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II International Conference on Chromosome 21 and Medical Research on Down Syndrome

Official Presentations
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

David Patterson
Eleanor Roosevelt Cancer Research Institute, Denver, CO 80206

In May of 2000, the entire genetic code of chromosome 21 was determined. This work reported that there are less than 230 genes on the chromosome. Ultimately, it is the presence of the extra copy of these genes in individuals with Down syndrome that leads to the challenges that they face. This is an unexpectedly small number of genes and offers hope that understanding the role of the genes in Down syndrome may be less difficult than expected. If we can identify the specific genes on chromosome 21 that are associated with specific aspects or features of Down syndrome, this should aid immeasurably in developing early intervention programs, assistive technologies, and therapies to ameliorate the challenges faced by individuals with Down syndrome.
Before the existence of this information, researchers had to attempt to pick specific genes known to be on chromosome 21 and to relate these one at a time to the features of Down syndrome. While this is still a viable approach, the availability of the gene catalogue of chromosome 21 enables other, more systematic, approaches. Now, one can use a variety of tools to determine experimentally which genes are most likely to be involved without any prior bias. This can be done in several ways, all of which are likely to be pursued. For example, one can determine which of the genes on chromosome 21 actually is expressed differently in individuals with and without Down syndrome. This can be done using microarray technology in which expression of each gene on chromosome 21 is measured. Similarly, one can determine alterations in expression of each chromosome 21 gene using other approaches, for example, quantitative PCR techniques to measure the levels of the mRNA's for each gene. These approaches levels of mRNA's for each gene. These approaches measure levels of mRNA, not the levels of protein expression

William I Cohen MD,
Director, Down Syndrome Center of Western PA, Children's Hospital of Pittsburgh; Associate Professor of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh PA USA

Down syndrome (DS) has a well-known association with a variety of medical conditions, secondary to the presence of a third copy of Chr 21. The early detection and treatment of these conditions has led to a significant improvement in the quality of life of individuals with DS, in addition to a decreasing both morbidity and mortality. Since first proposed by Dr Mary Coleman and published as the "Preventive Medical Check List" in 1981, these recommendations have been revised an updated periodically. Many different national and international groups have published their own sets of guidelines, reflecting local needs and customs. This presentation will discuss the areas of medical vulnerability associated with DS and discuss the process of development of the guidelines by the Down Syndrome Medical Interest Group (US). DSMIG recognizes that the resources available in different areas are dramatically different. Nevertheless, each country must bear in mind the need for using its resources wisely to identify and ameliorate the serious, life-threatening medical problems associated with DS. [Note: the 1999 revision of the Health Care Guidelines is available on the World Wide Web. The original version, in English, on the Down Syndrome Quarterly page at http://www.ndsccenter.org/resources/healthcare.pdf and in Spanish at http://empresas.mundivia.es/downcan/Programa_Salud.html

Jean M. Delabar
CNRS UMR 8602, Faculté Necke-Enfants Malades, Paris.

Despite a vast amount of investigation of the pathology, biochemistry, and physiology of Down syndrome it is still not known how the individual genes on HSA21, either singly or in concert, produce the anatomical and functional alterations associated with the trisomic state. However, what is known is that the phenotypic features that define the syndrome are attributable to the genes on HSA21 and are not the result of a non specific effect of aneuploidy. The ultimate goal of constructing a phenotypic map is to define molecularly the chromosomal region and ultimately the genes that are responsible for particular phenotypic features. Due to the variability of the phenotypic expression of a given trait among patients with full trisomy 21, the molecular data from many individuals must be combined to define small regions, optimally less than 2-3 Mb, that are suitable for molecular analysis. Such a study has been performed on ten patients with partial trisomy 21 . After gene dosage characterization of the extent of the duplications, a genotype-phenotype correlation study allowed to define regions duplications of which might be associated with given features : a region of 2.3 Mb mapping in 21q22.2 (DCR-1) should contain genes involved in 13 features including facial and hand features, joint hyperlaxity and mental retardation. This association is also observed for a set of data obtained on 50 patients and collected in different laboratories (ANEU21 database). Some of the genes from this region (DCR-1) present a strong expression restricted to specific areas of the central nervous system and might be considered as good candidate genes for some features. A second approach to relate specific components of the trisomic phenotype to the increased expression of genes is to construct mouse models with segmental trisomies or overexpressing specific genes. First studies on these models seem to indicate that it will be possible to identify genes responsible for many components of the phenotype.

Michael B. Petersen
Dept. of Genetics, Institute of Child Health, Athens, Greece.

Chromosomal aneuploidy is a fundamental characteristic of the human species. Our knowledge about the mechanisms underlying chromosomal nondisjunction in man is, however, surprisingly poor. During the last decade new insight into the origin and mechanisms of nondisjunction in human trisomy 21 has been gained by the use of DNA polymorphism analysis. The first molecular correlate of nondisjunction in humans is altered recombination, meiosis I errors being associated with reduced recombination and maternal meiosis II errors with increased recombination between the nondisjoined chromosomes 21. As recombination takes place in meiosis I, virtually all maternal meiotic errors seem to be initiated in meiosis I, which profoundly affect our undersfanding of the etiology of trisomy 21. Advanced maternal age remains the only well documented risk factor for maternal meiotic nondisjuction, but several factors have been suggested in a few recent population-based studies, where the origin of nondisjunction was determined by DNA analysis. These factors include apolipoprotein E and presenilin-1 gene polymorphisms, maternal cigarette smoking and oral contraceptive use, all in association with young mothers with meiosis II errors. Another recent study, which was not population-based and without determination of the origin of nondisjunction, suggested abnormal folate metabolism in Down syndrome mothers. The proposed risk factors might support the same hypothesis of compromised microcirculation in the ovary but need to be confirmed from other studies.

