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Chromosome 21 and Down syndrome: from genomics to pathophysiology
Author: Stylianos E. Antonarakis
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"Genomic aneuploidy, defined as an abnormal number of copies of a genomic region, is a common cause of human genetic disorders. Classically, the term aneuploidy was restricted to the presence of supernumerary copies of whole chromosomes (trisomy), or absence of chromo- somes (monosomy), but we extend this definition to include deletions or duplications of subchromosomal regions. Trisomy 21 is a model of all human disorders that are the result of supernumerary copies of a genomic region. In this review, we focus on Down syndrome (DS) and human chromosome 21 (HSA21) to show the effect that genomics has had on our understanding of the ?disorders of the genome?. We discuss the recent advances in genome sequencing, comparative genome analysis, functional genome exploration, use of model organisms and lessons from models of gene overexpression. We also discuss the consequences of genomic dosage imbalance owing to an extra copy of a genomic segment (trisomy). These are exciting times for genomic disorders, such as trisomy 21, because we now have the necessary tools to understand how three copies of a functional genomic element result in abnormal phenotypes. Trisomies According to the size of the triplicated genomic region, trisomies can be divided into four categories: complete, or whole-chromosome, trisomies; partial trisomies; microtrisomies and triplication of single genes or single functional genomic elements. Whole-chromosome trisomies. Whole-chromosome tri- somies that result from meiotic or mitotic non-disjunc- tion events are common in humans; they account for ~0.3?0.5% of live births. Trisomy for HSA21, which results in Down syndrome and occurs at ~1 in 750 live births, is the most frequent event. Trisomies are often observed in a significant proportion of spontaneous abortions; for example, trisomy 16 is found in 1 out of 13, and trisomy 21 in 1 out of 43 such abortions 1 . Partial trisomies. Partial (or segmental) trisomies that involve a genomic region of more than one chro- mosomal band (usually larger than 5 Mb) are much less frequent than whole-chromosome trisomies. They usually result from abnormal meiosis and seg- regation in individuals with balanced chromosomal rearrangements. One in about 1,800 newborns have an unbalanced, non-robertsonian rearrangement and approximately half of these are partial trisomies. Unbalanced ROBERTSONIAN TRANSLOCATIONS with trisomies of the long arms of ACROCENTRIC CHROMOSOMES occur in 1 of about 14,000 newborns 1 . Microtrisomies. This type of trisomy is defined here as the partial trisomy of a genomic segment that is shorter CHROMOSOME 21 AND DOWN SYNDROME: FROM GENOMICS TO PATHOPHYSIOLOGY Stylianos E. Antonarakis*, Robert Lyle*, Emmanouil T. Dermitzakis ? , Alexandre Reymond* and Samuel Deutsch* Abstract | The sequence of chromosome 21 was a turning point for the understanding of Down syndrome. Comparative genomics is beginning to identify the functional components of the chromosome and that in turn will set the stage for the functional characterization of the sequences. Animal models combined with genome-wide analytical methods have proved indispensable for unravelling the mysteries of gene dosage imbalance. ROBERTSONIAN TRANSLOCATIONS Occurs when the long arms of two acrocentric chromosomes fuse at the centromere and the two short arms are lost. A non- robertsonian rearrangement is a chromosomal rearrangement other than a Robertsonian translocation. NATURE REVIEWS | GENETICS VOLUME 5 | OCTOBER 2004 | 725 *Department of Genetic Medicine and Development, University of Geneva Medical School and University Hospitals of Geneva, 1 rue Michel-Servet, 1211 Geneva, Switzerland. ? Population and Comparative Genomics, The Wellcome Trust, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. Correspondence to S.E.A. e-mail: Stylianos.Antonarakis@med ecine.unige.ch doi:10.1038/nrg1448 REVIEWS � 2004 Nature Publishing Group ACROCENTRIC CHROMOSOMES A chromosome, the centromere of which is located near one end. 726 | OCTOBER 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS lead to microtrisomies and micromonosomies. Microduplications are seen, for example, in many cases of Charcot?Marie?Tooth disease, type 1A (CMT1A). This neurological disorder is caused by a ~1.4 Mb dupli- cation of chromosome 17p12, the result of recurrent non-allelic homologous recombination between dupli- cons that flank the duplicated segment 3 .Another more recent example of a microtrisomy is that of SHFM3 than 3?5 Mb and that is not detectable by routine high- quality cytogenetic analysis. It is also known as segmental duplication. The incidence of microtrisomies is, at pre- sent, unknown. Most are due to unequal crossovers in meiosis, mediated by the presence of interchromosomal duplicons or low copy repeats (LCRs; 10?100 Kb each). These duplicons, which make up ~5% of the human genome 2 ,promote unequal recombination events that Box 1 | Hypothesis for Trisomy 21 phenotypes We might consider that there are two categories of genes on human chromosome 21 (HSA21); those that are dosage sensitive (that is, three copies result in phenotypic effects; shown in red) and contribute to the phenotypes of Down syndrome (DS), and those that are not dosage sensitive (green) and therefore do not contribute to any of the phenotypes. This could be true for both protein-coding genes and non-coding RNA (ncRNA) genes (including microRNA genes). The effect of some dosage-sensitive genes on the phenotypes might be allele specific. According to this hypothesis, certain combinations