six patients (8.7%, see Table). Three cases were familial. In addition we detected a common familial polymorphism del(2)(q37.3) in four patients (5.8%).
Patient Karyotype Parents
1 46,XX,der(2)t(2;10)(q37.1;q26.3) maternal balanced translocation
2 46,XY,der(9)t(9;19)(q34;p13.3) paternal balanced translocation
3 46,XX,der(18)t(2;18)(p25.3;q23) normal karyotype
4 46,XX,del(1)(p36) normal karyotype
5 46,X,der(X)ins(X;9)(q22;p24.2p24.3) maternal 46,X,der(X)ins(X;9)(q22;p24.2p24.3)
6 46,XY,del(20)(q13.3) normal karyotype

Detailed clinical findings of the patients and their relatives will be presented.
Subtelomeric FISH analysis proved to be a useful method in the detection of cryptic subtelomeric changes and provides a means for genetic counselling and prenatal diagnostics.



A novel method for the detection of subtelomeric rearrangements: Subtelomere COBRA FISH 

H. Engels 1 ,2, A. Ehrbrecht 1, S. Zahn 1, K. Bosse 1, J. Vrolijk 2, P. Propping 1, G. Schwanitz 1, A. K. Raap 2, J. C. A. G. Wiegant 2, H. J. Tanke 2;
1Institute of Human Genetics, University of Bonn, Bonn, GERMANY, 2Laboratory for Cytochemistry and Cytometry, Dept. of Molecular Cell Biology, Leiden University Medical Center, Leiden, NETHERLANDS. 


Cryptic subtelomeric chromosome rearrangements have recently been implied in the aetiology of mental retardation. Systematic FISH screening techniques to detect such rearrangements using subtelomeric probes have been introduced. To facilitate a more efficient analysis, multi-colour hybridisation techniques for subtelomeric probes are being developed. COBRA (COmbined Binary RAtio labelling) is a recent multi-colour FISH labelling technique which combines ratio and combinatorial labelling to attain especially high multiplicities [Ref. 1, 2].
With the novel "Subtelomere COBRA FISH", the specific detection of all necessary 41 BAC and PAC FISH probes (second generation probe panel, Ref. 3) is possible in only two hybridisations. By strict probe selection and characterisation and by using the ULS / Universal Linkage System ® labelling technique, high specificity and hybridisation efficiencies could be reached. This allows the unequivocal analysis of 21 respectively 20 probes per hybridisation with only two to three metaphases each. In contrast to other subtelomeric multi-colour techniques, the high multiplicities of Subtelomere COBRA FISH make it possible to differentiate e. g. long arm and short arm subtelomeric regions of a given chromosome thus permitting the diagnosis of cryptic pericentric inversions in addition to translocations and deletions.
The technique, its validation and first results will be presented.
References: [1] Eur J Hum Genet (1999) 7: 2-11, [2] Genome Res. (2000) 10:861-865, [3] Am J Hum Genet (2000)67:320-332
Supported by Deutsche Forschungsgesellschaft / DFG, Doktor Robert Pfleger-Stiftung, Richard Winter-Stiftung and the University of Bonn (BONFOR program)



Studies Of Telomeric Fish Screening And High Resolution Cgh In Populations With Recurrent Miscarriages Or Mental Retardation 

B. Benzacken 1, E. Pipiras 1, J. P. Siffroi 2, C. Bauman 3, L. Burglen 4, D. Heron 5, T. Billette de Villemeur 4, Y. Alembik 6, J. Gaudelus 1, J. P. Wolf 1, P. Bitoun 1;
1CHU Paris-Nord, Bondy, FRANCE, 2Hopital Tenon, Paris, FRANCE, 3Hopital Robert Debre, Paris, FRANCE, 4Hopital Trousseau, Paris, FRANCE, 5Hopital de La Pitié, Paris, FRANCE, 6Hopital de Hautepierre, Strasbourg, FRANCE. 


Aberrations of chromosomal telomeres are a frequent cause of mental retardation, particularly if they are associated with dysmorphism. It has been postulated that they could also account for recurrent miscarriages.
The goal of this study is to use and compare both Fluorescent In Situ Hybridisation (FISH) and high resolution Comparative Genome Hybridisation (CGH) in 2 populations: children with mental retardation, and couples having had at least 3 unexplained miscarriages.
200 children with idiopathic mental retardation and 57 couples (114 patients) were studied after informed consent according to the Helsinki convention. Of 200 patients 29 were excluded because they had abnormal karyotype, non-telomeric microdeletion or a sib in the study.
FISH was performed using the Cytocell or Adjenix probes. CGH was performed using the Adjenix Nick Translation kit and the Cytovision Image Analyser.
In the mental retardation group 13/171 anomalies were found or 7.6% using FISH. Anomalies were often familial and 5 involved chromosome 22q.
CGH was performed in 50 patients: 39 picked at random and 11 to validate the method. One familial microdeletion was found involving chromosome 17.
7 couples had an abnormal Karyotype but no FISH anomalies were found in the miscarriage group.
Telomeric FISH screening is useful for diagnostic and prognostic purposes in children with mental retardation and normal karyotype resulting in 7.6% anomalies. It does not seem useful for couples with recurrent miscarriages.The value of CGH will be discussed.



FISH characterization of 16p11.2-p12.2 tandem duplication in a dysmorphic patient with severe mental retardation and autistic behaviuor. 

F. Natacci 1, P. Finelli 2, G. Gottardi 2, C. Zuccarini 1, D. Giardino 2, M. Sala 1, L. Larizza 3;
1Foundation Institute Sacra Famiglia, Cesano Boscone, ITALY, 2Cytogenetic Laboratory,Istituto Auxologico Italiano, Milan, ITALY, 3Department of Biology and Genetics, University of Milan, Milan, ITALY. 


Chromosome 16p partial trisomy is a rare anomaly, described in the literature in less than 30 patients. We report on the genotype/phenotype correlations drawn on a 23 years old dysmorphic male affected by mental retardation and autistic behaviour, who was found to have a 46,XY,add(16)(p13) karyotype, redefined as 46,XY,dup(16)(p12.1p12.2). The detailed clinical evaluation showed the presence of hypertelorism, broad nasal bridge and tip, short phyltrum, macrostomia, thick lips, short stature, joint laxity. FISH analysis by using a WCP and a PCP chromosome 16 probes confirmed duplication of 16p11.2-12 region. FISH studies with YAC probes belonging to WC16.2 contig and BAC probes mapping in the region between 16p12.3 and 16p11.2 allowed to restrict the duplication to the 16p12.1-12.2 region; the inclusion of 16p11.2 band in the duplicated fragment is still under test. The comparison between clinical features of 16p trisomic patients in the literature and those of our patient carrying a precisely characterized chromosomal abnormality can improve the karyotype/phenotype correlations for 16p imbalances. The absence of microcephaly, heart and genital abnormalities in our patient and in the other one with a similar16p anomaly, previously reported, suggests that distal 16p is involved in the pathogenesis of these anomalies, while the presence of severe mental retardation with autistic behaviour, may be attributed to more centromeric 16p sequences.



Clinical, cytogenetic and molecular analyses of partial 21q monosomy in a girl with mental retardation, marfanoid habitus and minor dysmorphic features 

J. Wirth 1, B. Lemcke 2, I. Kennerknecht 2, R. Exeler 2, D. Ehling 1 ,3;
1Developmental Biology and Molecular Pathology, University of Bielefeld, GERMANY, 2Westfaelische Wilhelms-Universitaet Muenster, Institut fuer Humangenetik, GERMANY, 3Praenadia GmbH, Muenster, GERMANY. 


Phenotypic and molecular analysis of patients with partial monosomy 21 resulting from translocations, ring chromosomes or pure partial monosomy 21 allows to define which regions of chromosome 21 contribute to the generation of specific aspects of the 21q- syndrome. Here we report on the clinical, cytogenetic and molecular characterization of a `pure´ de novo partial monosomy 21 with a deletion of 21q22.2-qter. FISH mapping of cytogenetically and genetically anchored YAC and BAC clones resulted in the identification of a breakpoint spanning BAC clone. Our FISH results clearly showed that the deletion breakpoint is located distal to ETS2 gene, in the proximal part of the region 21q22.2. Furthermore molecular studies using polymorphic markers supported these findings and showed that the derivative chromosome 21 was of paternal origin. The patient who presented with mild mental retardation, marfanoid habitus and minor dysmorphic features, is lacking most of the typical features seen in the 21q- phenotype and thus is quite unique. Our findings support the suggestion that the loss of the region at 21q22.2-qter is critical for only some minor aspects of the 21q- syndrome. Genotype-phenotype correlations of our case and other reported cases will be discussed.



Unbalanced subtelomeric translocation 11q;16q in a mildly retarded boy with severe speech delay and minor dysmorphic signs 

S. Zahn 1, A. Ehrbrecht 1, D. Seufert 1, G. Schwanitz 1, H. Engels 1, B. Albrecht 2;
1Institute of Human Genetics, Bonn, GERMANY, 2Institute of Human Genetics, Essen, GERMANY. 