Mercè Armelles
Direcció General de Salut Pública. Departament de Sanitat i Seguretat Social. Generalitat de Catalunya. Barcelona. SPAIN

INTRODUCTION In Catalonia, infant mortality have gone down in the last years to a very low level. In 1998 the value was 4.1 infant deaths per 1000 livebirths. This fact have been possible because of the control of communicable diseases, the general administration of vaccines in childhood and the widespread of the each time more efficient antibiotics to the general population.
These facts have changed the main causes of infant mortality and if in the middle sixties infectious diseases were the first cause of infant mortality, now congenital anomalies are the second cause of mortality during the first year of life of a child, very close to perinatal affections that are the first cause.
From the Maternal and Child Health Programme of the Public Health General Direction of the Health Department of Catalonia we know about the growing interest of the general population and of the pregnant women about congenital anomalies and their consequences. This interest also affects to the future parents and the families with someone diagnosed of a disease labelled as a congenital anomaly.
The aetiology of congenital defects is unknown in more than 50% of cases and this is the case of Down's syndrome. This chromosomal anomaly characterized by a third chromosome in the 21 position gives mainly mental retardation and may have associated congenital hert disease.
This syndrome has no treatment and the life expectancy has increased around the world and is considered about 50 years.
In the year 1983 in the Health Department of Catalonia we have initiated a programme that includes prenatal diagnosis of congenital anomalies based on the criteria of maternal age. So in ages 35 and over, a diagnosis of chromosomal anomalies was offered to the pregnant women.
From then to now we have changed the guidelines of prenatal diagnosis of Down's syndrome and introduced the biochemical screening of this trisomy.
In order to evaluate the implementation of this public health programme and their acceptation in Catalonia we have done a systematic collection of all cases diagnosed of Down's syndrome between liveborn, deaths and terminations of pregnancy (TOP) with the aim of calculate the incidence and distribution of cases of this autosomal trisomy.
The data of 1987 is not a coincidence because this year is when in Spain was approved the law that allows pregnant women to finish their pregnancy in case of a confirmed diagnosis of a great foetal defect.
Epidemiological data about Down's syndrome is interesting to inform pregnants about their risk of having an affected child; families with members affected want to know the frequency of which is presented and also these data are interesting for health and educational planners of services to attend the needs of this population.
METHODS Diagnosed cases were obtained from 3 registries: liveborn cases from the hospital registry of the Catalan Health Service;TOP from the TOP registry of the Health Department and deaths from mortality registry of the Health Department.
All registries have an estimated coverage of 100% of the cases in the studied period (1992-1999).
Because of the abortion law dated from 1987, declaration of TOP by Down's syndrome are not exhaustive until the year 1992.
RESULTS Prevalence rate of Down's syndrome in Catalonia in the period studied is 1.2 x 1000 liveborns, increasing the rate from 1.1 in 1992 to 1.5 in 1999.
To evaluate the effect of the programme of prenatal diagnosis for Down's syndrome we have calculated the population rate of this trisomy in liveborns, observing a significant decreasing value in the period studied, from 7.7 per 10000 liveborn in 1992 to 3.5 per 10000 in 1999.
If we study the relationship between children born with Down's syndrome and TOP made by this diagnosis, we have that in 1992 it was a 38% of TOP, but now, in 1999 this percentage has increased to a 80% value.
DISCUSSION The rate of prevalence observed in Catalonia in the studied period is similar to the published by Martinez-Frias in Spain and other regions of Europe as Scotland.
The rate founded in Catalonia is higher than the rate of the city of Paris and lower of the values published for the city of Barcelona (Spain), El Valles county ( Spain) ; Saudi Arabia; Republic of South Africa and Israel.
So we think that our sources of information are good enough to make epidemiological studies and calculate population rates.
The decreasing value observed for children born with Down's syndrome is presumably due to the effect of the wide demand of pregnant women about prenatal diagnosis.
The distribution observed of our cases between liveborns and TOP suggest that women with a diagnosis of Down's syndrome choose the TOP in a very high percentage. This value is very different to those published by López for the city of Glasgow with a 19% of TOP described.
Joan Blanco, Ester Anton, Josep Egozcue and Francesca Vidal
Unitat de Biologia Cellular, Facultat de Ciències, Universitat Autònoma de Barcelona08193-Bellaterra (Cerdanyola del Vallès) Spain.
The controversy about the so-called interchromosomal effects (ICE) (Lejeune, 1963), that is, the increase in the number of gametes aneuploid for chromosomes not involved in the reorganization in carriers of structural reorganizations still remains open.
Several authors have performed epidemiological studies to detect the possible existence of ICE (Aurias et al. 1978; Mikkelsen 1971; Serra et al. 1990) and, although in some cases the results seemed to indicate a possible relationship between the presence of a balanced reorganization and trisomy 21, other epidemiological studies failed to confirm it (Schinzel et al. 1992). Likewise, several meiotic studies have shown that the presence of a structural reorganization can give rise to interchromosomal effects (Vidal et al. 1982; Saadallah and Hultén 1986). Nevertheless, the only valid method to determine the final result of any type of ICE is the chromosomal evaluation of the gametes produced.
FISH (fluorescent in situ hybridization) in decondensed sperm nuclei has been widely used to indirectly study the chromosome constitution of human spermatozoa, and it has proven to be a good method to analyze high numbers of spermatozoa, thus providing a statistically valid method to analyze the occurrence of a given interchromosomal effect.
Using this approach, we have analyzed the possible presence of ICE for chromosome 21 in three robertsonian translocation carriers, four reciprocal translocation carriers and one inversion carrier. A dual FISH methodology with a locus-specific probe for chromosome 21 (LSI 21, region 21q22.14-q22.3, Spectrum Orange, Vysis
Ò ) and a centromeric probe for chromosome 6 (CEP 6, Spectrum Green, Vysis
Ò ) were used for the study.
A highly significant increase (p<0.0001) of disomy 21 (1.90% vs. 0.37% in controls) that could be considered as a positive ICE was observed in one of the reciprocal translocation carriers analyzed (46,XY,t(3;15)(p25;q15); Blanco et al. 2000). All other carriers showed an incidence of 21 disomy within the limits described in controls. Detailed results of the FISH studies will be presented.
Data published so far by different groups are in good agreement with our own, with individual positive ICE (Rousseaux et al. 1995; Mercier et al. 1998) and negative ICE results (Van Hummelen et al. 1997; Martini et al. 1998; Cifuentes et al. 1999).
Theoretically, and according to the definition of ICE, this disparity could be the consequence of the severity of the meiotic anomalies induced by the reorganizations. The severity depends mainly of three factors: type of reorganization, chromosomes involved, and characteristics of the reorganized segments. For instance, in the carrier of the t(3;15), the length of the translocated segments of chromosome 3 are very short. Short segments give rise to the formation of asymmetrical quadrivalents at pachytene, which may produce chain configurations at metaphase I (Speed 1989) and, in consequence, to result in the presence of unpaired regions. It has been reported that the unsynapsed segments may associate to other bivalents, thus leading to synaptic anomalies (Vidal et al. 1987; Goldman and Hultén 1993) and increasing the rate of non disjunction of other chromosome pairs.
If we accept as a valid this argument, other types of meiotic anomalies could end in an increase of gamete aneuploidy. In fact, FISH studies in infertile patients, frequently associated with meiotic abnormalities (Vendrell et al. 1999), described a significant increase in the frequency of aneuploidy (Pang et al. 1999; Arán et al. 1999).
It seems that ICE could be one of the different factors related to an increased incidence of numerical chromosome anomalies in gametes. Other ways could have different origins (e.g. mutation in gene reparation DNA mechanisms). In any case, all situations would coincide in an intersection known as meiotic anomalies.
In conclusion, our results support the occurrence of ICE in particular cases of structural chromosome reorganizations, depending on the reorganization and on the chromosome and chromosome regions involved.
This work was supported by Fundació Catalana Síndrome de Down (Marató de TV3) and Fondo de Investigación Sanitaria project n° 96/1992-01.