of alleles might contribute to the phenotype, whereas others might be silent. The effect of the combination of alleles could be either qualitative (alleles with amino-acid variation) or quantitative (alleles with variation in gene expression level). A threshold effect of total transcript output or amount of protein could be envisaged in the following way: a phenotype is only present if the total transcript/protein level from the combination of the three alleles reaches a crucial amount. If the output from the combination of the three alleles does not reach the crucial amount, the associated phenotype is not manifested. The insert in the figure explains the hypothetical threshold effect of gene expression in DS phenotypes. The allelic combination in cases 3, 4 and 6 result in total expression levels beyond the threshold and in the appearance of the phenotype; by contrast, the allelic combination in cases 1, 2 and 5 are not sufficient for the necessary pathological overexpression. The effect of the dosage sensitive genes could have a direct or indirect effect on the phenotype. The indirect effect might be due to the interaction of HSA21 genes or gene products with non-HSA21 genes or gene-products. This interaction could be allele-specific; only certain combinations of alleles of non-HSA21 genes (shown in light and dark blue) would contribute to susceptibility of specific phenotypes. The individual genetic background could therefore contribute to the phenotypic variation. So, the global dysregulation of the individual transcriptome or proteome could contribute to the DS phenotypes. Finally, triplication of certain conserved functional non-genic sequences (CNGs ? shown in grey) on HSA21 (or in the entire genome) might contribute to the DS phenotypes, as might the triplication of other functional but non- conserved genomic regions. The molecular mechanism of this type of locus dosage imbalance is unknown but it will probably be determined after the functional characterization of CNGs and other important genomic elements. The picture of the trisomy 21 female was reproduced, with permission, from REF. 90 � (1998) Royal Society of Medicine Press. Direct effect of HSA21 gene 3� gene Sensitive 3� gene Non-sensitive CNG Allele-non-specific CNGs direct or indirect contribution HSA21?Non-HSA21 gene interactions HSA21 Down syndrome phenotypes Expression level Case 2 Case 5 Phenotype Case 1 Case 4 Case 6 Case 3 Allele-specific � 2004 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 5 | OCTOBER 2004 | 727 REVIEWS maps ~1 Mb from the sonic hedgehog (Shh) locus and causes PREAXIAL POLYDACTYLY in the sasquatch (Ssq) mouse mutant 10 . Genomic dosage imbalance frequently occurs in somatic cancer cells. Chromosomal instability of tumour cells, resulting in various trisomies and monosomies, is associated with tumour progression 11 ; this topic deserves a separate discussion and we do not return to it here. Below, we focus on constitutional trisomies, that is, those that are present in all cells, and in particular on trisomy 21 as it is the most frequent example and serves as a model for all constitutional trisomies. Origin of the extra HSA21 The parental origin of the supernumerary HSA21 in trisomy 21 was determined using highly informative polymorphic markers in DNA from parents and DS offspring. DNA markers near the centromere indicate the stage of meiosis during which the segre- gation error occurred. Homozygosity for all poly- morphic markers throughout 21q (the long arm of HSA21) is indicative of a mitotic, post-zygotic error. Supplementary information S1 (table) lists the various origins of human trisomy 21. (a form of ECTRODACTYLY), which is caused by a duplica- tion of ~0.5 Mb on chromosome 10q24 (REF. 4).New pathogenic or polymorphic microtrisomies will proba- bly be identified using diagnostic methods such as BAC ARRAY CGH (comparative genomic hybridization) 5 . Single-gene duplications. Duplications of only one gene or one functional genomic element can also be pathogenic; for example, duplicated PLP1 causes Pelizaeus?Mertzbacher disease, and duplicated PMP22 causes CMT1A 6,7 .The genomic duplications that include these genes also encompass additional coding sequences, but studies of transgenic mice that carry sin- gle gene duplications (either of Plp1 or Pmp22) indicate that the abnormal phenotype is caused by duplication of these two genes 8,9 . Evidence from experiments using transgenic mice indicates that single gene dosage imbalance might cause abnormal phenotypes. For example, strain B6;SJL- Tg(EPO33)72Ptc/J,with a single copy of human EPO. Finally, the duplication of a non-genic, but func- tional, genomic element might also be pathogenic. A recent example of this comes from the duplication of ~30 Kb containing a conserved regulatory element that ECTRODACTYLY Limb malformations characterized by digit loss. BAC ARRAY CGH A microarray that contains DNA from bacterial artificial chromosomes, which is used in comparative genomic hybridization to determine copy number differences of DNA sequences. PREAXIAL POLYDACTYLY Polydactyly (additional fingers or toes) on the thumb side of the hand and the big-toe side of the foot. CNGs > 100 Mb ?1? X21 80 Y 0 160 CNGs 35-99 Mb ?1? 1721 40 Y 0 80 SNPs Mb ?1 * 2000 21 22Y 0 4000 CpG Islands Mb ?1? 20 1921Y 0 40 Genes Mb ?1 * 1921 12.5 Y 0 25 UniGene ESTs Mb ?1� 2221 70 Y 0 140 G+C% || 19 21 4 42.535 50 LINEs Mb ?1? 