Combined assessment of DYRK1A, BDNF and homocysteine levels as diagnostic marker for Alzheimer's disease.

Early identification of Alzheimer's disease (AD) risk factors would aid development of interventions to delay the onset of dementia, but current biomarkers are invasive and/or costly to assess. Validated plasma biomarkers would circumvent these challenges. We previously identified the kinase DYRK1A in plasma. To validate DYRK1A as a biomarker for AD diagnosis, we assessed the levels of DYRK1A and the related markers brain-derived neurotrophic factor (BDNF) and homocysteine in two unrelated AD patient cohorts with age-matched controls. Receiver-operating characteristic curves and logistic regression analyses showed that combined assessment of DYRK1A, BDNF and homocysteine has a sensitivity of 0.952, a specificity of 0.889 and an accuracy of 0.933 in testing for AD. The blood levels of these markers provide a diagnosis assessment profile. Combined assessment of these three markers outperforms most of the previous markers and could become a useful substitute to the current panel of AD biomarkers. These results associate a decreased level of DYRK1A with AD and challenge the use of DYRK1A inhibitors in peripheral tissues as treatment. These measures will be useful for diagnosis purposes.

Plasma DYRK1A as a novel risk factor for Alzheimer's disease.

To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-ß42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD.

DYRK1A promotes dopaminergic neuron survival in the developing brain and in a mouse model of Parkinson's disease.

In the brain, programmed cell death (PCD) serves to adjust the numbers of the different types of neurons during development, and its pathological reactivation in the adult leads to neurodegeneration. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in neural proliferation and cell death, and its role during brain growth is evolutionarily conserved. Human DYRK1A lies in the Down syndrome critical region on chromosome 21, and heterozygous mutations in the gene cause microcephaly and neurological dysfunction. The mouse model for DYRK1A haploinsufficiency (the Dyrk1a(+/-) mouse) presents neuronal deficits in specific regions of the adult brain, including the substantia nigra (SN), although the mechanisms underlying these pathogenic effects remain unclear. Here we study the effect of DYRK1A copy number variation on dopaminergic cell homeostasis. We show that mesencephalic DA (mDA) neurons are generated in the embryo at normal rates in the Dyrk1a haploinsufficient model and in a model (the mBACtgDyrk1a mouse) that carries three copies of Dyrk1a. We also show that the number of mDA cells diminishes in postnatal Dyrk1a(+/-) mice and increases in mBACtgDyrk1a mice due to an abnormal activity of the mitochondrial caspase9 (Casp9)-dependent apoptotic pathway during the main wave of PCD that affects these neurons. In addition, we show that the cell death induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that activates Casp9-dependent apoptosis in mDA neurons, is attenuated in adult mBACtgDyrk1a mice, leading to an increased survival of SN DA neurons 21 days after MPTP intoxication. Finally, we present data indicating that Dyrk1a phosphorylation of Casp9 at the Thr125 residue is the mechanism by which this kinase hinders both physiological and pathological PCD in mDA neurons. These data provide new insight into the mechanisms that control cell death in brain DA neurons and they show that deregulation of developmental apoptosis may contribute to the phenotype of patients with imbalanced DYRK1A gene dosage.

Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice.

BACKGROUND: Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started. MATERIALS AND METHODS: In order to establish a threshold of plasma homocysteine concentration we have analyzed the hepatic cystathionine beta synthase and paraoxonase 1 activities in a moderate to intermediate murine model of hyperhomocysteinaemia. Using wild type and heterozygous cystathionine beta synthase deficient mice fed a methionine enriched diet or a control diet, we first studied the link between cystathionine beta synthase and paraoxonase 1 activities and plasma homocysteine concentration. RESULTS: Among the animals used in this study, we observed a negative correlation between plasma homocysteine level and cystathionine beta synthase activity (rho=-0.52, P=0.0008) or paraoxonase 1 activity (rho=-0.49, P=0.002). Starting from these results, a homocysteine cut-off value of 15 microm has been found for both cystathionine beta synthase (P=0.0003) and paraoxonase 1 (P=0.0007) activities. CONCLUSIONS: Our results suggest that both cystathionine beta synthase and paraoxonase 1 activities are significantly decreased in mice with a plasma homocysteine value greater than 15 microm. In an attempt to set up preventive treatment for cardiovascular disease our results indicate that treatments should be started from 15 microm of plasma homocysteine.