M. Garcia, A. Ortega, I. Roig, I. Martínez, J. Egozcue
Dept Biologia Cellular, Fisiologia i Immunologia. Universitat Autònoma de Barcelona. SPAIN

For many years, one of the objectives of our research group has been to contribute to the understanding of chromosomal pairing and of the origin of aneuploidy in the human specie.
In this work we described the results from a preliminary study on the behaviour of chromosome 21 during meiotic prophase in human females with trisomy 21 and in controls.
Ovaries were obtained from three female fetuses prenatally diagnosed as 47,XX+21, as well as from three control female fetuses. All of them were collected after legal interruptions of pregnancy (LIP) in the second trimester of gestation. Chromosome preparations of germ cells were obtained by cytocentrifugation (Martinez-Flores et al, submitted) and were analysed by fluorescent "in situ" hybridisation (FISH) using a slight modification of the technique described by Cheng and Gartler (1994). The probes used were WCP 21, LSI 13/21 and WCP X (Vysis, U.S.A.)
In total, 7598 oocytes I were analysed. The hybridisation efficiency ranged from 67% to 82% for chromosome 21. And from 63% to 84% for the X chromosome. This efficiency does not significantly differ from the one obtained by other authors, which ranged from 66% to 95% (Cheng and Gartler, 1994; Cheng et al, 1995).
The fetuses with Down syndrome and the control fetuses, did not show significant differences in the proportions of the different stages of prophase I (leptotene, zygotene and pachytene). The slight differences observed in the proportions of leptotenes (29.83% in control fetuses and 34.5% in trisomic ones) and pachytenes (52.0% in control fetuses and 45.7% in trisomic ones) were related to the different mean age of gestation of the down fetuses (20.5 weeks) and the controls (22 weeks). This shows that the presence of an extra chromosome 21 does not visibly affect the proportion of the different meiotic stages, indicating that the presence of one extra chromosome does not result in any delay of prophase I in trisomic fetuses. In both trisomic and normal fetuses, we observed what could be described as a deficit of zygotenes (17.9% of control fetuses and 18.0% of trisomic fetuses) compared to other mammalian species. However, this is probably related to the difficulty of identifying stages of short duration in the absence of any marker chromosome, which can be identified in some other mammalian females. In control fetuses we analysed 975 pachytenes. The efficiency of homologous pairing for chromosome 21 was very high just under 100% (99.73%) in agreement with the figures (99%) obtained by (Cheng et al, 1998). Therefore, only 0.27% of the cells at pachytene had unpaired chromosomes 21 (univalents). These oocytes could well be in the origin of disomic or nullisomic secondary oocytes II for this chromosome pair. Furthermore, in controls fetuses 0.24% of oocytes analysed in leptotene had three hybridisation signals, suggesting the existence of possible premeiotic non-disjunctionary events.
In trisomic fetuses we analysed 910 pachytenes. A total of 61.8% of oocytes I analysed showed full pairing after synaptic adjustment of the three chromosomes (trivalent), 38.2% had two chromosomes paired and one unpaired (bivalent + univalent) and none of the oocytes had three signals corresponding to three univalents. This means that the pairing efficiency of the three chromosomes 21 to produce a bivalent or, after partner exchange, a trivalent is also very high, because of the absence of oocytes I at pachytene with three independent signals. The relatively high proportion of pachytenes with bivalent + univalent control also give rise to univalent loss, a well kwon phenomenon in other species which results in an increase of the proportion of normal oocytes produced by trisomic females.
Some of our data are clearly different from those obtained by Cheng et al (1998) who found 4.33% of oocytes analysed with three hybridisation signals. We think that the different number of oocytes studied (1993 us 6136) does not explain this difference because our study includes a high enough number of pachytenes.
We are grateful to Dr Ll. Cabero
from the "Servei de Ginecologia i Obstetrícia" from the Hospital de la Vall d'Hebron and to Dr S. Barambio
from the Clínica Tutor Médica for providing the specimens used in this study.
This work received financial support from FIS 96/1992-02 , 1999 SGR00104 and Fundación Salud 2000.