9 500 21X 0 1000 SINEs Mb ?1? X21 700300 Y 1100 Length Mb * 0 22 21 150 1 300 Figure 1 | Features of human chromosome 21 (HSA21). Each line describes a particular feature shown on the left. The position of HSA21 in each scale is shown as a red triangle. All other chromosomes are shown as blue triangles. The identity of chromosomes with extreme values per feature is also shown. The sources of information are: *ENSEMBL 34; ? GALA 34 ; � NCBI 34; || REF. 20. The high SNP density is probably a biased estimate owing to the updated estimate provided by REF. 41. CNGs, conserved non-genic sequences; LINEs, long interspersed nuclear elements (such as L1 repeats) are retroelements present in over 100,000 copies in the mammalian genome; SINEs, short interspersed-repeat transposable elements. � 2004 Nature Publishing Group 728 | OCTOBER 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS Phenotypic variability of DS There are two types of phenotypes that are observed in trisomy 21: those seen in every patient and those that occur only in a fraction of affected individuals (see Supplementary information S2 (table)) 15,16 .For exam- ple, cognitive impairment is present in all patients with For an extensive discussion of the origin of aneu- ploidy in all human trisomies, and the link between recombination and non-disjunction, the reader is referred to the excellent recent review by Hassold and Hunt 12 .The origin of translocation trisomy 21 is discussed in REFS 13,14. Table 1 | Characteristics of human chromosome 21 Property Description Comments References Physical size 21p 5?15 Mb 91 21q 33.5 Mb 18 % human genome 21q = ~1% 19 Genetic size Sex average 61.7?67.3 cM 92,93 Male meioses 47.3?54.3 cM 92,93 Female meioses 76.4?80.1 cM 92,93 Recombination rate Sex average 500 kb/cM 93 Male meioses 420 kb/cM 93 Female meioses 620 kb/cM 93 Range 250?2125 kb/cM 93 Repeat content 40.06% SINEs 10.84% 18 LINEs 15.15% 18 LTR elements 9.21% 18 DNA transposons 2.4% 18 Paralogous regions Intra HSA21 1.4% 18,19 Interchromosomal 1.6% 18,19 Variability Nucleotide diversity 5.19 x 10 ?4 SNPs 105,334 Includes 335 coding synonymous, * 355 coding non-synonymous, 27,433 intronic, 7,827 in UTRs Microsatellites 5,987 These are short sequence repeats, 18 potentially polymorphic Genes (protein coding) Swiss-Prot 212 (including 21 KRTAP) ? Ensembl 261 (244 known and 17 novel) � NCBI 263 (including 44 KRTAP) || Berlin list 284 (including 27 KRTAP) 240 confirmed, 26 predicted by ESTs, � 18 computationally predicted Denver analysis 364 (170 well conserved in mouse, 25 83 minimally conserved in mouse, 111 non-conserved) Pseudogenes Ensembl 31 � NCBI 49 || Berlin list 83 � Yale analysis 77 processed, 72 non-processed 94 Non-coding RNAs miRNAs 5 E.T.D.& S.E.A., unpublished data rRNA genes Estimated ~40?50 in 21p (RNR4A and RNR4B) 95 Other CNG content 2,978 >100 bp; >70 ungapped identity 35 # CpG islands 149 In blood cells: 103 non-methylated (Me);31 fully Me; 7 composite Me; 3 monoallelic Me 96 Mouse orthologous MMU16; 76.7?100 Mb ** genomic regions MMU17; 30.6?31.7 Mb MMU10; 78.4?80.6 Mb CNG, conserved non-genici; HSA, human chromosome; kRTAP, keratin-associated proteins; LINEs, long interspersed nuclear elements (such as L1 repeats) are retroelements present in over 100,000 copies in the mammalian genome; LTR, long terminal repeat; miRNA, micro RNAs; NCBI, National Center for Biotechnology Information; SINEs, short interspersed-repeat transposable elements. *http://www.ncbi.nlm.nih.gov/SNP/; ? http://au.expas.org//cgi-bin/lists?humchr21.txt; � http://www.ensembl.org/Homo_sapiens/mapview?chr=21; || http://www.ncbinlm.nih.gov/mapview/map.cgi?ORG=hum&MAPS=idiogr,est,loc&LINKS=ON&VERBOSE= ON&CHR=21; � http://chr21.molgen.mpg.de/chr21_catalog9.html; # http://globin.cse.psu.edu/gala/ ; **http://www.ensembl.org/Homo_sapiens/syntenyview. � 2004 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 5 | OCTOBER 2004 | 729 REVIEWS genome sequences will further increase our ability to rec- ognize additional genes and validate initial gene predic- tions. The 33.5 Mb chimpanzee chromosome 22, which is homologous to HSA21, has recently been sequenced 30 . There are 1.44% single nucleotide substitutions between the human and chimpanzee sequences, and nearly 68,000 insertions and deletions (indels); more than 99% of the indels are shorter than 300 bp. Remarkably, 86% of the 231 unambiguous coding sequences in both species show amino-acid differences. Indels within cod- ing regions represent one of the main mechanisms that lead to protein diversity. An interesting approach to improving the functional annotation of HSA21 involves studying transcriptional activity of the entire chromosome. In one study, oligonucleotide arrays that contain probes spaced, on average, 35 bp apart and that cover the entire 21q were used to estimate mRNA expression from 11 different human cancer cell lines 31 .As many as 9.7% of the probes showed positive hybridization signals in 5 of the 11 cell lines 31 ,which indicate that the potentially tran- scribed genome is ~10-fold larger than the current genic annotation. This transcription potential could be due to additional unidentified genes, RNA transcripts without protein-coding capacity, alternative RNA isoforms of previously annotated genes, or ?illegitimate? non- functional transcription. Subsequent analysis of these data for chromosomes 21 and 22, showed that ~49% of the observed hybridization signal (transcription) was outside the known annotation 32 ; 65% of these tran- scripts were verified by reverse transcriptase PCR (RT- PCR). These results emphasize that the annotation of HSA21 sequence is far from complete. Another study combined chromatin immunopre- cipitation and high-density oligonucleotide arrays to map more than 300 binding sites of the three transcrip- tion factors, Sp1, cMyc, and p53 on HSA21 (REF. 33).As expected, these sites clustered near 5? promoters of pro- tein-coding genes and CPG ISLANDS but many were found in 3? ends of known protein-coding genes. Most of the binding sites identified