Classification of human chromosome 21 gene-expression variations in Down syndrome: impact on disease phenotypes.

Down syndrome caused by chromosome 21 trisomy is the most common genetic cause of mental retardation in humans. Disruption of the phenotype is thought to be the result of gene-dosage imbalance. Variations in chromosome 21 gene expression in Down syndrome were analyzed in lymphoblastoid cells derived from patients and control individuals. Of the 359 genes and predictions displayed on a specifically designed high-content chromosome 21 microarray, one-third were expressed in lymphoblastoid cells. We performed a mixed-model analysis of variance to find genes that are differentially expressed in Down syndrome independent of sex and interindividual variations. In addition, we identified genes with variations between Down syndrome and control samples that were significantly different from the gene-dosage effect (1.5). Microarray data were validated by quantitative polymerase chain reaction. We found that 29% of the expressed chromosome 21 transcripts are overexpressed in Down syndrome and correspond to either genes or open reading frames. Among these, 22% are increased proportional to the gene-dosage effect, and 7% are amplified. The other 71% of expressed sequences are either compensated (56%, with a large proportion of predicted genes and antisense transcripts) or highly variable among individuals (15%). Thus, most of the chromosome 21 transcripts are compensated for the gene-dosage effect. Overexpressed genes are likely to be involved in the Down syndrome phenotype, in contrast to the compensated genes. Highly variable genes could account for phenotypic variations observed in patients. Finally, we show that alternative transcripts belonging to the same gene are similarly regulated in Down syndrome but sense and antisense transcripts are not.

C21orf5, a human candidate gene for brain abnormalities and mental retardation in Down syndrome.

Mental retardation represents the more invalidating pathological aspect of trisomy 21 and has a hard impact on public health. The dosage imbalance of chromosome 21 genes could be the cause of neurological alterations and mental retardation seen in Down syndrome. We studied C21orf5 that we have demonstrated to be overexpressed in Down syndrome tissues, as a candidate gene for trisomy 21. A new optical technology (Rachidi et al., 2000) was used to compare signal intensity and cell density in presumptive embryonic brain compartments, at their boundaries and in higher specialized brain centres during fetal lifespan. We showed a developmentally regulated transcriptional activity of C21orf5 and a regional and cellular specific distribution of gene transcripts during human embryonic and fetal development. A wide but differential expression was detected in the nervous system during embryogenesis with a relatively lower level in the forebrain than in the midbrain and hindbrain and the highest transcription intensity in the future cerebellum. This developmentally regulated expression is maintained during post-embryogenesis and evolves selectively in fetal cerebral, hippocampal and cerebellar areas. Differential and cellular specificity were detected in hippocampus with higher C21orf5 mRNA level in the pyramidal cells compared to granular cells of the dentate gyrus. The expression pattern detected in cortical and cerebellar structures correlates well to the altered cortical lamination and to the lower size of the cerebellum observed in Down syndrome patients. In addition, the patterned differential expression detected in the medial temporal-lobe system, including hippocampal formation and perirhinal cortex, working as control centres of the memory circuits and involved in cognitive processes and memory storage, also corresponds to abnormal brain regions seen in Down syndrome patients. The C21orf5 selective expression in the key brain structures for learning and memory suggests that C21orf5 overexpression could participate in mental retardation pathogenesis in Down syndrome patients.

Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus.

In several neurological disorders including hyperhomocysteinemia, homocysteine (Hcy) accumulates in the brain, and acts as a potent neurotoxin. However, the molecular mechanisms induced by increased levels of Hcy in brain are not well understood. Here we show an activation of the extracellular signal-regulated kinases (ERK1 and ERK2) and the downstream nuclear targets Elk-1 and calcium/cAMP response element binding protein, in the hippocampus of cystathionine beta synthase deficient mice, a murine model of hyperhomocysteinemia. An ex vivo model of hippocampal slices allowed us to reproduce Hcy -induced ERK activation and to unravel the mechanisms responsible of this activation. Of interest, N-methyl-d-aspartate (NMDA), non-NMDA and metabotropic glutamate receptor antagonists all blocked Hcy -induced ERK activation. Moreover, the ERK activation was blocked in the presence of Na+-channel blocker tetrodotoxin, indicating the existence of a trans-synaptic activity in ERK activation by Hcy in hippocampal slices. The effects of Hcy on ERK cascade activation were also dependent on calcium influx, CaMK-II, PKC as well as PKA activation. Thus, altogether these data support a role of Hcy on ERK activation, via complex mechanisms, starting with a control of glutamate release, which in turn activates ionotropic and metabotropic receptor subtypes and produces increases in intracellular calcium levels.

