Posters April 6-7, 2001 Barcelona, Spain

Textos cedidos por la: Fundació Catalana Síndorme de Down, Katy Trias Trueta, Directora General c/ València, 229, pral. 1ª, 08007 Barcelona, España +34 93 215 74 23. Fax: +34 93 215 76 99 E-mail: integra@fcsd.org

Jürgen Groet¹, Lan Chen¹, Karin Blechschmidt², André Rosenthal², Marie-Laure Yaspo³ and Dean Nizetic¹

1Centre for applied Molecular Biology, School of Pharmacy, University of London, 29/39 Brunswick Square, London, WC1N 1AX, UK; ²Department of Genome Analysis, Beutenbergstraße 11, D 07745, Jena, Germany; ³Max-Planck-Institute for Molecular Genetics, Ihnestraße 73, 14195 Berlin, Germany.

A number of disease-related genetic changes have been associated with the 21q11 region. These include allelic loss in non-small cell lung carcinoma, linkage to familial early onset Alzheimer's disease and acute non-lymphocytic leukaemia of the M7 immunotype and transient abnormal myelopoiesis (TAM) in Down syndrome (DS) infants. We report the identification of a fourth novel full-length gene which is localised within this region. The gene codes for a protein with unknown function, which contains two putative nuclear localisation signals, a predicted sterile alpha motif (SAM) and a possible Src homology 3 (SH3) domain, and it was named SAMSN1. A mouse homologue is also described. A principal interacting protein for SAMSN1 in human brain has been discovered using Yeast-Two-Hybrid approach. The interaction has been so far re-confirmed by one-to-one yeast-two-hybrid approach. Expression and co-immunoprecipitation studies are in progress, as well as the sequence analysis in DS cases with TAM and leukaemia.

INTERSECTIN 1 SH3 DOMAINS: ALTERNATIVE SPLICING EVENTS REGULATE PROTEIN FUNCTION

Carles Pucharcos, Maria L. Arbones, Xavier Estivill and Susana de la Luna

Down syndrome Research Group, Medical and Molecular Genetics Center, IRO, Hospital Duran i Reynals, Avia. de Castelldefels Km 2.7, L'Hospitalet de Llobregat, 08907-Barcelona, Spain

Human Intersectin 1 (ITSN1) is a gene mapping on human chromosome 21 that codifies for a multimodular protein composed by two EH (Eps15-homology) domains, a central coiled-coil region and five consecutive SH3 domains. Moreover, it exists as a brain specific isoform that contains an extended carboxy-end region composed by a DH (Dbl-homology), a PH (Pleckstrin-homology) and a C2 domain. This isoform is overexpressed in Down syndrome fetal brain. Intersectin 1 interacts, through its SH3 domains, with dynamin and synaptojanin, two proteins involved in clathrin-mediated endocytosis, and with mSos, a protein implicated in signal transduction pathways.

We have identified several splicing events affecting intersectin 1 domain composition. Thus, skipping of exons 24 and 25 results in a protein lacking the SH3C domain. Both, intersectin 1 long and short isoforms, may exclude this protein domain. We have studied the tissue distribution of these variants and showed that they are present in all the tissues tested. On the contrary, exon 19, which affects the SH3A domain disrupting its structure when included, is present exclusively in adult and foetal brain and only appears in intersectin 1 long isoform transcripts. The deletion of the SH3 domains of intersectin 1 promotes the nuclear accumulation of the truncated protein, suggesting that these domains may be involved in cytosolic retention and therefore, contribute to the proper protein localization.

Supported by: European Union (BIOMED2 BMH4-CT98-3039), the Spanish CICYT (SAF99-0092-CO2-01), and the Fundació Catalana Síndrome de Down/Marató de TV3-1993

MODULATION OF CATECHOL -O- METHYLTRANSFERASE (COMT) PHENOTYPE BY OXIDATIVE STRESS STATUS IN DOWN'S SYNDROME

L. Breitenfeld¹, C. Monteiro², A. Varela², M.J. Laires² e M. Bicho¹

1-Laboratório de Genética da Faculdade de Medicina de Lisboa, PORTUGAL. 2- Laboratório de Bioquímica, FMH, Universidade Técnica, Lisboa, PORTUGAL.