We report on a mildly retarded boy, whom we investigated at the ages of 2 and 4 1/2 years.
He is the first child of a healthy non-consanguineous couple. The mother is currently pregnant.
After an uneventful pregnancy and delivery by caesarean section, measurements were normal (3130g , 47,5cm, 34cm). Neonatally, the boy developed meningitis. His craniofacial dysmorphism included a high forehead, slightly depressed, broad nasal bridge and tip with anteverted nares, thick lips, small mandible and high arched palate. He had pes equinovarus, mild muscular hypotonia and right kryptorchidism.
His motor development supported by physiotherapy was normal (crawling with 6 months, sitting with 8 months, walking with 13 months). He suffered from repeated otitis media and his hearing was severely impaired. His active and passive verbal development is severely delayed.
Screening for metabolic diseases and repeated chromosomal analyses were normal as were parental chromosomal analyses. Brain MRI showed periventricular leucodystrophy.
A screening for cryptic subtelomeric chromosome aberrations (Vysis ToTelVysion) detected a cryptic unbalanced translocation 11qter / 16qter causing partial monosomy 11q25 and partial trisomy 16q24. Parental analyses demonstrated a maternal balanced translocation t(11;16)(q25;q24)(VIJyRM2072-,16QTel013+;VIJyRM2072+,16QTel013-). Both findings were confirmed by the novel Subtelomere-COBRA multi-colour FISH method. Further family studies including the analysis of the ongoing pregnancy will be presented.
Given the proband's relatively mild phenotype, the results emphasise the importance of subtelomere studies in mildly retarded patients.
Supported by Deutsche Forschungsgesellschaft / DFG, Richard Winter-Stiftung, Doktor Robert Pfleger-Stiftung and the University of Bonn (BONFOR program).



Molecular cytogenetic characterization of two cases with cryptic rearrangements of the 6q subtelomeric region. 

P. Grammatico 1 ,2, A. Novelli 3, M. Poscente 1, C. Ceccarini 3, C. De Bernardo 2, B. Dallapiccola 3 ,1 ,2;
1Medical Genetics, University "La Sapienza", Rome, ITALY, 2Medical Genetics, San Camillo-Forlanini Hospital, Rome, ITALY, 3CSS Mendel, Rome, ITALY. 


We report two cases with cryptic rearrangements of the 6q subtelomeric region characterized by using FISH techniques.
Case n. 1 is a 28 years old male with: severe mental retardation, hypoplasic malar regions, cupid bowed upper lip, everted lower lip, micrognatia, large, anteverted and low set ears with prominent anti-helix, surgically corrected nasal dysmorphisms and slight bilateral clinodactyly. Standard karyotype and molecular test for FMR1 gene appeared to be normal.
FISH analysis for subtelomeric regions using the Cytocell Multiprobe T System showed the 6q deletion that was revealed to be de novo. A further characterization of the breakpoint was carried out with 6q27 region YAC and BAC probes.
Case n. 2 is a 9 years and 9/12 male child showing severe psycomotor retardation, patent ductus arteriosus, vescico-ureteral reflux, frontal bossing, short palpebral fessures, lateral displacement of inner canthi, flat nasal bridge, big and low set years with prominent anti-helix, triangular mouth, long upper lip, micrognatia, barrel thorax, scoliosis, wide spaced and low set nipples, bilateral hip dislocation, clubfoot, campto-clinodactyly of toes. Standard karyotype was normal while the FISH analysis by using the Cytocell Multiprobe T System evidenced a 2q deletion and a 6q duplication.
These two cases stress the necessity of using specific subtelomeric FISH probes to detect cryptic rearrangements in patients with syndromic mental retardation.
The precise characterization of the involved regions by using molecular techniques may be useful in order to clinically define new microdeletion/microduplication syndromes.



FISH analysis of replication and transcription of chromosome X loci in Rett syndrome 

I. Y. Iourov 1, A. D. Kolotiy 2, S. G. Vorsanova 2, P. M. Sheinson 1, I. V. Soloviev 1, Y. B. Yurov 1;
1National Center of Mental Health, Moscow, RUSSIAN FEDERATION, 2Institute of Pediatrics and Children Surgery, Moscow, RUSSIAN FEDERATION. 


Differential replication staining using BrdU + Hoechst 33258 technique has been carried out on a series of 60 girls with Rett syndrome (RTT). The results indicated that regions Xq23 and Xq28 of inactive chromosome X could contain early replicating and, therefore, transcriptionally active loci at RTT. Interphase fluorescence in situ hybridisation (FISH) studies of replication timing, using chromosome X specific genomic DNA probes, was applied to determine the loci with altered replication and transcription at RTT. 14 randomly selected PAC clones for Xp, Xcen and Xq were used. Two clones from Xq28 (anonymous clone PAC 24.23.0 and PAC 671D9, containing MeCP2 locus), probably, escape inactivation in late-replicating chromosome X in RTT patients. Therefore, region Xq28 could contain the genes escaping X- inactivation and expressing from the human active and inactive chromosomes X. These results support the hypothesis proposing the disturbances in dosage compensation effect due to aberrant activation of genes in inactive chromosome X at RTT genes (di-allelic expression instead of mono-allelic). Our results indicate that MecP2 itself could escape X-inactivation and reduce the pathogenic effect of mutated allele at RTT. Supported by Copernicus2 and INTAS grants.



Molecular-cytogenetic studies of Rett syndrome (RTT) in Russia: the investigation of 4 boys and 81 girls 

S. G. Vorsanova 1, Y. B. Yurov 2, V. Y. Ulas 1, I. A. Demidova 1, A. D. Kolotiy 1, A. K. Beresheva 1, V. O. Sharonin 1, M. Giovanucci-Uzielly 3, L. Giunti 4, I. V. Soloviev 2;
1Institute of Pediatrics and Children Surgery, Moscow, RUSSIAN FEDERATION, 2National Center of Mental Health, Moscow, RUSSIAN FEDERATION, 3Centre of Genetics and Molecular Medicine, University of Florence, Frorence, ITALY, 4Centre of Genetics and Molecular Medicine, University of Florence, Florence, ITALY. 


Rett syndrome (RTT) is a severe neurodevelopmental disorder with the incidence of 2.5% in mentally retarded girls in Russia. We have performed cytogenetic studies of 85 patients (81 girls and 4 boys) with clinical picture of RTT. Molecular analysis in 30 randomly selected RTT patients revealed that 84% of them have mutations in MeCP2 gene. Among 85 patients: 81 girls with clinical picture of RTT were with normal female karyotype (46,XX); two boy was with normal male karyotype in cells of blood (46,XY) and two boys were with mosaic forms of Kleinfelter’s syndrome (47,XXY/46,XY) in blood and muscle cells. 24 mothers and parents of RTT girls were with normal karyotype, three mothers - with mosaic form of Turner syndrome (45,X/46,XX), mosaic form of trisomy X syndrome (46,XX/47,XXX) and one - mosaic karyotype -47,XX,+mar/48,XXX,+mar. We analysed chromosome X in lymphocytes of 81 affected girls with clinical picture of RTT using BrdU + Gimsa staining technique. Specific type of inactive chromosome X (so-called type “C”) with unusual staining of chromatin in long arm of the chromosome X was found in 76 (93%) girls with RTT. This technique was positively used for presymptomatic diagnosis of RTT in five girls in affected families. We believe that the phenomenon of altered chromatin conformation in inactive chromosome X could be used as laboratory test for preclinical diagnosis of the RTT. Supported by Copernicus2 and INTAS grants.



Crytic translocation resulting in Angelman syndrome: implication for genetic counselling 

C. Missirian 1, P. Malzac 1, J. Arfi 1, C. Vo Van 1, X. Chauve 1, M. A. Voelckel 1, M. G. Mattei 2, A. Moncla 1;
1Department of Medical Genetics, Marseille, FRANCE, 2Unité INSERM 491, Université de la Méditerranée, Marseille, FRANCE. 


Angelman syndrome is a well-characterised neurobehavioral disorder, associated with different abnormalities: large de novo maternal deletions of 15q11q13, paternal uniparental disomy of chromosome 15, mutations in the UBE3A gene and imprinting defect. Most Angelman syndrome cases result from de novo deletion related to the presence of repeat elements, duplicons, flanking 15q11q13 region.
However, few reported cases demonstrate that deletions may be the result of cryptic structural chromosomal abnormality which involve the 15q11q13 region. This notion led us to systematically control the maternal chromosome 15 structure with molecular cytogenetic method. This strategy allowed us to identify a patient with 15q11q12 deletion resulting from malsegregation of cryptic maternal reciprocal translocation between chromosome 15 and 22: 46, XX, t(15;22)(q12;q11.2).
This observation illustrates the necessity to currently used molecular cytogenetic method to detect such rearrangement taking account their high recurrence risk.



Methylation-sensitive multiplex FRAXA-FRAXE PCR assay is a powerful non-invasive neonatal screening method capable of detecting genetic abnormalities in 1:500 newborn boys. 

V. Strelnikov 1, V. Zemlyakova 2, V. Artamonov 3, E. Vasil'ev 2;
1Institute for Molecular Medicine, Moscow Medical Academy, Moscow, RUSSIAN FEDERATION, 2Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, RUSSIAN FEDERATION, 3Russian State Medical University, Moscow, RUSSIAN FEDERATION. 


Fragile X syndrome (FRAXA mental retardation) is the most frequent form of inherited mental retardation. Discovery of molecular defect causing this disease in 1991 allowed development of precise laboratory diagnostics methods. Nevertheless, for many years these methods remained expensive, time- and labor-consuming. This hampered the development of fragile X screening programs, the necessity of which is widely accepted.
We present a molecular test for fragile X syndrome as well as for FRAXE mental retardation based on FMR1/FMR2 promoter methylation detection via methylation-sensitive PCR. It may be used not only for fragile X diagnostics, but also as a non-invasive screening test on umbilical cord blood to identify fragile X (FRAXA and FRAXE) patients and those carrying extra X chromosome(s) among newborn boys (47,XXY; 48,XXXY; 49,XXXXY; 46,XX male karyotypes and mosaic variants). It may become a powerful tool for detecting fragile X carriers alternative to overall screening of young women, for the latter is associated with serious technical, ethical and financial problems. The methylation-sensitive test proposed here is one of the most efficient and cost-effective screening methods, for it allows detection of genetic abnormalities with a total incidence of approximately 1:500 and is at least tenfold less expensive then conventional molecular detection of fragile X or extra X chromosomes in males. Introduction of such a test would allow early adequate therapeutic and psychological activities towards boys with frequent forms of X-linked genetic abnormalities and, in combination with cascade screening in families, reduce considerably the incidence of the fragile X syndrome.