are, in fact, associated with ncRNAs of as yet unknown function. Intriguingly, this study provided initial evidence for widespread antisense transcription, the functional significance of which remains unknown. Functional analysis of the HSA21-encoded proteins is one of the research priorities for the understanding of DS. Analysis of Swiss-Prot-listed HSA21-encoded pro- teins with Interpro for protein families, domains and functional sites, gave 207 entries (see Online links box). Analysis of the same proteins using the Gene Ontology Annotation (see Online links box) shows that they are involved in 87 different biological processes, have 81 dif- ferent molecular functions and are localized in 26 differ- ent cellular components. The most frequent molecular function is DNA binding and transcription factor activ- ity (15 proteins); the most common cellular localiza- tions are in the nucleus and the plasma membrane (19 and 15 proteins, respectively); and the most common biological process that they are involved in is signal transduction (11 proteins). DS, whereas congenital heart defect occurs in ~40% and ATRIOVENTRICULAR CANAL in ~16% of patients. DUODENAL STENOSIS/ATRESIA, Hirschsprung disease and acute mega- karyocytic leukemia occur 250-, 30- and 300-times more frequently, respectively, in patients with DS than in the general population. In addition, for any given phenotype there is considerable variability (severity) in expression. For example, the extent of cognitive impairment varies widely in individuals with DS 17 . There are several working hypotheses that attempt to explain the phenotypic variability in trisomy 21 (or other trisomies; see BOX 1). The gene (or genomic func- tional unit) dosage imbalance is the main molecular mechanism that requires further investigation. Genomic content of HSA21 The almost complete, high-quality sequence of 21q was published in May 2000 (REF. 18).There are three cloning gaps on 21q, each of which is only 20?30 Kb long. There are, therefore, 4 contigs for 21q (from centromere to telomere): 28,602; 229; 1,378; 3,432 Kb for a total length of 33,642,989 nucleotides (NCBI Build 34). Only a region of 281 Kb has been sequenced from the short arm of HSA21, 21p. FIGURE 1 provides a comparison of chromosome 21 features with those of other chromo- somes. The comparisons are based on the recent NCBI builds, and on published sequences 19,20 . HSA21 is among the smallest of human chromo- somes and its 33.6 Mb-long arm represents ~1% of the total sequences obtained (3020.3 Mb of NCBI Build 34 version 1). TABLE 1 lists other characteristics of HSA21. The total number of genes (protein-coding and non-cod- ing RNAs (ncRNAs)) on 21q has not yet been conclu- sively determined. A total of 225 genes was estimated when the initial sequence of 21q was published 18 , 35% of those are homologous to Drosophila melanogaster, 35% to Caenorhabditis elegans and 17% to Saccharomyces cerevisiae genes. Subsequent on-going analysis based on computational methods, EST sequencing, laboratory ver- ification and comparative genome analysis, resulted in an estimated 261?364 protein-coding genes (TABLE 1,refer- ences wherein and REFS 18,21?28). These include potential transcripts that are supported by only one ?spliced? EST. There are gene-rich and gene-poor regions on 21q, which correlate with the G+C content. The so-called L isochores (G+C <43%) contain few genes (1 per 300 Kb), whereas the H3 isochores in GIEMSA light chromosomal bands (G+C >48%) contain most of the genes (1 per 58 Kb, according to the first annotation). More research is needed for the complete and correct genic annotation of HSA21. This is particularly true for single exon genes, ncRNAs and rare transcripts. An international collaborative effort to re-annotate HSA21 is in progress. Computational analysis has provided ini- tial evidence for 5 microRNAs (miRNAs) on 21q, although their function and potential involvement in DS remain unknown. Comparative sequence analysis of human and other genomes, particularly that of the mouse, has resulted in the discovery of novel genes and verification of putative transcripts 25,29 .The availability of other mammalian ATRIOVENTRICULAR CANAL A complex congenital heart defect characterized by atrial septal defect, ventricular septal defect and abnormalities of the tricuspid and mitral valves. DUODENAL STENOSIS/ATRESIA Narrowing (stenosis) or complete obliteration of the duodenal lumen. GIEMSA BANDS Chromosome banding pattern produced by Giemsa staining. CPG ISLANDS A genomic region of about one kilobase that contains close to the theoretical, expected frequency of the CpG dinucleotide. � 2004 Nature Publishing Group 730 | OCTOBER 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS remaining 2,262 conserved sequences mapped preferen- tially to the gene-poor regions of 21q. About 80% of these sequences are located in intergenic regions, with the remaining 20% in introns. Experimental, bioinformatic and evolutionary analysis strongly suggested that these sequences are not ?functionally? transcribed, and do not correspond to protein-coding genes 35,36 .These sequences, which we call conserved non-genic (CNG), account for ~1% of the HSA21 sequence. The CNGs are highly con- served in mammals; from primates to monotremes, to marsupials 37?40 .The remarkable conservation, over The completion of the sequencing of HSA21 (REF. 18) and of the mouse genome 29,34 provided the first oppor- tunity to compare DNA sequences of entire chromo- somes of two mammalian species, so that conserved genomic elements that are likely to be functional can be identified. The comparison of the 33.5 Mb of HSA 21q with the orthologous mouse genomic regions on MMU16, MMU17 and MMU10 revealed 