Catechol -O- Methyltransferase gene whose locus is positioned on chromosome 22q11.2, expresses am enzyme (COMT; EC 2.1.1.6) that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the m- or p- hydroxy group of a catechol substrate (catecholamines, catechol steroids, α-methyldopa L-DOPA) therefore protecting cells from redox cycle toxic metabolites. Three-to fourfold differences in human COMT activities are atributed to codon 158 polymorphism that encode either a valine 8GTG) that produces an enzyme with higher activity or a methionine (ATG) that produces a markedly lower activity variant. It has been described variations in COMT activity in patients with Down's Syndrome, an inborn pathology coursing with an increased adenosine deaminase activity, an increased CuZuSOD (superoxide dismutase) production and with a decreased dopamine-β-hydroxilase activity. The main objective of our study was to understand the influence of the oxidative stress status in Down's syndrome on COMT activity. To achieve our purpose we determined COMT activity in 18 young adults with Down's syndrome (mean age 21.3 ± 2.8 years) and in 80 healthy adults (59,2 ± 9,94 years) by HPLC-EC methods, and determined by spectrofotometric method the epinephrine oxidation plasma capacity as an oxidative stress marker. "In vitro" studies in heathy volunteer erythrocytes were also performed to understand the response of COMT activity towards differents concentrations of SOD.

Results revealed a significantly decreased of COMT activity (p<0.05) in Down's syndrome and a decreased of the ratio between m- and p- hydroxy derivatives. The epinephrine oxidation plasma capacity is significantly increased (p<0.001) at the same individuals. "In vitro" results (higher p-derivative with higher SOD and Cu2+) levels allowed us to conclude that exists a Modulation of Catechol-O-Methyltransferase (COMT) phenotype by oxidative stress status in Down's syndrome.

THYROID FUNCTION IN A DOWN'S SYNDROME ADULT POPULATION

Benavent R, Beltrán M, Puertos D, Martí E, Plaza A, Rosado I, García R y Fernández-Delgado R. Down's Syndrome Unit. Department of Pediatrics. Hospital Clínico Universitario. University of Valencia.

Departamento de Pediatría. Hospital Clínico Universitario. Avda. Blasco Ibáñez 17. 46010. Valencia. ESPAÑA.

Hypothyroidism is a prevalent problem in persons Down's syndrome. In the setting of a Down's Syndrome medical multidisciplinary group, we have analysed thyroid function (Free T4 and TSH) of 142 adult persons (76 males and 66 females) in the last 8 years.

Thyroid dysfunction was defined, 1) Subclinical Hypothyroidism: TSH > 8

m U/ml and FT4 between normal limits; 2) True Hypothyroidism: FT4 < 0.8 ng/ml; 3) Hyperthyroidism: FT4> 1.7ng/ml.

Results: Males Females Total

- Euthyroydism: 41 28 69 (48.5%)

- Subclinical Hyphothyroidism: 25 28 53 (37.5%)

- True Hypothyroidism: 7 6 13 (9.1%)

- Hyperthyroidism: 3 4 7 (4.9%)

Conclusion: These data shows, once again, the high prevalence of thyroid dysfunction, either hyper- or hypo- in Down's Syndrome persons.

BASIC STUDY OF CELLULAR AND HUMORAL IMMUNITY IN CHILDREN WITH DOWN'S SYNDROME.

Plaza A, Martí E, Puertos D, Rosado I, García R, Vázquez P, Benavent R, Garnelo A and Fernández-Delgado R. Down's Syndrome Unit Department of Pediatrics. Hospital Clínico Universitario de Valencia.

Departamento de Pediatría. Hospital Clínico Universitario. Avenida Blasco Ibáñez 17. 46010. Valencia

Infections are especially frequent in children with Down's syndrome. Until now, the specific defect of immunity has not been recognised. To study this problem, we have examined 155 children with Down's syndrome who were followed-up in the setting of Down's Syndrome Unit of Pediatrics Department of Hospital Clínico Universitario de Valencia. CD4-T cell number and CD4/CD8 ratio, IgG, IgA, IgM and IgE levels, neutrophil count, and complement proteins (C3 and C4) and activity (CH50) were examined. Children with serious infectious conditions (suspects of immunodeficiency) and extreme values were excluded. The mean values of each group were:

1-2 yr 2-3yr 3-5yr 6-8yr 9-11yr 12-16yr

CD4-T cells Down's synd. 994 936 202 354 727 616

(cells/10³/l)

± 523 689 323 479 351 298

Control 1880 1880 1510 1080 1080 1130

CD4/CD8 ratio Down's synd. 1.2 1.31 0.77 0.74 0.91 0.88

Control 1.89 1.89 1.22 1.2 1.2 1.3

IgG level (mg/dl) Down's synd. 746 947 1074 1241 1363 1381

Control 640 700 990 990 990 990

IgM level (mg/dl) Down's synd. 67.6 79.1 62.8 64.9 66.4 71.6

Control 130 130 130 120 110 120

IgA level (mg/dl) Down's synd. 62.7 88.8 116.5 160.5 210.3 257.2

Control 70 80 100 130 140 190

IgE level (UI/ml) Down's synd. 8.6 7.0 62.2 80.7 36.5 29.5

Control 8 8 8 15 18 26 (median values)

Reference values from Hinchliffe RF in Pediatric Hematology. Lilleyman J, Hanh I and Blanchette V. (eds.)Churrchill Livingstone 1999. London

HYPOTHYROIDISM IN DOWN'S SYNDROME

Puertos D, Martí E, Plaza A, Rosado I, Torres C, Beltrán M, López MJ, Alpera R y Fernández-Delgado R.

Down's Syndrome Unit and Endocrinology Unit. Department of Pediatrics. Hospital Clínico Universitario. University of Valencia.

Departamento de Pediatría. Hospital Clínico Universitario. Avda. Blasco Ibáñez 17. 46010. Valencia. ESPAÑA.

Thyroid dysfunction is a prevalent problem in persons Down's syndrome. In the last 8 years, we have seen 550 individuals with Down's syndrome, in the setting of a Down's Syndrome medical multidisciplinary group.

A longitudinal analysis has been made of the first 283 children whose thyroid hormones were determined. For some of them, antithyroid antibodies were determined and celiac disease screening was also performed. Thyroid dysfunction was defined, 1) Subclinical Hypothyroidism: TSH > 8

m U/ml and FT4 between normal limits; 2) True Hypothyroidism: FT4 < 0.8 ng/ml. Subclinical hypothyroidism was found in 84 (29.6%). True hypothyroidism was found in 7 (2.4%). Family history was not found in any case. Some of the children who showed subclinical hypothyroidism became normal spontaneously in the subsequent determinations. These data shows that the decision of treating transient hypothyroidism needs further discussion. In borderline cases, without symptoms, we propose waiting until the following determination.

HEALTH IN TRISOMY 21 : EXPERIENCE OF FOLLOW UP OF 1400 PATIENTS IN THE JÉRÔME LEJEUNE MEDICAL CENTER-PARIS

Mircher C., Conté M., Ravel A., Bléhaut H.,Rethoré M.O.

The Jerôme Lejeune Medical Center was created in 1997, and is a referent center for clinical follow up, research, and vocational training concerning people with intellectual disability.Among these patients, 75% are trisomic for 21 chromosome: about 1400 trisomic patients (newborn to 60 year old) are coming regularly in our center for clinical evaluation, psychological testing and follow up, and participation in research protocols (epidemiological, therapeutic, and basic research). Some medicals disorders (congenital or appearing during life in cardiology , orthopedics, etc...) are more frequents in this population, and are important to diagnose, in order to prevent more disability in this population. We present here medical condition of our population, and the distribution of complications according to patient's age.

CONGENITAL HEARTH DEFECTS IN NEWBORNS WITH DOWN SYNDROME IN MARACAIBO-VENEZUELA. PRELIMINARY STUDY.

Cedeño-Rincón Rosa, Vegas América, Vergara-Márquez Gloria, Caraballo Adela and Romero Rosalinda.

Registro de Malformaciones Congénitas. Unidad de Genética Médica. Facultad de Medicina. La Universidad del Zulia (LUZ). Maracaibo-Venezuela.