José Manuel Vidal-Taboada, Albert Lu, Maria Piqué, Gabriel Pons, Joan Gil, Verónica Ramos and Rafael Oliva
Laboratori Genoma Humà. Facultat de Medicina. Universitat de Barcelona. SPAIN

Two complementary approaches have been followed to characterise the function of the novel gene DSCR2 (Down Syndrome Critical Region gene 2): the isolation and characterisation of the mouse gene homologue to the human DSCR2 gene, and the analysis of the expression of the gene in different human cell lines. The mouse cDNA has a length of 1012 bp and has a high homology to the human DSCR2 gene. The predicted mouse dscr2 protein has an identity of 85.4% to the human protein, indicating that the DSCR2 protein has been conserved during the evolution. The dscr2 gene is expressed throughout all the stages of the mouse embryo development. In adult mouse tissues, the gene is expressed in testis, kidney, liver, brain, heart, skeletal muscle, and pancreas. The levels of the DSCR2 mRNA correlate with cellular growth of T98G and Jurkat cells in response to different treatments. The expression pattern throughout the foetal development together with the correlation observed with the cell cycle indicates a possible function for the DSCR2 gene related to cell proliferation.
Supported by Fundació Catalana Síndrome de Down and EU-BIOMED2 to RO.

Roser González
Departament de Genètica. Facultat de Biologia. Universitat de Barcelona

Deubiquitinating (UBP) enzymes are thought to play an essential role in protein degradation via the 26S-proteasome pathway. We have recently identified, USP25, a specific-ubiquitin protease gene spanning over 150 kb at 21q11.2, a genuinely genepoor region of the human genome. Characterization of the homologous murine gene, mUSP25, allowed to obtain probes for expression analysis in mouse. In situ hybridization assays on embryonic brains showed a clear correlation with proliferative neuroepithelial cells and postmiotic neurons. Moreover, a high level of expression was detected in the seminiferous tubules of adult mouse testis. In human, Northem analysis revealed two USP25 transcripts, of approximately 4.6 and 6.5 kb, in most adult and foetal tissues, except in testis, where the highest level of expression and only one transcript of 4.6 kb was observed.
Lately, data base homology searches and subsequent cDNA library screenings have allowed the identification of a new human UBP member, USP28, at 11q23. Genomic and protein sequence comparisons has suggested that USP 25 and USP28 constitute a new subfamily of UBP. Tissue-specific alternatively spliced products have been shown for both cytosolic enzymes.
Although the ubiquitin system is essential to all eukaryotic cells and several deubiquitinating enzymes have been shown to contribute to development and differentiation, the specific funtion of most family members remains unknown. In our case, USP25 overexpression in Down syndrome foetal brains with respect to control samples would agree with the dosage effects described for other UBP members involved in aneuploidy syndromes, USP9X (DFFRX) in Turner syndrome and USP18 in DiGiorge syndrome, and with the in vitro data showing that either overexpression or inhibition of UBP leads to programmed cell death.

M. Claude Potier, G. Golfier, N.Gibelin, J. Rossier, K. Toyama, P.M. Sinet
Neurobiologie, CNRS UMR7637, ESPCI, 10 rue Vauquelin 75005 Paris, France.
Faculté Necker-Enfants Malades, 156, rue de Vaugirard 75015 Paris, France.

DNA microarrays are used to monitor changes in gene expression in response to any physiological, pathological or pharmacological variations. Microarrays containing 200 genes involved in neurotransmission (precursors for neuropeptides, enzymes involved in the synthesis of neurotransmitters, calcium-binding proteins, subunits of receptors for neurotransmitters: glutamate, GABA, acetylcholine, serotonine, dopamine, adrenaline, somatostatine) were designed for mice, rat and human samples.
In collaboration with various groups, we studied gene expression in brains of mice in which genes involved in neurotransmission were knocked out (KO). Because the KO animals appeared phenotypically normal with very slight behavioral modifications, we used the neurotransmission microarrays for studying compensations in gene expression. Results from a serie of nine different KO will be presented. In the parvalbumin-calretinin and parvalbumin-calbindin KO mice several genes involved in GABAergic transmission are downregulated. Conversely in the KO for GABA-A receptor subunits calretinin and calbindin are downregulated. In addition we show on the GABA- 2 KO mice that expression variations vary with the age of the animals and do not appear as constitutive variations.
The DNA microarray technology has proven to be very useful in showing expression compensations on different transmission systems in KO mice.
Recently brain samples from trisomic 16 mice (Ts1Cje) were applied to the neurotransmission DNA microarrays. Results show that genes that are present in three copies are overexpressed, together with other unexpected genes mapping to other chromosomes than chromosome 16. ßßßßß

George T Capone
Kennedy Krieger Institute. Baltimore, Maryland, USA.

The past two decades have witnessed unprecedented growth and development in the neurosciences. Several adult neurologic and psychiatric disorders, once considered untreatable, have become the targets of active biomedical intervention in an attempt to reduce impairment and improve function. Historically, there has been less interest within the biomedical community for making childhood cognitive disorders a legitimate priority for neurobiologic intervention.
The prospect of pharmacologic treatment for cognitive impairment is frequently discussed and actively pursued by many families. Because these interventions are still in the development stage, recommendations for drug treatment are premature and cannot be routinely prescribed at this time.
Since 1995, when many parents first became interested in giving Piracetam to their young children with Down syndrome, it became obvious that no research data was available regarding either safety or efficacy in this population. Piracetam is classified as a nootropic agent with demonstrated benefits on learning and memory in rodents. Human studies have been considerably less convincing. We have conducted a small pilot study of Piracetam in 10 children with Down syndrome between the ages of 5-7 years.
Two other classes of medicine also offer the potential for cognitive enhancement in persons with Down Syndrome: anti-cholinesterases, which increase the level of the neurotransmitter acetylcholine in the brain; and anti-amyloid compounds, designed to prevent or reduce the deposition of amyloid plaques in the aging brain.
As new "cognitive medicines" are developed it is imperative that they be thoroughly and carefully tested to ensure safety and efficacy, especially in young children.