3,491 sequences of ?100 bp and ?70% identity without gaps. Unexpectedly, only 1,229 of those corresponded to exons of previously known genes 35 .Remarkably, the MMU16 MMU17 MMU10 HSA21 41-42 cM 78.4-80.6 Mb Mouse partial trisomies 47-70 cM 76.7-100 Mb 21q11 21q21.1 21q21.2 21q21.3 21q22.11 21q22.12 21q22.13 21q22.2 21q22.3 17-17.4cM 30.6-31.7 Mb RBM11, ABCC13, STCH, SAMSN1 NRIP1, Q9H386 USP25, Q8TDA7 CXADR, BTG3 Q96PM7, Q9BXE6 C21orf91, CHODL PRSS7, PPIA NM_030970, NCAM2 Q9NSI7, Q96G90 C21orf74, C21orf42 MRPL39, JAM2 ATP5J, GABPA APP, Q96PM8 CYYR1, ADAMTS1 ADAMTS5, C21orf94 C21orf100, N6M1_HUMAN ZNF294, C21orf6 C21orf6, USP16 CCT8, C21orf7, BACH1 C21orf41, Q8TCZ6 CLDN17, CLDN8 KRTAP cluster, TIAM1 SOD1 SRA4_HUMAN, HUNK C21orf45, C21orf61 C21orf108, C21orf119 C21orf63, C21orf63 C21orf77, TCP10L C21orf59, SYNJ1 C21orf66, Q9BSL5 C21orf62, OLIG2 OLIG1, IFNAR2 IL10RB, IFNAR1 IFNGR2, C21orf4 C21orf55, GART SON, DONSON CRYZL1, ITSN1 ATP5O, MRPS6 C21orf82, KCNE2 C21orf51, Q8TCY0 KCNE1, DSCR1 CLIC6, RUNX1 C21orf18, CBR1 CBR3, C21orf5 Y136_HUMAN, CHAF1B CLDN14, PSMD4P SIM2, HLCS DSCR6, DSCR5 TTC3, DSCR9 DSCR3, DYRK1A KCNJ6, DSCR4 DSCR8, DSCR10 KCNJ15, ERG ETS2, C21orf104 DSCR2, WDR9 C21orf87, Q96N32 WRB, CU13_HUMAN SH3BGR, C21orf88 B3GALT5, Q9NSI5 PCP4, Q9NSI4 BACE2, Q9NSI3 FAM3B, MX2 MX1, TMPRSS2 NM_152506, ANKRD3 PRDM15, C21orf25 ZNF295 K179_HUMAN H2BFS HSF2BP C21orf84 SNF1LK CRYAA U2AF1 CBS PKNOX1 NM_030970 Q9BSD2 NDUFV3 WDR4 PDE9A SLC37A1 TSGA2 UBASH3A TMPRSS3 TFF1 TFF2 TFF3 ABC G1 NM_173568 HRMT1L1, S100B DIP2_HUMAN, PCNT2 C21orf58, C21orf57 MCM3APAS, MCM3AP C21orf85, Q8NAP6 LSS, C21orf56 FTCD, COL6A2 COL6A1, Q9NSH7 PCBP3, Q9NSH8 SLC19A1, COL18A1 Q8N8W7, Q9NSH9 C21orf93, C21orf86 C21orf80, Q9BW55 C21orf89, Q9NSI0 Q9NSI1, ADARB1 C21orf122, C21orf70 C21orf67, ITGB2 PTTG1IP, Q9BXE6 SMT3H1, UBE2G2 KRTAP10-10, C21orf90 C21orf29, C21orf30 LRRC3, TRPM2 C21orf2, PFKL AIRE, DNMT3L ICOL_HUMAN, C21orf33 PWP2H, TMEM1 Q8TEB0, YU01_HUMAN AGPAT3, NNP1, CSTB, C21ORF97, PDXK 23 genes, 1.1 Mb 58 genes, 2.2 Mb 154 genes, 23.3 Mb 132 genes 85 genes 46 genes T s1Cje Ms1T s65 Ts 6 5Dn Figure 2 | Regions of synteny between human chromosome 21 (HSA21) and mouse chromosomes (MMUs) 16, 17 and 10. There are three partial trisomy mouse models of human trisomy 21, all trisomic for a portion of MMU16. The gene content of these partial trisomies is shown on the right. The list of gene names in Ts65Dn is from REF. 43. Sequence and gene data are taken from ENSEMBL. � 2004 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 5 | OCTOBER 2004 | 731 REVIEWS generated by long-range PCR. A total of 24,386 SNPs were identified with the rare allele observed at least twice (in addition to 11,603 nucleotide variants that were seen only once). There were 4,135 inferred HAPLOTYPE BLOCKS with an average length per block of 7.83 Kb (REF. 41).Such studies describing the chromo- some-wide linkage disequilibrium maps provide the genomic infrastructure for the determination of SNPs involved in gene expression variation, and in the predis- position to different variable phenotypic manifestations of DS and to common, complex phenotypes. Mouse models and phenotypes HSA21 is homologous to mouse chromosomal regions that map three different chromosomes. From 21cen to 21qter, about 23.2 Mb are homologous to MMU16, 1.1 Mb to MMU17, and 2.3 Mb to MMU10 (FIG. 2). Importantly, none of the existing mouse models per- fectly mimic the chromosomal abnormality that is observed in DS. more than 150 million years of evolution since the common mammalian ancestor, strongly indicates that most CNGs are functional, although their function is unknown. Genomic variability The variability of HSA21 might be partially responsible for the different DS phenotypes. It is therefore necessary to determine the common and rare DNA variants on this chromosome. Approximately 1.26% of HSA21 con- sists of short sequence repeats that might be polymor- phic 18 .In addition, 105,334 probable SNPs have been identified (see Online links box). The initial assessment of LINKAGE DISEQUILIBRIUM structure of polymorphic sites involved re-sequencing 20 haploid human genomes from different ethnic groups using tiled oligonucleotide arrays of the non- repetitive fraction of 21q (REF. 41).For this study, HSA21 from each individual was separated in somatic cell hybrids and haploid DNA hybridization probes were LINKAGE DISEQUILIBRIUM Refers to the fact that particular alleles at nearby sites can co- occur on the same haplotype more often than is expected by chance. HAPLOTYPE BLOCKS Long stretches (tens of megabases) along a chromosome that have low recombination rates (and relatively few haplotypes). Adjacent blocks are separated by recombination hot spots (short regions with high recombination rates). Table 2 | Mouse models of Down syndrome* Model Genotype Neurological phenotype Segmental trisomy Ts16 Trisomy 16 Reduced brain size and some structural changes Ts65Dn Trisomic for App-Znf295 (~132 known genes) Learning and behavioural deficits Reduction of the cerebellar volume and granule cell density Reduced cell number and volume in the hippocampal dentate gyrus Reduction in excitatory (asymmetric) synapses in the temporal cortex at advanced ages Age-related degeneration of basal forebrain cholinergic neurons Astrocytic hypertrophy and increased astrocyte numbers Failure of retrograde NGF signalling Ts1Cje Trisomic for Znf295- Learning and behavioural deficits Sod1 (~85 known genes) (less severe than in Ts65Dn) Cerebellar dysmorphology (a subset of Ts65Dn features) Ms1Cje/Ts65Dn Trisomic for App-Sod1 Learning deficits (less severe than in Ts1Cje) (~46 known genes) Cerebellar dysmorphology (a subset of Ts65Dn features) C10-2 Chimeric mice with free HSA21 Learning and behaviour impairment Single genes TgSod1 Transgenic for SOD1 Learning defects TgPfkl Highly overexpressed cDNA TgS100? 