The genetic and epidemiology of Down Syndrome (DS) was studied at Maracaibo, area which is covered by the Registry of Congenital Malformations at Castillo Plaza Maternity (MCP) through the Program of Down Syndrome of Survival (investigation supported by CONDES). For the period of January 2000 - January 2001 23 new cases of DS were detected, obtaining a frecuency of 82.6% of DS for the MCP and 17.4% for the others institutions. The incidence of MCP was 1.88 x 1000 liveborn, 100% of the cases were live birhts and for the first period of study (1^st. Year) 21.73% died. Kariotipes were found in 34.78% of children out of 100% numerical trisomics. 20 newborns were evaluated from the cardiovascular examination and 17 (85%) of them were detected with congenital heart defects and distributed in the following ways: 41.66% had atrial septal defects, 26.92% persistent ductus arteriosus, 12.5% had atriovascular defects, 8.23% ventricular septal defects and 8.23% vasculars defects. The results suggest that approximately cardiac anomalies are observed in 85% of the children with DS in Maracaibo who required medical attention that guarantees a better quality of living.

A 23-YEAR-OLD WOMAN WITH BOTH DOWN SYNDROME AND TYPE I NEUROFIBROMATOSIS AND BREAST CARCINOMA.

D. Satgé ¹, A.J. Sasco ², M.J. Vekemans ³, M-O. Réthoré ⁴.

1) D. Satgé, Laboratoire d'Anatomie Pathologique, Centre Hospitalier Tulle, France. 2) A.J. Sasco, International Agency for Research on Cancer, Lyon, France. 3) M-O. Réthoré, Centre Médical Jérome Lejeune, Paris, France. 4) M.J. Vekemans, Laboratoire de Cytogénétique, Hopital Necker, Paris, France.

A 23-year-old female patient with Down syndrome (DS) and type I neurofibromatosis (NF1) presented with an in situ adenocarcinoma of her right breast. No history of BC was noted in her family. Although BC is the most frequent neoplasm in woman in the general population, a review of the literature found only 5 cases in patients with DS. Futhermore two epidemiological studies conducted in the USA and in Denmark showed a decreased incidence of BC in DS. Here we suggest that in the present case the oncogenic action of NF1 could not be modified by trisomy 21.

A better knowleddge of the mechanism(s) protecting patients with Down syndrome against BC could contribute to understand the pathogenesis of the phenotype of DS and the oncogenesis of breast carcinoma.

A grant from the Fondation Jérôme Lejeune supported this study.

DOWN SYNDROME AND NEURAL TUBE DEFECT ASSOCIATED WITH ABNORMAL FOLATE METABOLISM AND GENETIC POLYMORPHISMS IN THE FOLATE PATHWAY

1Al-Gazali LI, ²Padmanaban R, ³Melnyk S, ³Yi P, ³Pogribny, IP,³ Pogribna M, ⁴Bakir M,⁴Hamid ZA,¹Abdulrazzaq Y, ¹Dawodu A, and ³James SJ

1Departments of Paediatrics, ²Anatomy, FMHS, UAE University, ³National Center for Toxicological Research, Division of Biochemical Toxicology, Jefferson, USA, ⁴Department of Paediatrics, Al Ain Hospital, Al Ain UAE.

We report here the case of a male child with trisomy 21, cervical meningomyelocele, agenesis of corpus callosum, hydrocephaly, cerebellar herniation into the foramen magnum, and shallow posterior cranial fossa. Molecular analysis of the MTHFR gene revealed homozygosity for the mutant 677C

® T polymorphism in both the mother and child. The plasma homocysteine of the mother was highly elevated and was associated with a low methionine level. Her SAH level was three times that of reference normal women resulting in a markedly reduced ratio of SAM to SAH and significant DNA hypomethylation in lymphocytes. The child had low plasma levels of both homocysteine and methionine and a reduced SAM/SAH ratio that was also associated with DNA hypomethylation. We suggest that a low folate status plus homozygous mutation in the MTHFR gene in the mother could promote chromosomal instability and meiotic non-disjunction resulting in trisomy 21. Low folate status and homozygous TT mutation in the MTHFR gene in both mother and child would be expected to increase the risk of neural tube defects. The presence of both trisomy 21 and postclosure NTD in the same child strongly supports the need for an extended periconceptional period of maternal folate supplementation to achieve greater preventive effects for both NTD and trisomy 21.

IDENTIFICATON AND CHARACTERIZATON OF GENES IN THE DOWN SYNDROME

CRITICAL REGION 2 .

Verónica Ramos de Galván, Jose M. Vidal-Taboada, Rafael Oliva.