X inactivation and fragile X methylation in human placentas 

P. Steinbach 1, S. Jakubiczka 2, T. Bettecken 3;
1Universitätsklinikum, Ulm, GERMANY, 2Institut für Humangenetik, Magdeburg, GERMANY, 3GSF Forschungszentrum, Neuherberg, GERMANY. 


In female somatic cells X inactivation is associated with differential CpG methylation on the X chromosomes, e.g., the LINE1 element of the DXS255 minisatellite is extensively methylated only on the active X whereas CpGs in the androgen receptor and the FMR1 gene are methylated only on the inactive X chromosome. Full expansion of the FMR1 CGG repeat in male and female fragile X patients is usually associated with promotor hypermethylation. In chorionic villi of female placentas, X inactivation methylation differs significantly from somatic cells. Also, differences between X inactivation and full mutation methylation of FMR1 have been reported. We have evaluated the methylation status of FMR1 and other X linked loci in chorionic villi from first trimester and full term female placentas of normal and fragile X pre/full mutation individuals. In contrast to somatic tissues, X inactivation methylation was absent from DXS255 and from the FMR1 promotor at any stage of development while methylation was present on other loci. In contrast to X inactivation methylation, fragile X full mutation hypermethylation has been detected in a 13 week old male fetus but was frequently not seen in the first trimenon. Human chorionic villi are hypomethylated. Differential methylation and inactivation of X-linked genes seems to depend on tissue and locus specific methylase interaction as proposed for fragile X hypermethylation.



Prenatal diagnosis of X-linked Opitz G/BBB syndrome 

C. N. Yapijakis 1 ,2, I. Konstantopoulou 3, T. Antoniadi 1, D. Yannoukakos 3;
1Dept. of Molecular Biology and Genetics, Bioerevna Diagnostic Center, Athens, GREECE, 2Dept. of Neurology, University of Athens Medical School, Athens, GREECE, 3Lab. of Molecular Diagnostics, Research Center Demokritos, Athens, GREECE. 


Opitz G/BBB syndrome (OS) is an inherited disorder characterized by midline defects and psychosomatic retardation. OS is genetically heterogeneous with a X-linked and an autosomal form known (respectively Xp22 and 22q11.2). We describe here the first (to our knowledge) reported case of prenatal diagnosis of X-linked OS.
A 9 year-old Greek boy presented with typical signs of OS, including mental/growth retardation, hypertelorism, strabismus, epicanthus,narrow palpebral fissures,flattened nasal bridge, flat filtrum, micrognathia, cranial assymetry and brachycephaly, prominent forehead, low set ears, oesophageal stenosis, heart defects, corpus callosum hypoplasia, seizures, hypotonia, and hypospadias.There was no family history with similar cases.The boys' parents were apparently normal, unrelated, and had no other child.Another pregnancy was terminated at 6 months, because US revealed that the male fetus presented with laryngotracheal cleft and clubfoot.The boy had a normal karyotype, and FISH analysis of the DiGeorge region of 22q11.2, revealed no abnormalities found in autosomaly inherited OS cases.We therefore searched for a mutation in the MID1 gene, which is responsible for X-linked OS. SSCP analysis of the whole gene revealed a child's DNA segment of 345 bp with an abnormal electrophoretic pattern compared to normal controls and his father.The boy's mother was heterozygous for the abnormal segment.
The parents asked for prenatal testing for OS. DNA from CVS was obtained at 12th week of pregnancy and screened for the mutant MID1, in addition to two chromosome Y genes.The fetus was male and had the OS mutation, so after genetic counseling, the parents decided to terminate the pregnancy.



A rheostat model for a rapid and reversible form of imprinting-dependent evolution 

A. L. Beaudet, Y. Jiang;
Baylor College of Medicine, Houston, TX. 


The evolutionary advantages of genomic imprinting are puzzling. We propose that genomic imprinting evolved as a mechanism that maximizes the inter-individual variability in the rates of gene expression for dosage-sensitive loci that can alter the phenotype over a wide continuum with minimal unrelated deleterious effects, and we refer to this as a rheostat model. We hypothesize 1) that genomic imprinting provides a haploid selective advantage (HSA) - not an original proposal; 2) that many imprinted genes have evolved genetic and epigenetic mechanisms that facilitate quantitative hypervariability (QH) of gene expression; 3) that the combination of haploid selective advantage and quantitative hypervariability makes possible a rapid and reversible form of imprinting-dependent evolution (IDE) that can mediate changes in growth, behavior, and perhaps other traits; 4) that this enhanced adaptability to a changing environment provides selective advantage as an assisted form of evolution; and 5) that these mechanisms have provided at least one of the driving forces for the evolution of genomic imprinting in mammals. The rheostat model suggests some nontraditional genetics including both genetic and epigenetic variants contributing to an integrated mechanism of mixed Mendelian and non-Mendelian inheritance, the possibility that the majority of variants are not intrinsically deleterious but are each potentially advantageous depending on the environment, a reversible form of assisted evolution, and the ability to protect a silent allele from selection for many generations but reactivate and expand it in the population quickly.



Characterization of a novel brain specific transcript as candidate for imprinting 

S. Worch, I. Hansmann, D. Schlote;
Institut fuer Humangenetik und Medizinische Biologie, Halle, GERMANY. 


We have identified a novel human gene on chromosome 20q13, a region known to be syntenic to distal mouse chromosome 2 containing imprinted genes. The human transcript is strongly expected to be brain specific as Northern analysis of 8 tissues revealed expression of a 3.2 kb and a 3.0 kb transcript in brain only. The corresponding cDNA (AJ311122) contains a 1680 bp ORF distributed on 13 exons spanning a genomic region of approximately 250 kb.
The homologous mouse cDNA (AK005136) contains a 1680 bp ORF either, and multiple tissue Northern analysis revealed 3 major transcripts of 3.3 kb, 2.9 kb and 2.5 kb visible in brain only emphasizing the human expression pattern. During detailed expression analysis using 8 different brain specific tissues a complex pattern has been detected concerning the quantity of every single transcript depending on analyzed tissue. A developmental specific expression pattern has been found during embryogenesis displaying weak signals from day 10 pc and strong signals from day 15 pc onwards to adult mice suggesting a function from late development.
Database analysis using translated human and mouse gene products revealed homology to several hypothetical proteins of yet unknown function sharing a striking homology of approximately 120 aa at the C-terminal end suggesting the existence of a shared domain, evolutionary conserved down to D. melanogaster and C. elegans.
This work was supported by DHGP.



Prevalence of fragile-X female carrier in Taiwan is lower than expected 

C. Tzeng 1, W. Chen 2, K. Huang 2;
1Department of Pathology, Chi-Mei Foundation Medical Center, Tainan, TAIWAN REPUBLIC OF CHINA, 2Department of Obstetrics and Gynecology, Chi-Mei Foundation Medical Center, Tainan, TAIWAN REPUBLIC OF CHINA. 


Fragile X syndrome (FXS) is not treatable today, but can be prevented by prenatal genetic examination. Identifying female mutant carrier before or at early pregnancy through a wide screening program is considered a practical approach. However, the carrier prevalence in a population and cost of screening test should be carefully evaluated prior to implementation of such a program. To ascertain the prevalence of FXS in Taiwan, we screened a total of 1002 pregnant women using a high-resolution Southern blot test to examine the pooled DNA, and a simple non-radioactive PCR test to identify heterozygous women (Tzeng et al; Diagn Mol Pathol 2001;10:34-41). From these women, we did not find any carrier of premutation and/or full mutation. There were 22 women with an allele exhibiting CGG-repeat between 40 and 52, including two with 48 and three with 52. Approximate one third of the women could be rapidly excluded from being a carrier with the simple PCR test by proving both their alleles were different and within normal CGG-repeat range. This is the first study reporting the female fragile-X carrier rate in Asia population, with a result indicating that the carrier rate in Taiwan is lower than that reported from Israel, Finland, Canada, and the United States, ranging between 1:113 and 1:320. Therefore, we do not recommend such a program to screen general population in Taiwan. Whether it is worthy for women with family history of mental retardation with undermined cause needs to be further investigated.



5 years of molecular diagnosis of Fragile X syndrome (1997-2001): a collaborative study of 22 laboratories in France. 

V. Biancalana 1, C. Beldjord 2, B. de Martinville 3, E. Bieth 4, M. Blayau 5, J. Bonnefont 6, I. Creveaux 7, V. Cusin 8, M. Doco-Fenzy 9, F. Fellmann 10, F. Gerson 11, A. Guiochon-Mantel 12, C. Houdayer 13, M. Kottler 14, G. Lesca 15, C. Philippe 16, F. Prieur 17, H. Puissant 18, M. Raynaud 19, P. Saugier-Veber 20, A. Taillandier 21, L. Taine 22, M. Voelckel 23;
1Laboratoire Diagnostic Génétique, Strasbourg, FRANCE, 2Laboratoire Biochimie et Génétique Moléculaire - Hôpital Cochin, Paris, FRANCE, 3Laboratoire de Génétique Médicale, Lille, FRANCE, 4Service de Génétique Médicale, Toulouse, FRANCE, 5Laboratoire de Génétique Moléculaire, Rennes, FRANCE, 6Département de Génétique Moléculaire - Hôpital Necker, Paris, FRANCE, 7Laboratoire Biochimie Médicale et Biologie Moléculaire, Clermont-Ferrand, FRANCE, 8Laboratoire Biochimie Génétique - Hôpital Debré, Paris, FRANCE, 9Laboratoire cytogénétique, génétique et biologie prénatale, Reims, FRANCE, 10Service de cytogénétique, Besançon, FRANCE, 11Laboratoire Génétique Moléculaire, Nantes, FRANCE, 12Laboratoire d'hormonologie et biologie moléculaire, Le Kremin-Bicêtre, FRANCE, 13Biochimie et Biologie Moléculaire - Hôpital Trousseau, Paris, FRANCE, 14Laboratoire biochimie B, Caen, FRANCE, 15Laboratoire Génétique Moléculaire, Lyon, FRANCE, 16Laboratoire de Génétique Médicale, Nancy, FRANCE, 17Laboratoire Génétique, St-Etienne, FRANCE, 18Service de génétique CHU, Angers, FRANCE, 19Service biochimie et génétique, Tours, FRANCE, 20Laboratoire Génétique Moléculaire, Rouen, FRANCE, 21Centre d'étude de biologie prénatale, Versailles, FRANCE, 22Service de génétique médicale, Bordeaux, FRANCE, 23Laboratoire de Génétique Moléculaire, Marseille, FRANCE. 