Mercè Boada, R. Novell

The study of ageing in people with intellectual disability is a recent subject of interest. Until a short time ago, living conditions for the majority of these people, irrespective of the gravity of their impairment, led them to premature death at an early age. There was little or no interest in their state of health, their adaptive competency and even less in changes made in accordance with the ageing process.
As with the general population, the ageing of people with mental deficiency is a consequent circumstance of a combination of better health care and improved living conditions that together increase life expectancy.
Life expectancy in people with Down's Syndrome has increased from less than 10 years of age at the beginning of the 20th century to 50 years of age now. In fact, around 20% of people with Down's Syndrome can now live longer than 55 years.
Age-related changes in people with Down's Syndrome prompt us to suggest that these people age prematurely. Age-linked changes that can be observed in the general population from 70 years of age can be observed in people with Down's Syndrome from the age of 30.
Among other evidence, which reinforces this fact, can be seen a greater risk of sensorial, visual and hearing impairment, thyroid disorders and Alzheimer like dementia at an earlier age in people with mental deficiency when compared those to other ethiologies.


Age-related changes in people with Down's Syndrome are becoming of greater interest, especially because these persons develop at an early age neuropathologic damage typical of Alzheimer's disease.
Neuropathologic studies have demonstrated that at the age of 30, several characteristics of Alzheimer's disease can be seen, such as amyloid defectation, senile plates, neurofibril balls, predominantly in the tonsils, the "hipocampo" and areas of cortical association in the frontal, temporal and parietal lobe.
Neuropathologic damage is of special significance if we consider it in conjunction with early ageing indexes, reduction in life expectancy in people with Down's Syndrome and evidence of the ever more outstanding relationship between Chromosome 21 genes and the developement of Alzheimer's disease.
Therefore, since deterioration in cognitive and behaviour functions is obvious, the anatomopathologic , neurophysiologic and neuroimaging findings suggest a great risk of developing on Alzheimer's disease from the 40's and 50's in a significant proportion of people with Down's Syndrome.

M.D. de la Calzada1, A. Queralt2, I. Casas3
Unitat del Son. Servei de Neurofisiologia Clínica. 1Hospital General y 2 Hospital Materno-Infantil Universitaris Vall d'Hebron. Barcelona. SPAIN

Sleep disorders have important impact on the quality of waking life for children and adolescents.
Patients with Down syndrome are a risk group for disturbed breathing during sleep due to tjeir structural characteristics affecting upper airway size.
Children with attention deficit disorders show sometime behavior disorders that can be related to sleep pathology.
The aim of the study is to know the existence of sleep disorders on patients diagnosed as having a Down syndrome and their repercussion over wake time.
Material and Method: Three pages screening questionnaire was administered to parents of Down syndrome patients and for comparison siblings of patients Down syndrome. The questionnaire included 14 items that focused on medical and structural characteristics, 12 items related to sleep hehavior and 7 items related to daytime behavior.
Both groups results were compared and showed significant statistical differences.

Jesus Carnicer
Servei de Pediatria. Hospital de la Santa Creu i Sant Pau de Barcelona. SPAIN

Celiac disease is an autoimmune condition characterized bt an immune mediated enteropathy of the small intestine. The condition occurs in genetically predisposed individuals who ingest gliadin and related proteins that are found in wheat, rye or barley. Celiac disease produces a spectrum of small intestinal mucosalchanges (increase in the number of intraepithelial lymphocites, flattening of the villi and crypt hyperplasia). Malabsortion of nutrients is a consequence of the intestinal damage, causing significant malnutrition when prolonged. Clinical symptoms of celiac disease differ considerably depending on the age at presentation. In children diagnosed whitin the first years of life intestinal symtoms (short stature, delayed puberty, anemia, enamel hypoplasia, osteopenia, etc). Celiac disease may also be clinically silent. Several serological tests are useful to identify individuals requiring intestinal biopsy for definitive diagnosis. The tests used measure antibodies to gliadin, reticulin, endomysium and tissue transglutaminase. Treatment of celiac disease requires lifelong adherence to a diet, complete recovery of the small intestinal damage can be expected with resolution of related symptoms.
The association between Down's syndrome and celiac disease, although occasionally observed in the past, has been reported with increasing frequency during the last decade. Prevalence rates of celiac disease in patients with Down's syndrome found in different studies range from 2.5 to 18.6%. These significantly higher prevalence rates of celiac disease in children and adolescents with Down's syndrome found by different authors indicate that the coexistence of these disorders is not fortuitous. it has been suggested that there are common pathogenetic factors in these diseases, such as the presence of common histocompatibility (HLA) antigens probably involved in the immune response. There is a high prevalence of immune-related disorders in patients with Down's syndrome. In patients with celiac disease, autoimmune disease are also reported. Common immunogenetic markers, particularly HLA antigens, have been discussed as a possible link for the association. The association between celiac disease and HLA-DQ2 haplotype (DQA1*0501 and B1*0201 alleles) has been clearly established.
In order to determine the real prevalence of celiac disease in Down's syndrome, a large series of patients with trisomy 21 of our geographical area from different settings (hospitals, special schools, occupational therapy centers) was studied. A total of 284 persons with Down's syndrome aged between 1 and 25 years were included in the study. In all cases, serum concentrations of antigliadin antibodies (AGAs) (Pharmacia CAP system ELISA), antiendomsysium antibodies (AEA) (indirectmmunofluorescence) of IgA class or of IgG class in cases of IgA deficiency were determined. In all patients, a clinical study was made to evaluate the presence and time-course of symptoms related to celiac disease. Jejunal biopsy was offered to all patients with AEA positivity and to those with suggestive clinical manifestations of celiac disease. In 18 of the 284 subjects WITH Down's syndrome, aged between 2 and 15 years, celiac disease was confirmed by jejunal biopsy. Accordingly, the minimum prevalence rate of celiac disease was of 6.3%. Ninety-four percent (17/18) and 78% (14/18) of patients with the association Down's syndrome and celiac disease showed AEA and AGA positivity, respectively. Fifteen patients with the association celiac disease and Down's syndrome (15/18) showed clinical manifestations compatible with celiac disease, with a predominance of intestinal symtoms (8/18) over those with atypical or extra-intestinal forms (7/18). Three patients had clinically silent forms of celiac disease (3/18). In conclusion, celiac disease should be included among the conditions related to Down's syndrome. Measurement of serum concentrations AEA (or autoantibodies against tissue transglutaminase) should be added to the list of screening tests for celiac disease in patients with Down's syndrome, otherwise definite association between both diseases may passed unnoticed and diagnosis of celiac disease considerably delayed.