2?12 copies Astrocytosis, neurite degeneration, slower to habituate to novelty, behavioural defects TgApp Low copy of YAC Cognitive/behavioural defects TgEts2 Highly overexpressed cDNA TgHmg14 2?6 copies TgDyrk1A 1?3 copies of YAC and Learning/memory defects highly overexpressed cDNA TgSim2 1?2 copies of BAC and Behavioural defects highly overexpressed cDNA *For references, see text and Refs 48,97,98. NGF, nerve growth factor. � 2004 Nature Publishing Group 732 | OCTOBER 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS mice with a particular HSA21 gene knocked out with the partial trisomy mouse. One such example has been pub- lished recently for Ifngr and Ifnar2 (REF. 55),which encode the interferon-? and -? receptor 2, respectively. The resulting mouse with trisomy 16, but only 2 instead of 3 copies of these genes, showed significantly improved fetal growth and cortical neuron viability. A more direct approach involves creating transgenic mice that overex- press one gene that is orthologous to HSA21. Several such transgenic mice have been developed, following the first such mouse that carried SOD1 transgenes 56 (TABLE 2).To achieve results that are biologically relevant to DS, the transgene (ideally the mouse gene) needs to be ?physiologically? regulated from its own regulatory ele- ments and only one extra copy needs to be expressed. Ideally, a total transcript level (endogenous and from the transgene) should be ~1.5 times that of the normal expression level. One such example has been described for Sim2; the mice that carry three copies of Sim2 are characterized by abnormal spatial exploration and social interactions, and reduced NOCICEPTION 57 . The best studied model is that of a partial (seg- mental) trisomy 16, named Ts65Dn (REF. 42).The tri- somy in this mouse mutant extends for at least 23.3 Mb from Mrpl39 to the Znf295 genes 43 (predicted to contain ~132 genes that are homologous to HSA21). Detailed morphological and behavioural characteriza- tion of the Ts65Dn mouse model revealed several abnormal phenotypes that are similar to those seen in human trisomy 21 (REFS 44?51).Additional partial tri- somies for MMU16 that extend from Sod1 to Znf295 (85 known genes) (Ts1Cje) 52 and from App to Sod1 (46 known genes) (Ms1Cje/Ts65Dn) 53 have been reported, and their phenotypic characterization is in progress. Neurological phenotypes in mouse models of DS are shown in TABLE 2.There is also a chimeric mouse model that has substantial numbers of cells that carry portions of HSA21 (REF. 54). The contribution of triplicated individual genes to the mouse phenotype could be further evaluated by the sequential deletion of one copy of the gene from the par- tial trisomy mouse. This could be achieved by crossing Figure 3 | Gene expression levels of trisomic genes in the Ts65Dn mouse model of Down syndrome. a | The Ts65Dn mouse is trisomic for a ~16 Mb region of mouse chromosome 16 containing ~132 genes. b | Summary of gene expression data of trisomic genes in Ts65Dn. Total data from 79 genes, 2 developmental stages and 6 tissues are shown. The percentages of genes with expression relative to the values 1.0 and 1.5 are also shown. c | Examples of expression data for the heart. The majority of genes present in 3 copies are expressed close to 1.5. A notable exception is the gene Ankrd3, which is highly overexpressed in both developmental stages P30, postnatal day 30;Ts, the trisomy state; Eu, the euploid state. Data are from REF. 60. 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ab cd HSA21 Expression category 21q11 21q21.1 47-70 cM 18% 37% 9% 36% 21q21.2 21q21.3 21q22.11 21q22.12 Ts65Dn Trisomic for ~132 genes 21q22.13 21q22.2 21q22.3 MMU16 Expression ~ 1.5 Expression ~ 1.0 Expression < 1.5 Expression > 1.5 6.0 P30 heart 11-month-old heart Ankrd3, 2.9x Ankrd3, 5.1x Three copies, mean expression 1.4 Normalized r elative expr ession ratio T s/Eu Two copies, mean expression 1.0 Three copies, mean expression 1.5 Two copies, mean expression 1.0 5.0 4.0 3.0 2.0 1.0 0.0 � 2004 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 5 | OCTOBER 2004 | 733 REVIEWS studied by RNA in situ hybridization, by RT-PCR on adult and fetal mouse tissues, and by in silico mining of ESTs (REFS 61,62). Initial attempts to study the global transcriptome differences between trisomy 21 and euploid state using microarrays or serial analysis of gene expression (SAGE) have also been published 63?66 (TABLE 3).A meta- analysis of the existing data, and additional data col- lection, are needed to draw biologically meaningful conclusions and to develop further hypotheses about the aetiology of DS. Finding candidate genes for DS phenotypes Given the extent of sequencing data and the efforts to gather functional genomic data, how can we identify and prioritize candidate genes for their contribution to DS phenotypes? Several criteria could be used on the basis of present and future functional analysis of HSA21. The spatial and temporal pattern of expression is one criterion. Another is the potential functional clas- sification of the predicted protein. Genes in which the expression varies widely between individuals might contribute to the variable phenotypes, whereas genes with no expression variation might underlie the pheno- types that are present in all individuals with DS. Abnormal phenotypes in transgenic mice with 1.5-fold overexpression would provide strong evidence for the role of such candidate genes. The map position of a gene in a HSA21 interval that is associated with a given phenotype is another strong criterion. The study of rare patients with partial trisomy 21 defined the