Human Genetic Research Group, institut de Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), faculty of medicine. University of Barcelona, Casanova 143,08036 Barcelona Spain.

The characterisation of the genes located within the Down critical region 2 (DCR2) from the chromosome 21 is an essential step towards the understanding of the mechanisms involved in the DS features associated to this region. As part of a physical and transcription effort in order to characterise the region between the markers EST2 and HMG14, we have constructed high resolution maps and have located and characterised several genes in this region such as the SH3BGR gene, the WRB gene, and the DSCR2 gene. Thanks to the availability of the genomic sequence, we are presently characterising additional genes in this DCR 2 region through the determination of the intron / exon boundaries involving RT-PCR, EST characterisation, sequencing experiments, and comparison of the cDNA sequences to the genomic sequence.

Work supported by Fundació Catalana Síndrome de Down, Marató de TV3 -08 to R.O.

IDENTIFICATION OF DS DIFFERENTIALLY EXPRESSED GENES BY SSH.

Sánchez-Font M:F; González-Angulo O; Gonzàlez-Duarte R & Marfany G.

Dept. Genètica, Facultat de Biologia, Universidad de Barcelona.

Avda. Diagonal 645, 08028, Barcelona, SPAIN

In complex diseases, as the Down Syndrome, it is difficult to identify the genes responsible for the basic phenotypic traits because the cummulative effect of the chromosome 21 genes may also affect the expression of genes located on other chromosomes. Therefore, the analysis of differential gene expression in Down vs control individuals can be regarded as a complementary approach to the cloning and characterization of individual chrom. 21 genes.

Mental retardation is a common phenotypic trait in all DS patients. We have focussed on the analysis of differential expression in DS vs control foetal brains through suppression substractive hybridization (SSH). The overexpression of one gene located on 6q22-23 in Down brain samples and its effects on in vitro cultured cells will be reported.

VII RAMON TRIAS FARGAS AWARD

MOLECULAR-CYTOGENETIC STUDY OF DOWN SYNDROME: GENOTYPE-PHENOTYPE CORRELATIONS IN CASES OF PARTIAL TRISOMY OF CHROMOSOME 21

Marga Nadal

Down Syndrome Research Group

Centre de Genètica Mèdica i Molecular-IRO

Down syndrome (DS) patients with partial trisomy of chromosome 21 have historically been studied to establish correlations between the genotype and the phenotype. Thus, cytogenetic, molecular and clinical characterisation of these patients is essential. Given the high phenotypic heterogeneity and the lack of penetrance of most of the features, to make reliable comparisons, it is crucial to define the breakpoints and the clinical features of each patient. We have identified six patients with DS due to partial trisomy of chromosome 21, four of them involving only this chromosome (making the correlations more reliable because there is no other chromosomal rearrangement that may affect the phenotype). Two of the patients present a translocation involving a partial monosomy of chromosome 13 and 15 respectively; two other patients present a direct duplication from marker D21S302 to the telomere; another patient presents a translocation between the short and the long arm of chromosome 21; and finally, we have identified a patient with an interstitial duplication of YAC 876d4 which contains the gene GART. The breakpoints were characterised by FISH using a contig of YACs from 21q previously assessed for chimerism. The clinical evaluation was performed in each case by the clinical geneticists of the referring hospitals, all according to the protocol of Epstein (1991). The six patients define four different segmental trisomies: from D21S302 to the telomere, from D21S226 to telomere, from CBR to the telomere and finally, one case only encompasses the region from D21S216 to D21S323. For the traits not fully penetrant, the clinical comparisons are based only in the presence of the trait. Our results indicate that most of the clinical alterations seen in DS map to the distal third of the long arm of chromosome 21 as it has already been postulated in the literature. Comparing our results with the published ones, we may conclude that: 1/ the duodenal stenosis may be restricted from D21S59 to D21S302, 2/ the furrowed tongue maps from the centromere to D21S302, and 3/ hypotonia may be excluded from the region encompassed from D21S216 to D21S323. In addition, since APP is always excluded from the trisomies studied and two of the patients are older than 30 years and do not show any sign of Alzheimer's type dementia, it may be inferred that trisomy of APP is necessary to develop Alzheimer disease in DS. The characterisation of the partial trisomies has enabled the genetic counselling to the families of the patients, specially, when the chromosomal rearrangement was of familial origin.