The fragile X syndrome is the most common cause of inherited mental retardation. A preliminary study of the efficiency of the screening in mentally retarded probands with no previous familial diagnosis was done in Strasbourg with a comparison between data from 1991-1994 and 1997-2000 (1058 and 2771 families analyzed respectively). This comparison showed a quite stable efficiency of the diagnosis since 1994 (between 1.5 to 3.2% of positive cases), and some improvements in its precocity and exhaustiveness with a decrease of the age at diagnosis of the proband (average :16 to 12 years) and an increase of the percentage of families detected with only a sporadic case (35 to 65%) or with a female proband (7.7 to 15%).
This study was enlarged to 23 laboratories and we will present the results covering 5 years of screening. The preliminary data from 18 laboratories are in agreement with the above results. 15517 families were studied, and allowed the identification of 351 FraX probands. The efficiency is 2.26%. The average age of FraX probands detected was 11. The detection concerned a sporadic case in 56% of the families, and a female in 12%.
225 of the 351 families were analysed further in each laboratory, leading to the testing of 463 females at risk, among whom 338 carriers of premutations or full mutations were diagnosed, and to 52 prenatal diagnoses.
The data from the 23 laboratories will be presented, with issues concerning premature ovarian failure and premutation discovered in a mentally retarded proband.



Five years experience with DIG labeled probes on Southern blots applied in Fragile X diagnostics 

W. H. Deelen, C. Jansen, S. R. Ramlakhan, I. Labrijn-Marks, R. Olmer, D. Dooijes, A. M. W. vanden Ouweland, D. J. J. Halley;
Erasmus University, Rotterdam, NETHERLANDS. 


The DIG labeling and detection system is a simple system to detect single copy genes on Southern blots. DIG labeled probes are at least as sensitive as 32 P labeled probes, they are very stable and faster and safer to work with. In our hands, incorporation of DIG-dUTP by PCR is the most preferred labeling method, which generates highly specific probes. The hybridized probe can be detected after hybridization with an alkaline-phosphatase antibody and CDP-star as a substrate. We have been using the DIG technique in our diagnostic tests for fragile X syndrome. Since this is a triplet expansion syndrome with a wide range of fragment lengths, it is a critical application for Southern blotting. The DIG labeling method proved to be a reliable single test that discriminates between normal alleles, premutations, full mutations and mosaics. Postnatal screening involved > 2000 samples and we identified 30 patients and 31 carriers. We did 26 prenatal diagnoses and found 8 affected and 3 carrier fetuses.



Analysis of FMR 1 methylation in Fragile X Syndrome in Iranian Population 

M. Hasanzad 1, S. Hosseini Amini 1 ,2, F. Taghizadeh 1 ,2, S. Teimourian 1 ,2, R. Karimi-Nejad 2, Y. Shafeghati 1 ,2, m. Karimi-Nejad 2, H. Najmabadi 1 ,2;
1Genetics Research Center the Social Welfare and Rehabilitation University., Tehran, IRAN (ISLAMIC REPUBLIC OF), 2Karimi-Nejad Pathology and Genetic Center, Tehran, IRAN (ISLAMIC REPUBLIC OF). 


Fragile X syndrome is the most common cause of inherited mental retardation. This syndrome is knows to be the result of a dynamic trinucleotid mutation at the 5' UTR (Untranslated region) of the FMR1 (Fragile X Mental Retardation) gene.
We use PCR as a pre-screen and only to proceed to southern blot on those sample which fail to amplify (males) or show a single normal alleles (females). The remaining samples were subjected to southern blot analysis that often combined with methylation analysis by restriction enzyme digestion with a methylation-sensitive enzyme. Determining methylation status EcoR I is combined with a methylation sensitive enzyme Nru I was used.
In both procedures PCR and Southern Blot analysis non- radioactive protocol were used PCR product were detected by silver staining and digoxignin was used in Southern Blot.
Total of 275 individuals from 200 families with at least one mentally retarded child were examined 110 case had a full mutation, 17 with a permutation and 148 were normal. In prenatal diagnosis that was performed for 8 fetuses from these families 2 normal males, 1 normal females 3 fullmutation males and 2 fullmutation females were detected.



CGG-repeat expansion and metilation status of the promotor region of FMR1 gene analysis in the Fragile-X sindrome patients from Ukraine 

N. V. Gryschenko, S. G. Malarchuk, L. A. Livshits;
Institute of Molecular Biology and Genetics, Kiev, UKRAINE. 


FRAXA is folate sensitive fragile site, which associated with X-linked mental retardation. FMR1 gene, whose 1-st exon includes the FRAXA site on Xq27.3, accounts for nearly 20% of all X-linked forms of mental retardation. The fragility in this site is due to expansion of CGG-reports, which associated with hypermethylated CpG islands.
For this study we selected the group of patients with Fragile-X syndrome phenotype from Ukraine. We detected CGG-expansion in FMR1 gene by Southern blot analysis using pX6 probe and/or direct PCR analysis in 14 causes from 79. We performed the methylation analysis for group of 8 patients with detected CGG-expansion or negative PCR analysis (CGG-expansion or deletion). Hin6.1 is a methylation-sensitive enzyme that cuts only unmethylated recognition sites. The PCR of Hin6.1-restricted DNA by use of primers flanking the FRAXA CpG islands amplifies only the non-restricted (inactivated) copies of CpG islands. We detected hypermethylation in samples with CGG-expansion (5/8) as well as in samples with negative PCR (3/8). This test is very simple, inexpensive and effective method. At our mind, this test would be useful for performing diagnosis, postnatal and prenatal diagnostics as a control of Southern blot analysis and direct PCR analysis and for screening patients with mental retardation and newborn boys.



Mutation spectrum in Rett syndrome in Denmark 

J. B. Nielsen 1 ,2, K. Ravn 2, M. Schwartz 2;
1John F. Kennedy Institute, Glostrup, DENMARK, 2Rigshospitalet, Copenhagen, DENMARK. 


At present 88 patients have been registered in Denmark with the diagnosis Rett syndrome, 87 female patients and one boy. They were born in the period 1923 - 1999. Seven have died at ages between 11 and 65 years. Seventy-four have been screened for mutations in MECP2 by sequencing the exons 2-4. In 62 patients a mutation was identified (84 %), 48 single base substitutions, 12 small deletions and two small additions. The single base substitutions were 23 nonsense mutations: Y141X(2), R168X(5), R255X(7), R270X(3) and R294X(6) and 25 missense mutations: R106W(4), R133C(2), S134C(1), T158M(11), P302L(1), R306C(5) and R309W(1).
R309W has not earlier been reported. The patient has a very mild variant form of Rett syndrome with only a few of the typical traits.
There is a slight tendency towards a milder phenotype in the cases with a missense or a late truncating mutation. But it applies to all the recurrent mutations, R133C excepted, that the phenotype varies from mild to severe.
The oldest of the seven patients with the R255X mutation is 27 years old and still able to walk independently, while the youngest is 2 years old and probably will never be able to walk. The oldest of the 11 patients with the T158M mutation is 78 years old, she walked with support until she was 41 years old, while three of these patients, 2-9 years of age, have never walked independently.
For details about the boy, see abstract by Ravn et al.



Low frequency of MECP2 mutations in mental retardation of unknown origin: implications for routine DNA diagnostics 

H. G. Yntema, T. Kleefstra, A. R. Oudakker, B. B. A. de Vries, W. Nillesen, E. A. Sistermans, H. G. Brunner, B. C. J. Hamel, H. van Bokhoven;
Department of Human Genetics, Nijmegen, NETHERLANDS. 


Mutations in the methyl-CpG binding protein 2 (MECP2) gene are not always lethal in males. MECP2 mutations have been found in males with severe mental retardation with or without progressive encephalopathy, males with an Angelman-like phenotype, and males with mild nonspecific mental retardation. It was suggested that the frequency of mutations in MECP2 in mentally retarded males equals the frequency of the CGG expansion in the FMR1 gene. In order to determine if MECP2 screening should be implemented in a routine diagnostic setting for patients with nonspecific mental retardation, we tested a cohort of 500 male and 100 female mentally retarded patients who were negative for the expansions across the FMR1 CGG repeat. Furthermore, 70 mentally retarded patients with a clinical diagnosis of Angelman syndrome, but without a molecular abnormality on 15q, were included in this study. In each of these three patient groups only one causative mutation could be identified. Several amino acid changes appeared to be polymorphisms after testing unaffected male family members. Because one of these patients showed a Prader-Willi like phenotype, we performed MECP2 mutation analysis in 100 patients with a clinical but no molecular diagnosis of Prader-Willi syndrome. Until now no mutations have been found. We conclude that the incidence of MECP2 mutations in patients with mental retardation of unknown origin is low and we do not favour implementation of this gene in routine DNA diagnostics. Results of the mutation analyses and clinical findings in the patients with a MECP2 mutation will be presented.