Identification and characterisation of soluble epoxide hydrolase in mouse brain by a robust protein biochemical method.

The central nervous system is an important potential target for certain environmental prototoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. On the other hand, developments in the field of molecular biology and advances in high-throughput screening methods continue to increase the number and amounts of available proteins. We used thus a robust and reliable technique, two-dimensional gel electrophoresis coupled to matrix assisted laser desorption/ionisation mass spectroscopy followed by tandem mass spectrometry and identified for the first time soluble epoxide hydrolase and added other biotransformation enzymes in the hippocampal region of mouse brain. Soluble epoxide hydrolase has an Mr of 61.5 kDa, pI of 5.9, twenty-six matching peptides and sequence coverage of 56% and was unambiguously identified by MS/MS. Since localised biotransformation events in regions of the central nervous system may account for pathologies and/or toxicities initiated by exposure to certain endogenous and/or environmental chemicals, identification of these enzymes would present an opportunity for developing novel therapeutic targets or would have critical toxicologic significance.

Mass spectrometrical analysis of phosphoprotein enriched in astrocytes of 15 kDa in mouse hippocampi.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is a small protein that was first identified as an abundant phosphoprotein in brain. PEA-15 was characterised so far at the immunochemical level and by a microsequencing attempt. In order to update characterisation of this important structure by advanced methodology unambiguously identifying proteins independent of antibody availability and specificity, we used a proteomic method for this purpose: Performing protein profiling in mouse hippocampi using two dimensional gel electrophoresis with subsequent mass spectrometrical (MS/MS) identification we detected this protein and demonstrate proteomic characterisation of PEA-15 (Q62048). This study enables further specific and unambiguous determination serving as an analytical tool.

Chromosome 21 KIR channels in brain development.

Two KIR (K+ Inwardly Rectifying) channel genes have been identified on chromosome 21, in a region associated with important phenotypic features of trisomy 21, including mental retardation: KIR3.2 (GIRK2) and KIR4.2. We analysed the expression of these channel genes in developing human and mouse brains to determine the possible role of the corresponding channels in brain development and function. KIR3.2, which has been extensively studied in the mouse, was found to be expressed in the human cerebellum during development. The KIR4.2 channel is expressed later in development in both mice and humans. We compared the expression of these channels in terms of RNA and protein levels and discussed the potential synergy and consequences of the overexpression of these channels in Down's syndrome brain development.

Assessing implications between genotypic and phenotypic variables through lattice analysis.

A previous paper assessed a "Molecular Mapping of Twenty-Four Features of Down Syndrome on Chromosome 21" (Delabar et al., 1993), by analyzing the genotypes/phenotypes of patients suffering from partial trisomy. The mapping was defined through implications--each feature was mapped to the conjunction of cytogenetic bands that were shared by all patients having that feature. In the present paper, we extend that approach to determine how far those implications depart from defining equivalences. Finding equivalences is important. Local equivalences permit a genetic characterization of a feature. And if global equivalences held for all features, that set of bands would be sufficient to characterize the various phenotypes observed in individuals with partial trisomy 21. To extend the earlier approach, we examine the structure of equivalences as well as the structure of implications. We examine both conjunctions of bands and conjunctions of features. The use of Galois lattices permits simultaneous evaluation of both kinds of structures. Each Galois lattice is labeled with a basis (minimal generating set) of implications going from conjunctions of features into bands and those going from conjunctions of bands into features. Analysis reveals that about half of the conjunctions of bands that characterize the genetic structure embody equivalences. This allows us to improve the genetic description of features and to specify minimal sets of questions that need to be investigated to make the global genetic description more precise.

The CASK/Lin-2 Drosophila homologue, Camguk, could play a role in epithelial patterning and in neuronal targeting.