genomic regions that harbour genes associated with some DS phenotypes. For example, a region that is crucial for the heart defect (see Supplementary information S2) was identified in this way 67 ; and so searches for genes and genetic variation that contribute to this phenotype should focus on this region. A number of investigators have described a ?DS critical region? that specifically contains genes that contribute to cognitive defects or other DS features. However, the definition of these regions has been Multicopy overexpression of a transgene from an exogenous promoter can also provide insights into gene function 58 , although not necessarily into its role in trisomy. The crossing of these animal models into different genetic backgrounds might uncover contri- butions of modifier genes that affect certain DS phenotypes. Gene expression in trisomy 21 Characterizing the spatial and temporal expression of HSA21 genes is an important step towards understand- ing how gene dosage imbalance affects DS phenotypes. Since the discovery of trisomy 21 in 1959 (REF. 59),it has been hypothesized that the genes that are present in 3 copies are overexpressed 1.5-fold relative to the euploid state. This hypothesis has only recently been tested in the Ts65Dn partial trisomy mouse model of DS (REFS 43,60).In one study, a total of 78 genes present in 3 copies on MMU16 were tested by quantitative RT-PCR in 6 mouse tissues at 2 developmental stages (postnatal day 30 and 11 months old) 60 .Surprisingly, only ~37% of genes are expressed at the theoretical value of 1.5-fold; ~45% of the genes are expressed at levels significantly lower than 1.5-fold, 9% are not sig- nificantly overexpressed and 18% had expression lev- els greater than 1.5-fold (FIG. 3).Similarly, a second study using cDNA arrays on nylon filters and quanti- tative RT-PCR to examine nine adult tissues found that nearly all triplicated genes had elevated transcript levels in most tissues where they were expressed, whereas a few showed downregulation, compensation or strong overexpression in a tissue-specific man- ner 43,60 .These data not only provide candidate genes for contribution to DS phenotypes, but also highlight the complex regulation of gene expression related to genomic dosage imbalance. The detailed and systematic gene expression profiles of HSA21 genes (and, for that matter, all human genes) should be one of the research priorities of the functional analysis of our genome. First attempts towards this goal have been recently published. A considerable number of mouse orthologues of HSA21 genes (~160) have been NOCICEPTION The perception of pain. SAGE Serial analysis of gene expression: a method for comprehensive analysis of gene expression patterns using a short sequence tag (10?14bp) for each RNA molecule. Table 3 | Transcriptome studies of trisomy 21 (T21), or mouse model, versus euploid state Comparison* Method Results References Human amniocytes: cDNA microarrays 187 upregulated 63 T21 X3, normal X4; UniGene I Incyte; (>2 SD);85 downregulated (>2 SD) pooled RNAs 8,466 human transcripts Human astrocyte Affymetrix microarrays 679 differentially 64 cell lines: T21 X4, U95Av2 or U133A; regulated (ANOVA p<0.05) normal X4; individual RNAs 18,462 human transcripts Human frozen fetal Affymetrix microarrays 725 differentially regulated 64 cerebrum: T21 X4, U133A; 18,462 human (ANOVA p<0.05) normal X4; individual RNAs transcripts Mouse cerebellum: Affymetrix microarrays 29 with >2-fold 65 Ts65Dn X3, normal X3; U74Av2; 12 488 mouse consistent difference; individual and pooled RNAs transcripts 1,532 discriminating probes Mouse brains: 152791 SAGE tags; 330 differentially 66 Ts65Dn X3, normal X7; 45,856 unique RNA tags expressed RNA tags (p<0.05) pooled RNAs * X denotes the number of unrelated cases used in each experiment. SAGE, serial analysis of gene expression; SD, standard deviation. � 2004 Nature Publishing Group 734 | OCTOBER 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS Diagnostic methods: old and new Cytogenetic analysis of metaphase karyotypes remains the standard practice to identify not only trisomy 21, but also all other aneuploidies and balanced translocations. Over the past 10 years however, several other methods have been developed and used for the rapid detection of trisomy 21, either in fetal life or after birth (FIG. 4). The most widely used is fluorescent in situ hybridiza- tion (FISH) of interphase nuclei, using HSA21-specific probes or whole-HSA21 PAINTING 70 .An alternative method that is now widely used in some countries is quantitative fluorescence PCR (QF-PCR), in which DNA polymorphic markers (microsatellites) on HSA21 are used to determine the presence of three different alleles 70 .This method relies on informative markers and the availability of parental DNA. Additional methods to measure copy number of DNA sequences include the multiple amplifiable probe hybridization (MAPH) 71 , controversial as there are patients with partial triplica- tions outside this region who, nevertheless, manifest some features of DS (REFS 67?69).This issue should be re- examined now that the complete sequence of HSA21 and better diagnostic tools are available. Finally, non-HSA21 transcripts with significant expression differences, as measured by microarray analysis in cells, tissues and organs with trisomy 21 versus normal organs, constitute a class of candidates that not only are likely to contribute to DS phenotypes, but could also be targets for potential therapeutic interventions. Identifying and prioritizing CNGs that might con- tribute to DS phenotypes will pose a particular chal- lenge. CNGs that are cis- or trans- regulators, or that harbour variation with functional consequences, are obvious