Large Deletions of entire Exons of MECP2 gene, may represent the genetic defect in some RTT patients with no mutations found in the coding region by using DNA sequencing analysis 

M. L. Giovannucci Uzielli, L. Giunti, S. Guarducci, E. Anichini;
University of Florence, Firenze, ITALY. 


Rett syndrome (RTT) is an X-linked progressive neurodevelopmental disorder. Affected females develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, autism, ataxia, seizures, abnormal hyperventilation and stereotypic hand movements. Rett syndrome is caused by mutation in the MECP2 gene on chromosome Xq28 (Amir et al., 1999).
To date, mutations in the coding region of MECP2 account for RTT in 65-85% of the known cases.
We performed a long distance PCR coupled with long-read direct sequencing, to analize the entire MECP2 gene coding region in 101 unrelated RTT girls.
Mutations were identified in 79/101 patients, both with classic and non-classic phenotype.
Special attention was dedicated to the subjects with no mutation found in the coding region of MECP2 gene by sequencing analysis, using further approaches. In one family we found in the mother a polymorphic, common neutral variant C-A in the last codon, at the 3’ of exon 3. The father does’nt shows the variant, and the RTT daughter has apparently only the maternal allele.
We developed a quantitative PCR based densitometric dosage assay, on a Long Readir LICOR -4200, to demonstrate the deletion of entire exons of MECP2 gene in this RTT patient.
Other approaches (FISH, Southern blotting and RT-PCR analyses) are now in progress to confirm this hypothesis.



Comprehensive mutation analysis of the MECP2 gene and the analysis of 54 Rett syndrome suspected patients 

Y. J. Vos 1, G. J. J. Mol 1, J. H. Begeer 2, E. Verlind 1, P. C. van den Akker 1, C. H. C. M. Buys 1, R. Hordijk 1, R. M. W. Hofstra 3;
1Department of Clinical Genetics, University Hospital, Groningen, NETHERLANDS, 2Department of Neurology, University Hospital, Groningen, NETHERLANDS, 3Department of Medical Genetics, University of Groningen, Groningen, NETHERLANDS. 


Rett syndrome is an X-linked dominant neurodevelopmental disorder, affecting 1/10.000-15.000 girls, characterised by a period of early normal growth and development followed by regression with loss of speech and acquired motor skills, stereotypic hand movements and seizures. The disease-causing gene, mapped to Xq28, was identified as MECP2, encoding the methyl-CpG-binding protein 2.
To analyse this gene in patients suspected of Rett syndrome, we developed an efficient pre-screening method, based on denaturing gradient gelelectrophoresis (DGGE), followed by direct sequencing in case an aberrant band pattern is found. The DGGE system consists of 15 amplicons which all can be analysed under one single experimental condition.
Until now 54 patients have been analysed, whose clinical diagnosis varied from mental retardation to classic Rett syndrome. In 14 patients (26%) 10 different mutations have been identified, 3 nonsense mutations in 5 patients and 7 missense mutations in 9 patients. One nonsense mutation has not yet been reported, Q47X. In one patient two missense mutations have been found, both previously published as being the cause of Rett syndrome.
The overall mutation frequency is far below other published results. In view of our extensive experience in designing DGGE systems for mutation detection that result in the detection of virtually all possible mutations, this low frequency is most probably due to loose inclusion criteria. Clinical characteristics of the patients will be presented in comparison with the results of the mutation analysis.



Mutation analysis of MeCP2 gene in 36 patients with Rett syndrome of Slavic origin: Detection of two novel mutations and one new polymorphism 

R. Rosipal 1, J. Zeman 1, J. Hadac 2, N. Misovicova 3, P. Martasek 1;
1Center of Integrated Genomics, Department of Pediatrics, First Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, 2Department of Child Neurology, Thomayer University Hospital, Prague, CZECH REPUBLIC, 3Department of Clinical Genetics, Martin University Hospital, Martin, SLOVAKIA. 


Rett syndrome (RS) is an X-linked dominant neurodevelopmental disorder that almost exclusively affects girls. A prevalence is estimated to be 1:10, 000 to 1:15, 000 females. Patients with classic RS are characterized by a period of normal growth and development followed by regression with loss of speech and acquired motor skills. Neurologic abnormalities include spastic paraparesis, ataxia, intermittent hyperventilation, and epilepsy. Growth retardation, scoliosis, and autonomic dysfunction are common. RS is caused by mutations in X-linked MECP2 gene, encoding for methyl-CpG-binding protein 2. It plays an important role in the regulation of gene expression. The spectrum of mutations in MECP2 gene is known from numerous countries and ethnic groups and steps are being taken to determine the genotype/phenotype relationship in order to understand the disease process. Here we report mutation analysis of 36 patients with RS from the Czech and Slovak republics. Systematic sequencing of the entire coding sequence of MeCP2 gene revealed, in exon 4, thirteen different disease-causing mutations in 22 sporadic patients (61%). Two have not been previously published: a small deletion of 3 bp (1069delAGC), and a deletion of 172 bp along the insertion of 41 bp (1063del172bp+ins 41bp). Eleven patients had nonsense mutations (Y141X, R168X, S204X, R255X, R270X, R294X), eight carried missense mutations (R133C, K135E, T158M, R306C), and one had a frameshift mutation (1157del41bp). The novel polymorphism 587 C>G (T196S) was detected in a patient carrying the mutation 397 C>T (R133C). (Supported by Czech Granting Agency - GACR 301/01/P068 and LN00A079 from MSMT of Czech Republic)



Analysis of the MECP2 gene by Direct Sequencing in Hungarian Rett Syndrome Patients 

J. Kárteszi 1, J. Bene 2, É. Morava 1, M. Czakó 2, K. Hollódy 3, B. Melegh 1, G. Kosztolányi 1;
1Department of Medical Genetics and Child Development, University of Pécs, Pécs, HUNGARY, 2Department of Medical Genetics and Child Development, MTA-PTE Clinical Genetic Research Group, University of Pécs, Pécs, HUNGARY, 3Department of Pediatrics, University of Pécs, Pécs, HUNGARY. 


Rett syndrome is an X-linked neurodevelopmental disorder characterized by loss of acquired skills, stereotypical hand movements, microcephaly, trunk ataxia and hyperventilation. Epilepsy may also be present in some patients. Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) have been identified as cause of Rett syndrome. Based on these results we initialized mutation screening of MECP2 in Hungarian Rett syndrome patients. Patients of the Hungarian Rett Syndrome Association were involved in the present study. So far we have examined 19 patients, from various districts of Hungary, who were supposed to have Rett syndrome for MECP2 mutations. Initially a detailed clinical evaluation were performed including physical, neurological and orthopedical examinations, EEG and bone X-ray studies. Genetic studies included chromosomal analysis and FISH for Angelman syndrome. Routine metabolic screening and serum IEF for CDGS were also performed in every patient to exclude other underlying etiologies. Based on these scrutinized analysis, the clinical diagnosis of Rett syndrome could be supported in 15 patients out of the 19 cases. Mutations in MECP2 were detected in 8 cases. We found five already described mutations in six patients (R106W in two patients, P152R, R168X, R270X, R294X), a novel single base insertion in exon 3 (276insG) in a patient with a clinical history showing slow progression, and a large deletion in exon 4 in a patient with typical Rett syndrome. A new polymorhism (N126K) characteristic for the Hungarian population was also detected. This amino acid alteration was found in all examined patients so as in healthy controls.



A 10-year-old boy with classical Rett syndrome caused by a frameshift mutation the MECP2 gene. 

K. Ravn 1, J. Bieber Nielsen 2, P. Uldall 2, F. J. Hansen 3, M. Schwartz 2;
1Rigshospitalet 4062, Copenhagen, DENMARK, 2Rigshopitalet, Copenhagen, DENMARK, 3Rigshospitalet, Copenhagen, DENMARK. 


Rett syndrome (RS) is an X-linked dominant neurodevelopmental disorder, considered as exclusively affecting girls. Affected male fetuses were thought to be aborted spontaneously or to have a different phenotype. So far 17 males with a mutation in MECP2 have been reported so far. Five of these had the karyotype 47,XXY or were mosaic for the mutation. The remaining cases can be divided into two groups; a severe type, who dies within the first years of life, all caused by an MECP2 mutation, which in females has be found to be associated with classical RS, and a mental retardation type with mutations, which if present in females are compatible with a normal or mildly retarded development. Here we present a 10-year old boy with clinical RS, with a normal karyotype, no signs of mosaicism and a truncating mutation 816dup7 in the MECP2-region encoding the transcription repression domain, TRD.
The resulting MeCP2 protein is predicted to contain a functional methyl-binding domain, but lacking most the TRD and the site facilitating MeCP2 binding to DNA.
While comparing the genotype-phenotype correlation in female with mutations in MECP2 is hampered by X chromosome inactivation, the phenotype of males hemizygous for the same mutations should shred light over the effect of these mutations on the phenotype. However, our patients harbour a typical RS mutation, but have survived beyond early childhood in contrast to the expectation and the experience for these mutations. The hypothesis that other genes are interfering with the clinical features of RS is possible.



Mutation or polymorphism in the MECP2 gene in mentally retarded boys : diagnostic implications 

A. Grandemenge, V. Bourdon, C. Philippe, P. Jonveaux;
University Hospital of Nancy, Vandoeuvre les Nancy, FRANCE. 


Among the well characterized X-linked conditions causing mental retardation, mutations in the methyl-CpG binding protein 2 (MECP2) gene on Xq28 have been found in 70-80% of patients with Rett syndrome, a neurological disorder which, in addition to other symptoms, severely affects higher cognitive functions in females. Mutations in the MECP2 gene are involved in a broad spectrum of phenotypes from classical Rett syndrome to mild intellectual difficulties in feamles and neonatal encephalopathy in males .
Recently, few MECP2 mutations were reported in males with non specific mental retardation suggesting that defects in MECP2 are responsible for about 2% of X-linked mental retardation.
To assess the frequency of MECP2 mutations, we screened, by DHPLC, the coding sequence and flanking regions of the MECP2 gene in a cohort of 262 mentally retarded males found negative for fragile-X syndrome.
First results show one recurrent intronic polymorphism (IVS3-19delA) and a nucleotide variant P376S. None of these sequence modifications were detected in 200 controls. Nevertheless, we already identified the nucleotide variant P376S, in a girl presenting an atypic Rett syndrome. A detailed familial study on three generations showed that the substitution P376S was also inherited by a healthy uncle, thus ruling out its involvement in the etiology of the disease.
This finding clearly calls for a careful consideration of the pathogenicity of the MECP2 mutations identified in males before genetic counselling.