Drosophila Camguk (Cmg) is a member of the CAMGUK subfamily of the MAGUK family of proteins which are localized at cell junction and other plasma membrane specialized regions, from worms to mammals. The protein structure of Cmg, as the other CAMGUK proteins, is characterized by only one PDZ domain and an additional CaM kinase domain, similar to CaMKII. While the mammalian ortholog CASKs play an important role in synaptic protein targeting and in synaptic plasticity, the Drosophila Cmg role is unknown. To study its potential role, we reported a detailed analysis of mRNA distribution of the Drosophila cmg gene at cellular and developmental level, during embryonic, larval, pupal and adult stages. The transient cmg transcription in midgut and Malpighian tubules may suggest a potential function in cell junction formation and in epithelial tissue patterning. Interestingly, cmg transcription increases substantially during embryonic neuroblast proliferation, becoming predominant in the developing central nervous system (CNS) during embryonic and postembryonic development stages and in the mature brain. In addition, a high transcriptional level was detected in the eye imaginal discs and in the adult retina, demonstrating a specific and continuous expression of cmg in neuroblasts and photoreceptor neurons, from the onset of cytodifferentiation. Our findings suggest that Cmg could play a potential role in transmembrane protein targeting, particularly in synapses. These observations suggest the existence of a common highly conserved mechanism involved in forming and maintaining proper synaptic protein targeting, which are fundamental features of synaptic plasticity, learning and memory. Through its function, the CaM kinase domain-containing Cmg may be involved in signal transduction cascade. Its potential relation to Calmodulin and CaMKII is discussed.

C21orf5, a novel human chromosome 21 gene, has a Caenorhabditis elegans ortholog (pad-1) required for embryonic patterning.

To contribute to the development of the transcription map of human chromosome 21 (HC21), we isolated a new transcript, C21orf5 (chromosome 21 open reading frame 5), encoding a predicted 2298-amino-acid protein. Analysis of the genomic DNA sequence revealed that C21orf5 consists of 37 exons that extend over 130 kb and maps between the CBR3 (carbonyl reductase 3) and the KIAA0136 genes. Northern blot analyses showed a ubiquitously expressed RNA species of 8.5 kb. RNA in situ hybridization on brain sections of normal human embryos revealed a strong labeling in restricted areas of the cerebral cortex. In silico analysis of the deduced C21orf5 protein revealed several highly probable transmembrane segments but no known protein domains or homology with known proteins. However, there were significant homologies to several hypothetical Caenorhabditis elegans proteins and Drosophila melanogaster genomic sequences. To investigate the function of C21orf5, we isolated the cDNA of the C. elegans ortholog and performed double-stranded RNA-mediated genetic interference experiments. The major phenotype observed in the progeny of injected animals was embryonic lethality. Most of the tissues of the embryo failed to undergo proper patterning during gastrulation, and morphogenesis did not occur; thus we termed the ortholog pad-1, for patterning defective 1. These results indicated that pad-1 is essential for the development and the survival of C. elegans. This study provides the first example of the use of C. elegans as a model to study the function of genes on human chromosome 21 that might be involved in Down syndrome.

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Regional and cellular specificity of the expression of TPRD, the tetratricopeptide Down syndrome gene, during human embryonic development.

The TPRD gene (tetratricopeptide (TPR) containing Down syndrome gene) is one of the candidate genes in the Down syndrome chromosomal region-1. Duplication of this gene may be the cause of major phenotypic features of Down syndrome. Here we show that the TPRD expression is developmentally regulated during human embryogenesis. At the earliest stages of development (Carnegie 8-12) TPRD expression is ubiquitous. At later developmental stages (Carnegie stages 14, 16 and 18), it becomes restricted to the nervous system, as is the case for the mtprd gene during mouse development. We extended our analysis of TPRD expression during fetal development of the human nervous system (13, 22 and 24 weeks). A new oblique illumination technique was used to compare signal intensity and cell density. Some regions of the nervous system such as the external cortical layers of the brain, and the inner neuroblastic layer of the eye, strongly express the TPRD gene.

Characterization of a novel gene, C21orf6, mapping to a critical region of chromosome 21q22.1 involved in the monosomy 21 phenotype and of its murine ortholog, orf5.