Bonnie Patterson M.D.
Director Down Syndrome Clinic, Cincinnati Center for Developmental Disorders, Cincinnati, Ohio, USA

It is often thought that children with Down syndrome who also have characteristics of autism spectrum disorder are manifesting these patterns secondary to severe or profound mental retardation. We are beginning to understand that many of these children do meet diagnostic criteria for autism spectrum and the occurrence of the dual diagnosis is not rare. The Down Syndrome Clinic in Cincinnati reports an incidence of 6% diagnosed using the DSM-IV criteria for Pervasive Developmental Disorder/Autism.
It is important for the professional community to better understand children with this dual diagnosis in order to focus on effective medical and therapeutic interventions. Developing research protocols that combine information developed by scientists working in the fields of autism and Down syndrome research will be vital to our success in better understanding the complexity of this group of children. A recent study did this by evaluating prolonged EEG's in children with Down syndrome and autism spectrum disorder. The results were consistent with what is reported in the literature related to EEG findings in children with autism. Twenty percent of the children with dual diagnosis had epileptiform abnormalities on EEG's without a history of clinical seizures.
Early recognition and diagnosis of autism in children with Down syndrome has significant implications for both the family and the professionals working with the child. It is important to understand the needs of the child in regards to therapeutic, educational and medical evaluations and interventions. Continued research is needed in this area to explore environmental and familial risk factors, MRI findings, biochemical changes and the role of the extra chromosome and its genes. Research can help us gain a better understanding of Down syndrome and autism spectrum disorder which will reduce barriers to diagnosis and improve access to appropriate medical and educational interventions.

P. Kahlem, S. Schrinner, S. Henning, H. Lehrach and M-L. Yaspo
Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany.

The DNA sequence, clone map and gene catalogue of chromosome 21 opens new avenues for exploring gene function in the investigation of complex diseases such as Down syndrome (DS). Trisomy 21 represents a central issue in clinical genetics, affecting 1:1000 live births. Understanding how the presence of an extra chr. 21 leads to complex metabolic and developmental aberrations would provide a unique disease model whereby we could approach gene function from the perspective of gene-dosage effects. Systematic functional genomics strategies applied to the whole chr.21 gene collection will be essential to unravel gene function within a defined cellular context, as a key step towards molecular medicine applications. We are developing a comprehensive approach aimed at identifying pathological molecular profiles specific of trisomy 21 at the transcriptome and at the proteome levels. In parallel, functional analysis of the chr. 21 genes, that is isolating full-length cDNAs, expression patterns, cellular localization, protein structure and interactions, will be compulsory to the interpretation of the molecular phenotypic data. The chr. 21 gene catalogue provides the tool for initiating this integrated strategy that will serve as a basis for identifying candidate genes and molecular pathways involved in DS, hopefully shedding light for understanding the mechanisms underlying its pathogenesis.

X. Estivill, M. Dierssen, V. Fotaki, X. Altafaj, S. de la Luna, C. Baamonde, C. Martinez-Cué, M. Lumbreras, C. Casas, J. Guimerà, E. Martí, C. Fillat, J. Flórez and M.L. Arbonés
Medical and Molecular Genetics Center - IRO, Barcelona
Department of Physiology and Pharmacology, University of Cantabria, Santander

Down syndrome (DS) is a major cause of mental retardation, hypotonia and delayed development. The DYRK1A gene The DYRK1A gene, the human homologue of the Drosophila minibrain gene, is located on human chromosome 21 within the DS critical region, and is within the region minimally deleted in chromosome 21-linked microcephaly. Mutations in the Drosophila mnb (minibrain) gene cause marked size reduction of brain and perturbed motor behavior, along with cognitive defects. DS brains are smaller than normal with a decreased number of neurons in distinct regions. In the mouse brain DYRK1A is expressed in regions that are affected in DS. We have generated murine models with different levels of expression of Dyrk1A. 1) While Dyrk1A (-/-) mice are not viable, Dyrk1A (±) are viable to adulthood and fertile, but are smaller in size and weight than control littermates and present a marked reduction in brain size. Comprehensive evaluation of neurobehavioral development revealed a delay in the achievement of physical characteristics (eyelid and ear opening), in the emergence of adult-like responses in righting and auditory Preyer's reflex and poor performance in the homing test. Adult Dyrk1A (±) showed reduction in horizontal and vertical activity in the open field, without gross sensory-motor alterations. Dyrk1A (±) presented altered learning in the Morris water maze. 2) We have generated transgenic mice overexpressing specifically the full-length cDNA of Dyrk1A. Transgenic mice exhibit delay in neuromotor development with a persistence of immature locomotor patterns and retarded latency in walking activity. TgDyrk1A mice also showed an altered motor coordination during neurodevelopment, which was maintained in the adulthood. Hyperactivity was observed in the open field and in the plus maze. In the Morris water maze TgDyrk1A mice showed a significant increase in the average distance travelled in the first sessions of the acquisition phase. In the more complex paradigm of repeated reversal learning of the platform position, this defect turned out to be specifically related to reference memory, whereas working memory was almost unimpaired. These alterations are comparable with those detected in the Ts65Dn mice and clearly indicate that DYRK1A overexpression would be sufficient to cause some of the learning and behavioral deficits present in DS.
Supported by CEC/BIOMED2 GENE-CT96-0054, FIS 00/0795, SAF-99-0092, PM95-0106-C02, ERBFMBICT972278 (VF), Jerôme Lejeune Foundation and the Fundació Catalana Síndrome de Down.