Drosophila as a model to study the physiological pathway in which FMRP (Fragile X Mental Retardation Protein) is involved 

B. Bardoni, A. Schenck, V. van de Bor, A. Giangrande, J. Mandel;
IGBMC- Institute de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, FRANCE. 


The Fragile X mental retardation syndrome results from the absence of the protein (FMRP) encoded by the FMR1 gene. FMRP is an RNA binding protein, that has two close homologs, FXR1P and FXR2P. Absence of FMRP is thought to affect transport and/or translation of a subset of mRNAs and results in the formation of abnormal dendritic spines in patients and knock-out mice.
We have identified 4 novel proteins interacting with FMRP: NUFIP1, CYFIP1 and 2 (that share 95% of amino acids identity) and NUCIF1. Remarkably, CYFIP1 also interacts with Rac1, an important factor in neuronal maturation (1).
To study the physiological pathways in which FMRP and its partners are involved, we have chosen Drosophila, as its genome contains only a single FMR/FXR ortholog gene, dFMR (2), one dCYFIP gene and one dNUFIP gene. We found that in fly larvae brain, dFMR is specifically expressed in mushroom bodies, a structure involved in learning and memory. Analysis of the expression profile of dNUFIP and dCYFIP are in progress. We have generated dCYFIP null mutants and their phenotype is being analyzed. Preliminary results indicate a genetic interaction between dRac1 and dCYFIP mutants
1. Bardoni et al. (2001) Brain Bull. Res. 56: 375-382
2. Wan et al. (2000) MCB 20: 8536-8547



Towards an understanding of the Fragile X Syndrome: FMRP is translated at the synapses where it acts as a translational regulator 

F. Zalfa 1, B. Primerano 1, C. Lauro 1, M. Giorgi 1, A. Moro 1, G. Spinelli 1, E. Tongiorgi 2, B. Oostra 3, F. Amaldi 1, C. Bagni 1;
1Dipartimento di Biologia. Università di Roma, Rome, ITALY, 2Dipartimento di Biologia. Università di Trieste, Trieste, ITALY, 3Department of Clinical Genetics, Erasmus University, Rotterdam, NETHERLANDS. 


The Fragile X syndrome is an X-linked disorder and the most common heritable form of mental retardation, and results from the deficit or absence of the FMRP protein which is expressed mainly in the brain and has been implicated in translational regulation.
While the vast majority of mRNAs is restricted to the cell soma of neuronal cells, a number of them are also transported into the dendrites, where they are translated. Transport and translation of specific mRNAs in extrasomal locations, plays an important role in nerve cell development and synaptic plasticity. Here we show by RT-PCR that FMR1 mRNA that encodes FMRP is found in synaptoneurosomes. Furthermore, by electron microscopy studies we detected FMR1 mRNA in proximal and distal dendrites of the hippocampus.
To identify potential localisation signal on the mRNA, we started by analysing the 3’UTR. We observed that the FMR1 sequence contains several polyadenilation sites. DNA constructs were made expressing EGFP fused to the entire 3'UTR or to shorter region and transfected into mouse hippocampal neurons showing that alternative poly(A) sites are responsible for producing FMR1 mRNA molecules with different 3'UTRs differentially delivered into the cell. We have investigated the function of the FMRP protein at the synapses by analysing the translational efficiency of mRNAs in synaptosomal preparations. We find that some dendritic mRNA (a-CaMKII and Arc) are translated very efficiently in FMR1 K.O. mice as compared to wild type mice, whereas control RNA, (beta actin) is not affected showing that FMRP inhibits the translation of target mRNAs.



Differential gene expression in the fragile X mouse model 

I. Gantois, E. Reyniers, F. Kooy;
University of Antwerp, Antwerp, BELGIUM. 


Ten years of research showed that FMRP, the protein missing in fragile X patients, is an RNA binding protein that shuttles between the nucleus and cytoplasm. In neurons, the protein transports certain brain mRNAs towards the actively translating ribosomes near the synapses.
However, despite intense research it is still unclear why absence of the fragile X protein leads to the mental retardation, macroorchidism and specific behaviour problems observed in fragile X patients. In an attempt to unravel this mechanism, we performed gene expression analysis by means of the differential display method using the fragile X mouse model. In analogy to human patients, the fragile X knockout mouse shows a learning deficit and macroorchidism. The expression of approximately 95% of all genes in the hippocampus of control mice and fragile X knockout mice was compared. We isolated 224 sequences with a length range of 200-1100 bp. 143 sequences were underexpressed and 81 overexpressed in knockout mice. Using micro-arrays and real time PCR, for some of these sequences differential expression was confirmed. These partial cDNAs were sequenced, and homologies with known mouse and human ESTs and genes were searched on public and Celera databases.
The role of these genes differentially expressed in the fragile X animal model will now be investigated. These may help us to answer the question how the absence of fragile X protein relates to mental retardation in patients.



Quantitative analysis of DNA demethylation and trascriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine 

R. Pietrobono 1, M. G. Pomponi 1, E. Tabolacci 1, B. Oostra 2, P. Chiurazzi 3, G. Neri 1;
1Istituto di Genetica Medica, Università Cattolica del S. Cuore, Roma, ITALY, 2Department of Clinical Genetics and Center for Biomedical Genetics, Erasmus University, Rotterdam, NETHERLANDS, 3Dipartimento di Scienze Pediatriche Mediche e Chirurgiche, Università di Messina, Messina, ITALY. 


The Fragile X syndrome is the leading cause of inherited mental retardation, affecting approximately one in 5000 individuals. In fragile X cells carrying a full mutation , hypermethylation of the expanded CGG repeat and of the upstream promoter leads to transcriptional silencing of the FMR1 gene. Absence of the FMR1 protein results in the phenotypic manifestation of the syndrome. We previously proved that treatment with 5-azadeoxycytidine of fragile X cell lines results in reactivation of the FMR1 gene restoring the production of the specific mRNA and protein product. We now show that this treatment causes demethylation of the FMR1 gene promoter. We employed the bisulphite sequencing technique to detect the methylation status of individual CpG sites in the entire promoter region upstream of the CGG repeat. Lymphoblastoid cell lines of fragile X males with full mutations of different sizes were tested before and after treatment with 5-azadeoxycytidine at various time points. We observed that individual clones are either completely demethylated or not, with few relevant exceptions. We also investigated the extent of methylation in the full mutation (CGG repeat) itself by Southern blot analysis after digestion with methylation-sensitive enzymes Fnu4HI and McrBC and found that the CGG repeat remains at least partially methylated in many clones with a demethylated promoter. This may explain the quantitative discrepancy between the large extent of promoter demethylation and the limited levels of FMR1 transcriptional reactivation estimated by quantitative real-time fluorescent RT-PCR analysis.
Supported by FRAXA Research Fondation, Sigma-Tau and Associazione Anni Verdi.



Evidence for Skewed X Chromosome Inactivation in Females with the Fragile X Full Mutation 

K. B. McKelvie 1, A. Reynolds 1, F. Tassone 2, A. K. Taylor 3, R. J. Hagerman 2;
1The Children's Hospital, Denver, CO, 2M.I.N.D. Institute, University of California Davis Medical Center, Sacramento, CA, 3Kimball Genetics, Denver, CO. 


X chromosome inactivation is generally thought to be a random occurence in somatic tissue. Skewed X-inactivation has been observed in several human disorders in which there is a deletion or mutation on one of the X chromosomes. This skewing is thought to be the result of selection against cells with growth disadvantage. Previous research by Rousseau et al. and Taylor et al. revealed a skewed X-inactivation pattern for females with the Fragile X full mutation.
As part of a larger Genotype-Phenotype research study, we analyzed molecular data for 51 females with the full mutation and 43 females with the premutation status for FXS, with an age range of 4 to 65 years. In contrast to females with the premutation, females with the full mutation demonstrated skewed X-inactivation with a tendency toward a higher activation ratio or proportion of normal active X. The median activation ratio for females with the full mutation was significantly larger than that of premutation carriers. Also, linear regression analysis revealed a significant positive relationship between the activation ratio and age for females with the full mutation, but not for females with the premutation. These results support previous research. In addition, activation ratio in women with a maternally inherited premutation was compared to women with a paternally inherited premutation. There was no signigant difference found between these two groups.



Loss of mutation at the FMR1 locus : a gene conversion? 

A. Jouanolle 1, S. Bernard 2, V. David 1, M. Blayau 1;
1Laboratoire de Génétique Moléculaire, CHU Pontchaillou, Rennes, FRANCE, 2Gynécologie-Obstétrique, CH La Beauchée, St-Brieuc, FRANCE. 