Phenotypic and molecular analyses of patients with partial chromosome 21 monosomy enabled us to define a region, spanning 2.4 Mb between D21S190 and D21S226, associated with arthrogryposis, mental retardation, hypertonia, and several facial anomalies. The markers of the region were used to screen a total human PAC library (Ioannou, RZPD). We isolated 57 PACs, which formed primary contigs. EST clusters (UNIGENE collection) located in a 6-Mb interval, between D21S260 and D21S263, were mapped in individual bacterial clones. We mapped the WI-17843 cluster to the PAC clone J12100, which contains the two anchor markers LB10T and LA329. The open reading frame extends over 960 bp, with three putative start codons. The 1695-bp cDNA containing a polyadenylation signal should correspond to the full-length cDNA. From the genomic sequence, we deduced that the gene contained five exons and that there was a putative promoter sequence upstream from exon 1. In silico screening of DNA databases revealed similarity with a murine EST. The corresponding cDNA (1757 bp) sequence was very similar (>85%) to the human cDNA and had an open reading frame of 876 nucleotides. Somatic hybrid mapping localized the cDNA to mouse chromosome 16. EST analyses and RT-PCR indicated that the third exon in the human gene (exon 2 in the mouse) undergoes alternative splicing. Northern blot hybridization showed that the gene was ubiquitously expressed in humans and mice. The longest mouse clone was used to generate riboprobes, which were hybridized to murine embryos at stages E-9.5, E-10.5, E-12.5, E-13.5, and E-14.5-15, to study the pattern of expression during development. Ubiquitous labeling was observed, with strong signals restricted to limited areas of the telencephalon, the mesencephalon, and the interrhombomeric regions in the central nervous system, and other regions of the body such as the limb buds, branchial arches, and somites.

Developmentally regulated expression of mtprd, the murine ortholog of tprd, a gene from the Down syndrome chromosomal region 1.

The gene tprd, which contains three tetratricopeptide domains, has been recently localized in the Down syndrome (DS) chromosomal region 1. We have cloned a cDNA encoding part of the murine ortholog of tprd and used it to characterize the expression pattern of this gene during development and at the adult stage. At E8.5 the expression is uniform. In the later stages of embryogenesis, although expression remains ubiquitous, a pattern of tissues with particularly high expression develops: the strong expression is restricted to non proliferating zones of the nervous system such as the external layer of the cortex, the spinal cord, the cranial and root ganglia and the nerves. In the brain of adult mouse the strongest signals are observed in layers II-III and V-VI of the cortex, in the hippocampus and in the cerebellum, which correspond to the abnormal brain regions seen in DS patients.

Dynamic expression pattern of Ca(2+)/calmodulin-dependent protein kinase II gene in the central nervous system of Drosophila throughout development.

Calcium/calmodulin-dependent protein kinase II (CaM KII) is thought to be involved in the majority of the neuronal functions mediated by intracellular free Ca(2+), and has been implicated in long-term potentiation, learning, and memory. In this work, we have examined in detail the RNA expression pattern for the Drosophila CaM KII gene by in situ hybridization, during embryonic, larval, pupal, and adult stages. Our results indicate that expression of CaM KII was homogeneous in early embryos, but that during development the gene transcription rapidly became restricted to neuroblasts and their progeny in the nervous system. This predominant expression in the nervous system is maintained during late embryogenesis and post-embryonic development. A signal compartmentalization appeared in the larval central nervous system, where the CaM KII expression became progressively concentrated in the anterior ganglia. In the adult brain, a specific expression was more abundant in a subset of neurons around the central brain, particularly the mushroom bodies and the central complex, structures that play an important role in learning and memory.

Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms.

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.

High resolution physical mapping and identification of transcribed sequences in the Down syndrome region-2.

The identification and mapping of genes within the Down syndrome region is an important step toward a complete understanding of the pathogenesis of this disorder. The objective of the present work is to identify and map genes within the Down syndrome region-2. Chromosome 21 cosmid clones corresponding to "cosmid pockets" 121-124 have been first used as a starting material for generation of a single high resolution integrated cosmid/PAC contig with full EcoRI/SmaI restriction map. The integrated contig has been further anchored to genetic and physical maps through the positioning of 6 markers in the following order: ACTL5-D21S3-684G2T7-D21S71-D21S343-D21S 268. The entire contig covers 342 kb of the Down syndrome region-2 of chromosome 21. Subsequently, we have isolated, identified, and mapped four novel cDNAs which we have named N143, N144, CHD/333, and 90/3H1 and a potentially transcribed genomic sequence (E05133T7). Additionally, we have accurately located a previously described gene, the WRB gene, between the markers ACTL5-D21S268 within this Down Syndrome Region-2.