J. Flórez1, C. Martínez-Cué1, C. Baamonde1, M.A. Lumbreras1, M. Dierssen2
1Laboratorio de Neurobiología del Desarrollo, Departamento de Fisiología y Farmacología, Universidad de Cantabria, Santander; 2Centro de Genética Médica y Molecular, IRO, L'Hospitalet de Llobregat (Barcelona), Spain.
The strategies used for modeling Down syndrome (DS) in mice have been: a) to assess single gene contributions to DS phenotype, using transgenic techniques to create models overexpressing single gene or a small number of genes; b) to assess the effects of overexpressing large foreign DNA pieces, introduced on yeast artificial chromosomes (YAC) or bacterial artificial chromosomes (BAC), into transgenic mice; c) to assess the effects of the transference of a human chromosome into mouse ES cells (transchromosomal mice); and d) mouse trisomies that carry all or part of MMU16, which has regions of conserved homology with HSA21. In spite of their limitations, the combined information obtained from these and other new strategies is establishing firm groundwork to understand the molecular pathogenesis of DS and, even more important, to evaluate the efficacy of therapeutic interventions.
Transference of a gene or of large pieces of DNA that may encompass several genes (YAC and BAC) allows for the study of the effects of overexpressing single or multiple genes on phenotypic expression. The trisomy models have the advantage that they overexpress the orthologues of multiple genes located on HSA21. Their power lies in modeling and characterizing the syndrome (or part of it) and in testing therapeutic strategies. Three viable models bearing an extra copy of a part of MMU16 have been produced: a) Ts65Dn mice contain three copies of MMU16 from App to Mx1; b) Ts1Cje mice have three copies from Sod1 to Mx1; and c) Ms1Ts65 from App to Sod1. Differences in cognitive abilities among the three models have been reported, suggesting an increased learning deficit as the size of the trisomic segment increases1.
In experimenting with animal models an effort should be made continuously to address issues that have relevant implications in the management of DS. There can be suspected features in DS that can only thoroughly tested and confirmed in the mouse model. In addition, findings in the animal model can lead to document them in humans. Some of these issues have been addressed by our research group in the Ts65Dn mouse, and three significant examples will be discussed.
Anecdotal reports by parents and professionals suggested that individuals with DS had reduced responsiveness to pain. Ts65Dn mice showed reduced responsiveness to pain induced by thermal (tail-flick) and chemical (formalin) stimuli2. Based on these findings, Hennequin et al.3 have demonstrated that persons with DS express pain or discomfort more slowly and less precisely than the general population.
Studies performed in Ts65Dn mice documented severe changes in the activity of important brain signaling pathways, including the adenylyl cyclase and phospholipase C4,5. Consequently, we have analyzed the functioning of these signaling pathways in the brain of adult persons with DS and found a severe and consistent depression of both basal and stimulated adenylyl cyclase activity in cortex and hippocampus6.
Finally, early intervention is known to have positive short-term effects in infants and toddlers with DS, although the long-term effects are more debatable. We have studied the effects of environmental enrichment on the behavior and learning abilities of post-weaning Ts65Dn mice. Spatial learning and exploratory behavior were improved by enrichment, although the effects in the mice can be modulated by some factors such as gender7. Concurrent changes induced by enrichment on neurochemical and morphologic features of these mice are being evaluated. These results demonstrate that plasticity is conserved in the brain of the trisomic mice, and thus can be exercised by other therapeutic strategies.
G.M. Barlow1, X.-N. Chen1, G.E. Lyons2, J.R. Korenberg1
Dept. of Medical Genetics, Cedars-Sinai Medical Center and UCLA, Los Angeles, CA, 2 Dept. of Anatomy, University of Wisconsin Medical School, Madison, WI

Down Syndrome (DS) is a major cause of mental retardation and congenital heart disease (CHD), and is normally caused by trisomy for chromosome 21. Understanding the molecular genetic basis for these defects in DS may provide important clues to elucidate normal morphogenesis of the brain and heart, and to define possible modes of intervention. Through molecular studies of rare individuals with partial duplications of chromosome 21, we have previously established the candidate region for DS heart disease and candidate genes likely to have roles in DS mental retardation.
Congenital Heart Disease
DS-CHD now appears to be caused by genes located in the region defined by D21S3 through PFKL-c21orf1. This conclusion stems from our recent molecular data based on a panel of nineteen human subjects with partial trisomy 21, eight of whom have CHD. The importance of this result is twofold. First, as previously shown, duplication of the region of D21S55 is not required to generate some forms of DS-CHD, further emphasizing the need to parse DS into a number of well defined and frequently separable phenotypes. Second, the addition of a subject with DS-CHD (Tetralogy of Fallot) but no duplication of either Collagen VI (A1/A2) or Collagen 18 supports the exclusion of these genes from essential roles in generating this type of heart disease in DS. It is important to note that these and other genes may yet modify the variability of the DS-CHD phenotype.
The narrowed DS-CHD candidate region contains 45 known genes and 18 predicted genes and/or pseudogenes (Hattori et al., 2000). Many of these genes are newly identified and little is known about their expression patterns or function. At present, SH3BGR, WRB, DSCR2, DSCAM and HES1 are known to be expressed in the fetal heart and must therefore be considered potential candidates for DS-CHD. In order to investigate the potential roles of SH3BGR, DSCR2 and DSCAM in cardiac embryogenesis and function, we have analysed the temporal and spatial expression patterns of these genes using tissue in situ hybridisation (TISH) in fetal and adult mouse tissues. We propose these genes as candidates for Tetralogy of Fallot and VSD associated with DS.
Cognitive Dysfunction
Data from individuals with individuals with partial trisomy 21 suggests that almost all regions of chromosome 21 contain genes that when present in three copies result in mental retardation in humans. The brains of DS individuals show a variety of well-defined although subtle structural abnormalities which likely underlie aspects of the mental retardation. While none of the genes on chromosome 21 can as yet be related to these structural defects, genes expressed in the brain and peripheral nervous system, such as DSCAM, are reasonable candidates. The previously-described fetal expression pattern of DSCAM supported a potential role in DS mental retardation and, consistent with this, the Drosophila melanogaster DSCAM homolog has recently been shown to function in axon guidance (Schmucker et al., 2000). We now report studies of DSCAM expression using TISH in the newborn and adult mouse brain. In the newborn mouse, the transcript is expressed in the olfactory bulb and hippocampus. In the adult mouse through 21 months, DSCAM is expressed in the cerebellum in the Purkinje cell layer and in the deep cerebellar nuclei. Moreover, continued expression is seen in the adult cortex with highest levels in the hippocampus, CA3 region, dentate gyrus and olfactory bulb, all regions associated with adult neuronal plasticity, learning and memory. These results further support a role for DSCAM in DS brain function.
To further dissect the role of DSCAM and to provide a model with which to develop effective intervention, we have generated a mouse model using a 1.8 kb fragment from the putative DSCAM human promoter region. Mouse embryos expressing the lac Z gene under its control showed specific expression in parts of the brain and peripheral nervous system, representing a subset of the DSCAM expression pattern previously observed, as well as in the skeletal condensations of the developing fore- and hind-limbs. These results are consistent with a role for DSCAM in the development of the brain and peripheral nervous system and support it as a candidate for the cognitive defects of DS.