Fragile X syndrome is the most frequent inherited form of mental retardation. The mutation observed is almost exclusively an expanded (CGG) repeat in the first untranslated exon of the FMR1 gene. The repeat is polymorphic in length in the normal population (6 to 55 repeats) and may be unstable. Two types of mutations have been distinguished : premutation (up to 200 repeats) in the phenotypically normal carriers or full mutation (over 200 repeats) in the affected patients. In the latter case FMR1 is abnormally methylated and transcriptionally silent. Molecular diagnosis is based on the determination of the number of trinucleotide repeats by either Southern blot or fluorescent PCR. Transmission of the deleterious chromosome can also be established by haplotype analysis with flanking markers.
We have searched for the CGG expansion in a family comprising four siblings : two mentally retarded boys and their two sisters. Southern blot analysis revealed a large repeat expansion in the two boys and the oldest girl, whereas no expansion was observed in the youngest girl. These results were confirmed by PCR. Indirect analysis with three microsatellites (DXS548, FRAXAC1, FRAXAC2) allowed us to identify the maternal fragile X chromosome which is carried by the four children. In the youngest girl, the presence of this maternal fragile X chromosome is inconsistent with the absence of expansion. So, we discuss the hypothesis of a gene conversion where the segregation of flanking markers is dissociated from that of the CGG expansion.



Four novel mutations in the OFD1 (Cxorf5) gene in the Finnish patients with oral-facial-digital syndrome 1 

S. Ala-Mello 1, A. Rakkolainen 2, P. Kristo 2, A. Orpana 2, I. Järvelä 2;
1University of Helsinki, 00014 Helsinki, FINLAND, 2University of Helsinki, Helsinki, FINLAND. 


Oral-facial-digital syndrome type 1 (OFD1) is an X-linked dominant disorder characterized by malformations in face, oral cavity, and digits with a wide phenotypic variation. Recently, mutations in the OFD1 gene (Cxorf5) at Xp22 were found to underlie OFD1. We report here the identification of four novel mutations in the OFD1 gene in the Finnish families, two of which are familial and two sporadic.Three of the mutations in this study were located in the same exons as in the original study by Ferrante et al (Am J Hum Genet 2001;68:569-576). We also report the clinical findings of our patients. Our study confirms the causative role of the OFD1 gene in the pathogenesis of oral-facial-digital syndrome type 1.



Molecular basis of Oral-facial-digital type I (OFDI) syndrome 

G. Giorgio, A. Barra, A. Ballabio, B. Franco;
Telethon Institute of Genetics and Medicine-TIGEM, Naples, ITALY. 


Oral-facial-digital type 1 (OFD1) is part of the heterogeneous group of oral-facial-digital syndromes (OFDS). OFDI is an X-linked dominant condition lethal in males and is characterized by malformations of the face, oral cavity, and digits. Malformation of the brain and polycystic kidneys are commonly associated. By using a systematic mutation analysis approach we have identified the gene responsible for this genetic disorder, named OFDI, encoding a protein with unknown function. To gain insight into the pathogenesis of this disorder extensive mutation analysis and functional studies were undertaken. Twenty-seven OFDI patients were collected and mutations have been so far identified in 17 of them. Details on the newly identified mutations and on genotype/phenotype correlation will be presented. The presence of coiled-coil domains suggests that OFD1 may act via a protein-protein interaction mechanism. Interaction mating and two hybrid experiments are being performed to identify proteins potentially interacting with OFDI. Preliminary results show that OFDI does homo-interact through the central portion of the protein. Furthermore, subcellular localization experiments were performed on the wild type and mutated forms of the OFDI protein. Our study showed that the wild type protein concentrates in "cytoplasmic bodies" uniformly distributed in the cytoplasm while in the mutated forms, the cytoplasmic speckles become smaller, with a more diffuse distribution and tend to disappear. The functional characterization of the OFDI protein product will open the way towards understanding the molecular and cellular bases of OFDI as well as for the other forms of OFDs.



Partial deletion of the common 1,5 Mb critical region in an infant with classical Williams-Beuren syndrome. 

R. Heller 1, S. Endele 1 ,2, B. Schröder 1, C. Steglich 1, S. Lüttgen 1, A. Winterpacht 1 ,2;
1Institut für Humangenetik / UKE, Hamburg, GERMANY, 2present address: Institut für Humangenetik, F.-A.-Universität Erlangen-Nürnberg, GERMANY. 


Williams-Beuren syndrome (WBS; OMIM 194050) is a contiguous gene deletion disorder with a variable clinical phenotype that is caused in most cases by a heterozygous microdeletion in 7q11.23. Due to two highly homologous flanking ~300 kb duplicons, the microdeletion is usually of similar size in almost all cases and encompasses a common ~1,5 Mb interval that contains at least 17 genes mostly of uncertain pathogenetic relevance. Phenotype-genotype correlation studies for WBS are hampered by the uniform size of the microdeletion.
Here we report the case of a 1-year-old boy with a full WBS-phenotype that is caused by a partial deletion of the common ~1.5 Mb interval. Initial analysis with two sets of commercially available FISH-probes (Appligene/Oncor and Vysis) yielded conflicting results. We therefore carried out deletion mapping with microsatellite markers and an array of targeted FISH probes. Our data could help to redefine the critical region for the full WBS phenotype (WBSCR). Evidence from two other cases in the literature also suggests that the centromeric portion of the 1,5 Mb interval is not always deleted in patients with full WBS-phenotype.
The conclusions from our work concern diagnosis and molecular aetiology of WBS:
1.) Quantitative differences in signal intensity after FISH analysis with commercial probes for a WBS microdeletion should be followed up carefully if partial deletions are not to be missed.
2.) Even in patients with full WBS-phenotype the underlying molecular defect is more variable than previously thought.



Characterisation of genes in the region of mouse chromosome 5 orthologous to the region deleted in Williams-Beuren Syndrome (7q11.23)and their human homologues. 

P. D. Cunliffe, L. Heather, A. P. Read, M. Tassabehji;
University of Manchester, Manchester, UNITED KINGDOM. 


Williams-Beuren syndrome is a hereditary disorder caused by deletion of approximately 1.5Mb on human chromosome 7q11.23 and occurring in approximately 1/20000 live births. Symptoms include congenital heart disease, growth retardation, mild mental retardation with a distinctive cognitive profile and personality, facial dysmorphism and frequently infantile hypercalcaemia. The breakpoints of the commonly deleted region are flanked by highly homologous repeated regions of about 300kb. The flanking repeats are believed to mediate the disease causing deletion and have also caused difficulty in its genomic cloning and sequencing. The only aspect of the disease phenotye associated with a particular gene is the typical congenital heart defect, supravalvular aortic stenosis (SVAS) which is associated with deletion of the elastin gene. The orthologous region in mouse, on chromosome 5 (5G), does not have the flanking repeats, however the order of the genes within the commonly deleted region is conserved. Here we report expression profiles, gene structures and analysis of functional motifs for genes recently localised to the mouse equivalent of the commonly deleted region, including one gene, claudin13, which has not previously been reported to map to this region. Including these genes there are currently 29 transcripts shown to reside in this region of mouse chromosome 5. A comparison between mouse and human orthologues of the genes is also presented along with a complete physical map of the 1.4Mb mouse equivalent of the Williams syndrome region.



Identification of nine novel transcripts in the Williams-Beuren syndrome critical region 

G. Merla 1, C. Ucla 2, M. Guipponi 1, A. Reymond 1;
1Division of Medical Genetics, University of Geneva Medical School, Geneva, SWITZERLAND, 2Divisiobn of Medical Genetics, University of Geneva Medical School, Geneva, SWITZERLAND. 


Williams-Beuren syndrome (WBS) is a developmental disorder associated with haploinsufficiency of multiple genes at 7q11.23. Here, we report the characterization of WBSCR16, WBSCR17, WBSCR18, WBSCR20A, WBSCR20B, WBSCR20C, WBSCR21, WBSCR22 and WBSCR23, nine novel genes contained in the WBS commonly deleted region or its flanking sequences and of their murine orthologues. They were identified by mapping of previously undescribed human ESTs/cDNAs clusters to the WBS critical region. They encode an RCC1-like G exchanging factor, an N-acetylgalactosaminyltransferase, a DNAJ-like chaperone, NOL1/NOP2/sun domain-containing proteins, a methyltransferase, or proteins with no known homologies. Haploinsufficiency of these newly identified WBSCR genes may contribute to certain of the WBS phenotypical features.



Deletion breakpoint mapping in patients with Williams Syndrome using somatic cell hybrids 

M. Tassabehji 1, K. Metcalfe 2, D. Donnai 2, W. D. Fergusson 2, A. Karmiloff-Smith 3, A. P. Read 1, M. J. Carette 1;
1University of Manchester, Manchester, UNITED KINGDOM, 2St Mary's Hospital, Manchester, UNITED KINGDOM, 3Institute of Child health, London, UNITED KINGDOM. 


Williams-Beuren syndrome (WS) is a developmental disorder caused by a hemizygous microdeletion of ~1.5Mb at chromosomal location 7q11.23. Up to 28 genes have been identified within the critical region. Hemizygosity for ELN causes the heart defect SVAS, but there is no clear evidence implicating any of the other genes in the aetiology of the syndrome. To aid genotype-phenotype correlations it is important to define the deletion breakpoints in patients with classic and partial WS phenotypes precisely. Homologous recombination between flanking repeats accounts for the high incidence of de novo deletions and the deletion breakpoints lie within these repeated regions in WS patients making them difficult to map. We have therefore made somatic cell hybrids (segregating the normal and deleted chromosome 7 homologues) from 30 patients with classic or partial WS phenotypes, designed a series of specific PCR primers using SNP technology, and mapped the breakpoints in the centromeric and telomeric repeat regions by PCR analysis of DNA from these cells. Our results indicate that the breakpoints cluster at two main loci at the centromeric end and at least three loci at the telomeric end. This has allowed us to group our patients according to their genotype for further detailed phenotypic analysis that includes complex cognitive testing.



Prader-Willi-like phenotype caused by multiple dosage of maternal 15q11-q13 region. 

E. Monros 1, V. Català 2, E. Geán 3, P. Póo 3;
1Hospital Sant Joan de Deu, Esplugues, Barcelona, SPAIN, 2Prenatal Genetics, Barcelona, SPAIN, 3Hospital Sant Joan de Déu, Esplugues, Barcelona, SPAIN. 


Objective: Genetic diagnosis of Prader-Willi Sd. in a 13 years old male with normal motor development, obesity, moderate mental retardation, obsessive behaviour and small penis.
Methods: PWS methylation analysis was done by NotI-XbaI/pN09 hybridisation. Band densities were measured on a GS-700 Bio-Rad densitometer. Familial segregation analysis of seven 15q11-q13 linked markers was done by PCR. Karyotype was done by standard methods. Molecular cytogenetic studies included chromosome 15 painting using WCP15 probe and FISH analysis with probes LSI SRNPN/PML/CEP15 and LSI D15S10/PML/CEP15.
Results: Hybridisation showed the presence of both the paternal and maternal alleles, excluding a PWS methylation pattern. Maternal band intensity was 4 fold compared to normal controls. Familial segregation analysis of GABRA5 detected one paternal and two maternal alleles, suggesting a maternal trisomy of the region; D15S144 showed biparental inheritance. Karyotype: mosaicism 47,XY,+mar. Painting demonstrated the suspected chromosome 15 origin of the marker, which was present in 83% of metaphases. FISH results using specific probes were: 47,XY,+mar .ish der(15)(D15Z1++, D15S10++, SRNPN++). The marker showed the centromeric staining at both ends, and two adjacent signals for both SRNPN and D15S10 loci in the central part. The final cytogenetic result was mosaicism 47,XY,+ idic(15)(pter->q11.2 :: q11.2->pter).
Conclusions: The patient is a somatic mosaic for a marker chromosome of maternal origin which contains two inverted partial 15q11-q13 regions. Cells carrying the derivative chromosome are tetrasomic for genes contained in the region. The PWS-like features in the patient are caused by a multiple dosage of maternal 15q11-q13 genes.



Epsilon-sarcoglycan (SGCE), the gene mutated in myoclonus-dystonia syndrome, is imprinted. 

M. Grabowski 1 ,2, A. Zimprich 3, B. Lorenz-Depiereux 1 ,2, F. Asmus 3, M. Bauer 3, V. M. Kalscheuer 4, T. Gasser 3, T. Meitinger 1 ,2, T. M. Strom 1 ,2;
1Institute of Human Genetics, GSF National Research Center, Munich, GERMANY, 2Institute of Human Genetics, Klinikum rechts der Isar, Technical University Munich, Munich, GERMANY, 3Department of Neurology, Klinikum Großhadern, Ludwig-Maximilians-University, Munich, GERMANY, 4Max-Planck-Institute for Molecular Genetics, Department of Human Molecular Genetics, Berlin, GERMANY. 


Myoclonus-dystonia syndrome (MDS, DYT11) has been defined as an autosomal-dominant disorder characterized by variable combinations of bilateral, alcohol-sensitive myoclonic jerks and dystonia. Using a positional cloning approach, we have recently identified heterozygous loss-of-function mutations in the gene for epsilon-sarcoglycan (SGCE) in MDS families. Pedigree analysis showed a marked difference in penetrance depending on the parental origin of the disease allele. This indicates a maternal imprinting mechanism, which has been demonstrated for the mouse orthologue.
Bisulfite sequencing of the CpG-rich SGCE promotor showed a parent-specific methylation pattern in lymphoblasts. A rare single nucleotide polymorphism (SNP) in the promotor region allowed us to distinguish the parental alleles. The methylated strand showed the maternal polymorphism, while the paternal wildtype allele was unmethylated in all CpG dinucleotides examined.
Expression studies showed that SGCE is only paternally expressed in lymphoblasts.
Due to the fact that MDS is a non-degenerative central nervous system disorder we also investigated the promotor methylation in brain tissue. SNPs in the promotor region revealed that there is also differential methylation in human brain, suggesting regulation of expression in an allele-specific manner.
As imprinted genes are often located in clusters, we examined four adjacent genes by expression analyses in maternal and paternal UPD7 cDNA samples. SGCE and the adjacent PEG10 showed maternal imprinting in lymphoblastoid cell lines, whereas PP5 and BET1 were not imprinted. These results demonstrate that SGCE is also imprinted in humans and that chromosome 7q21 contains at least two imprinted genes.



Evaluation of a Mutation Screening Strategy for the Ube3a Gene in 33 Patients from 25 Families with Angelman Syndrome 

A. Moncla, P. Malzac, K. Pedeillier, C. Vo-Van, M. A. Voelckel;
Département de Génétique Médicale, Hopital des Enfants de la Timone, Marseille, FRANCE. 


Angelman syndrome (AS) is a severe neurodevelopmental disorder which results from deficiencies of the maternal ubiquitin protein ligase 3A (UBE3A) gene caused by heterogeneous genetic alterations. This gene remains the only gene in the 15q11-q12 region found to play a role in the pathogenesis of AS. All UBE3A mutations reported so far were randomly distributed over the 2.6-kb major coding region including exons 8 to 16. The detection rates are around 30 % in non deletion/ non UPD/ non imprinting defect index cases, with an incidence of UBE3A point mutations in total AS patients estimated around 2-10 %. In this study, we investigated 33 patients from 25 families with a definite clinical diagnosis of AS and we evaluated 4 methods to establish a mutation screening strategy for the UBE3A gene. Automated SSCP analysis was used to screen the UBE3A gene for point mutations in all patients, in combination with direct sequencing if mutation could not be detected. Since the majority of UBE3A identified mutations are truncating mutations, we developed the protein truncation test (PTT) as a possible alternative approach to rapidly detect such inactivating alterations in this large gene. The combination of the different techniques allowed us to identify 22 point mutations from 25 families (88%)which represents the highest percentage of mutation reported so far in AS.



CREBBP mutations in 10 cases of Rubinstein-Taybi syndrome, including a mild variant showing a missense mutation 

O. Bartsch 1, P. Meinecke 2, K. Locher 3, W. Kreß 4, M. Pilz 1, E. Seemanová 5, K. Ostermann 3, G. Rödel 3;
1Institut für Klinische Genetik, Technische Universität Dresden, Dresden, GERMANY, 2Abteilung Medizinische Genetik, Altonaer Kinderkrankenhaus, Hamburg, GERMANY, 3Institut für Genetik, Technische Universität Dresden, Dresden, GERMANY, 4Institut für Humangenetik der Universität, Würzburg, GERMANY, 5Department of Clinical Genetics, Motol, Charles University, Prague, CZECH REPUBLIC. 


Rubinstein-Taybi syndrome (RTS) is a well-defined autosomal dominant disorder of broad thumbs, broad halluces, short stature, microcephaly, facial anomalies and mental retardation. RTS can result from mutations of the gene for CREBBP. This nuclear protein controls chromatin structure (DNA accessibility) by acetylation of histones, and therefore assists in the initiation of DNA transcription. We report on 10 unrelated subjects, nine with RTS and one with a phenotype of possibly very mild RTS. Mutation analysis was performed using FISH and genomic sequencing. We identified five novel mutations (86del148nt, 1108C-T, IVS4+1G-A, IVS7+1G-A, 3524A-G), two gross deletions of the CREBBP gene and two single-nucleotide coding polymorphisms. The 1108C-T stop mutation was observed twice suggesting a mutational hotspot. The proband with the mild variant was very interesting. She presented a missense mutation (3524A-G) predicting a tyrosine-to-cysteine exchange (Y1175C) within a fully conserved (man vs. mouse) 79-aa segment of the protein domain that elsewhere was shown to confer the histone acetyltransferase activity. There has been one previous report of a CREBBP missense mutation in RTS (Murata et al., Hum Mol Genet 2001;10:1071-6). The "digito-facial" phenotype of this proband with clear-cut digital anomalies, subtle but typical facial changes, normal stature, normal head circumference and low but normal intelligence represents a mild RTS variant ("incomplete RTS") that provides insight into phenotypic variation with RTS.



Molecular analysis of the CREBBP gene in 65 patients with Rubinstein-Taybi Syndrome. 

B. Arveiler 1, I. Coupry 1, C. Roudaut 1, M. Stef 1, M. Delrue 2, M. Marche 1, I. Burgelin 1, L. Taine 2, D. Lacombe 2;
1Université Victor Segalen Bordeaux 2, Bordeaux, FRANCE, 2CHU de Bordeaux, Bordeaux, FRANCE. 


The Rubinstein-Taybi syndrome (RTS) is characterized by mental and growth retardation, broad thumbs, broad big toes and facial abnormalities. RTS is associated with mutations in the CREB-Binding protein (CREBBP) gene. Gross chromosomal rearrangements and microdeletions detected by fluorescence in situ hybridization and truncating mutations revealed by protein truncation test or Western blot analysis, account for only 20% of RTS cases. We report the use of molecular tools to thoroughly analyse the CREBBP gene in a cohort of 65 patients. These include cDNA probes to search for gross rearrangements by Southern blot analysis and to identify mRNA of abnormal size by Northern blot, intragenic microsatellite markers to look for intragenic deletions, as well as a complete set of primers to amplify each of the 31 exons of the gene for mutation search by direct sequencing. We analysed 62 patients and identified 29 mutations : 3 gross rearrangements by Southern blot and/or microsatellite analysis, 1 truncated RNA by Northern blot, 1 small intragenic deletion by RT-PCR and 24 point mutations resulting in either stop codons, aminoacid substitutions or abnormal splicing of the CREBBP RNA. Three additional patients were found to be deleted by FISH. Taken together, these results showed that the combination of the various techniques allowed us to identify a CREBBP mutation in 49.2% of RTS cases, which represents the highest percentage of CREBBP mutations reported sofar in RTS patients. These molecular tools will be useful to search for CREBBP mutations in other developmental pathologies with cancer predisposition and mental retardation.