Professor Shlomo Wientroub,
Head of Department of Pediatric Orthopaedics, Dana Children's Hospital
Tel Aviv Medical Center

Several musculoskeletal abnormalities and problems have been observed to be more common in persons with Down syndrome, such as subluxation and dislocation of the cervical spine, hip and patella, scoliosis, metatarsus primus varus, and instability of the atlanto-occipital and the atlanto-axial region, etc.
Today, the life expectancy of persons with Down syndrome has increased to 55 years and, to investigate the natural life history of orthopedic problems in this population with special attention to their relevance to the functional level and laxity of the person, the European Paediatric Orthopaedic Society decided, in 1997, to conduct a multicenter study on musculoskeletal disorders in persons with Down syndrome.
Separately, common musculoskeletal pediatric problems such as clubfoot, Perthes disease, etc, were studied in the Down population. This study will describe the findings of the European Paediatric Orthopaedic Society survey conducted in 1998-2000.

Bonnie Patterson M.D.
Director Down Syndrome Clinic, Cincinnati Center for Developmental Disorder, Cincinnati, Ohio, USA.

Individuals with Down syndrome have an increased incidence of atlantoaxial instability, with incidence ranging from 6.8-22%. C1-2 instability, defined as a predental space of greater than or equal to 4.5 millimeters, is thought to be secondary to laxity of the transverse ligament of C1. This may be combined with bony abnormalities of the cervical spine such as hypoplasia or malposition of the odontoid process leading to potential spinal cord compression. The importance and significance of cervical spine radiographic findings in Down syndrome have been debated by both pediatric radiologist and clinicians over the years, with one recurrent question being if radiological screening if necessary or whether a neurological examination could identify early evidence of spinal cord involvement.
A study was done to assess a screening test for diagnosis of atlantoaxial instability (cervical spine x-rays) in Down syndrome by using masked comparison of radiographs, MRI scans, and neurologic examinations. Thirty-eight subjects with Down syndrome between the ages of 3 and 21 years participated in the study. Lateral radiographs and MRI's of the cervical spine were obtained in neutral, 45-degree flexion, and 45-degree extension. All of the subjects in the study also had a neurologic examination by two developmental pediatricians. Six of the children had a predental space of 4.5 mm or greater on plain film, and four had an atlanto-dens change of 2 mm or greater from flexion to extension. None of the MRI's indicated spinal cord compression, but 6 of the MRI's did show incidental abnormalities. Neurologic examinations included measurements of tone, strength, reflexes, gait, and balance. Of the children who had mild abnormalities on plain films 4 had brisk patellar reflexes, 2 had increased tone, 3 had a slap-foot or shuffling gait, and 2 had difficulty balancing on one foot.
Recommendations for assessments of individuals with Down syndrome in relation to atlantoaxial instability remain confusing. It is unclear whether asymptomatic atlantoaxial instability is a precursor to symptomatic atlantoaxial instability, and though this study does not answer that question, it does support the fact that the atlas-dens interval measurement is not truly reflective of what is happening in the subarachnoid space with regard to spinal cord compression.

Ira T. Lott, MD
Departments of Pediatrics and Neurology- University of California, Irvine

Individuals with Down Syndrome (DS) are at risk for developing the dementia of Alzheimer's Disease (AD) over age 40 years. The gene for amyloid precursor protein is located on chromosome 21 and is overexpressed in DS. As a result, amyloid is deposited in brain from an early age in DS. We have applied a physical-chemical method to measure the age of amyloid and have found that the earliest deposits in DS appear to be in the superficial layers of the frontal lobe. This is of interest because the earliest symptoms of dementia in DS may be referable to the same structure.
Between ages 35-40 years, many individuals with DS show an exponential rise in the concentration of plaques and tangles (the neuropathological signs of AD). Cholinergic brain systems are targeted as they are with AD in the general population. The prevalence of the clinical disease in DS rises at the same time but does not affect all individuals. Even after age 60 years, the prevalence of dementia of the AD type is still significantly less than 100%. What determines whether an individual with DS will develop signs of dementia?
It seems likely that at least two factors may be involved. First, brain changes in DS suggest that an inflammatory component involving the complement system (C1q) is associated with plaques and tangles after age 40 years. C1q appears to be the first step in an inflammatory cascade which accelerates the pathology associated with amyloid. For example, the cored or mature AD plaque stain positive for C1q as do glial inflammatory cells in the brain. Inflammatory cells in DS show more positive staining than that observed in AD within the general population. The second factor appears to involve the oxidation of amyloid (A
b ), an early event in formation of the neuritic plaque. We feel that oxidative changes in the brain work together with the inflammatory components to propagate AD changes.
These observations suggest that chronic therapy with anti-inflammatory agents and anti-oxidants may be useful in delaying the onset of AD or slowing its course in DS. Once the clinical manifestations of the disease begin, drugs designed to elevate the levels of acetylcholine are effective (at least temporarily) in reversing early signs of dementia. A recent study in our group has shown some reversal in measures of dementia following administration of the acetylcholinesterase medication donezepil.

Revised: July 22, 2002.