candidates for dosage-related phenotypic abnormalities. PYROSEQUENCING A method for DNA sequencing, in which the inorganic pyrophosphate (PPi) that is released from a nucleoside triphosphate on DNA chain elongation is detected by a bioluminometric assay. a b c d 0 10 20 ES GTCAGTGAC 59% HSA21 HSA5 51% 0 10 20 ES GTCAGTGAC 0 50 100 150 D21S1270 D21S1270 325 320 0 50 100 150 D21S1270 D21S1270 325 320 D21S1270 330 Relative intensity Relative intensity Relative intensity Relative intensity Size Size Figure 4 | Trisomy 21 diagnostic methods: old and new. a | G-banded karyotype of a trisomy 21 female, showing three copies of human chromosome 21 (HSA21). b | Fluorescent in situ hybridization (FISH) of interphase nuclei of a trisomy 21 fetus. In each cell there are two green spots (LSI 13 SpectrumGreen probe, Vysis) and three red spots (LSI 21 SpectrumOrange probe, Vysis) marking the 13q14 and 21q22.13-q22-2 chromosomal regions, respectively. c | Quantitative fluorescence PCR (QF-PCR) of marker D21S1270 of a trisomy 21 proband (bottom line) and his mother (top line). There are three different alleles in the patient DNA (alleles of sizes 320, 325 and 330 bp), but only two in the patient?s mother (alleles 320 and 325 bp). d | Paralogous sequence quantification (PSQ). Two paralogous sequences, one mapping to HSA21 and the other to HSA5, which differ by a single nucleotide (in the example shown here, T on HSA21 and C on HSA5), are amplified and sequenced quantitatively (by PYROSEQUENCING) to determine the quantity of the variable nucleotide in a trisomy 21 proband (bottom line) and a parent (top line). Assuming similar amplification of both paralogous sequences, ratios of 0.5 for HSA21/HSA5, the two variable nucleotides in a normal control, and ratios of 1.5 in a trisomy 21 sample are expected. In this example, we observe 51% T and 49% C content in the parent, and 59% T and 41% C content in the trisomy 21 child. � 2004 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 5 | OCTOBER 2004 | 735 REVIEWS Cell surface receptors and ligands. Triplication of genes that encode receptors and ligands might also result in phenotypic differences. An illustrative example comes from Notch signalling in cell-fate determination in the epidermis of D. melanogaster.Cells that contained three copies of the wild-type Notch,which encodes a trans- membrane receptor, always adopted an epidermal fate, whereas cells with two copies of Notch had a 50% chance of adopting neural or epidermal fate 83 . A good example of dosage effects of genes encod- ing ligands is that of erythropoietin (EPO), which is the primary protein that regulates mammalian erythropoiesis. Transgenic mice with an extra copy of EPO develop POLYCYTHAEMIA with HAEMATOCRIT of ~80% (normal ~45%) 84 . Transporter molecules. Transporters such as members of the transmembrane ABC transporter family could also have gene dosage effects. ABCA1 transporter affects intracellular cholesterol transport, and pathogenic mutations in this locus cause high-density lipoprotein deficiency type 1. BAC transgenic mice with an extra copy of ABCA1 have significantly increased cholesterol efflux in different tissues, and elevated high-density lipoprotein (HDL) cholesterol 85 . Cell adhesion molecules. Cell adhesion molecules might also cause dosage sensitive phenomena. For example, the rate of aggregation of synthetic vesicles carrying the neural cell adhesion molecule (NCAM) is proportional to the concentration of NCAM 3.5 .Therefore, a 50% increase or decrease in NCAM concentration causes a 4-fold increase or 90% decrease in cellular adhesiveness, respectively 86 . Morphogens. Alterations in the concentration of mor- phogens can have considerable effects on development. One of the first examples to be described is bicoid (bcd) in D. melanogaster. Embryos derived from mothers that carry one, two, four or six copies of bcd-generated thresh- old concentrations of the homeodomain-containing BCD protein at progressively more posterior positions in these embryos 87 .These threshold concentrations of a morphogen dictate distinct developmental outcomes as a function of distance from the source; in the case of BCD protein gradient, the outcome is the development of the anterior body pattern of the fly. Regulatory elements. Duplication of regulatory elements could result in altered gene expression and specific pathological phenotypes. For example, tandem duplica- tion (owing to a transgene insertion) of a regulatory element ~1 Mb upstream of the Shh locus in the Ssq mouse, causes ectopic Shh expression resulting in preaxial polydactyly 10 .Therefore, three copies of any functional element in the genome might be responsible for specific trisomy-related phenotypes. Future directions of research The dissection of the genomic infrastructure of HSA21, the appreciation of its genomic variability and the con- servation of functional sequences in model organisms, and multiplex probe ligation assay (MLPA) 72 .A recent method, termed paralogous sequence quantification (PSQ), uses paralogous sequences to quantify the HSA21 copy number 73 . Finally, comparative genomic hybridization (CGH) on BAC chips can be used for the diagnosis of full trisomy or monosomy, and for partial (segmental) aneuploidies 5,74 . Examples of protein dosage imbalance How does a supernumerary (third) copy of normal (wild-type) alleles result in an abnormal phenotype? This question has, for a long time, interested researchers in this area 75?77 .Below, we provide some examples (mostly from model organisms) of potential molecular mechanisms that deal with this question. Subunits of multimeric proteins. Many protein complexes consist of different subunits that are encoded by genes on different chromosomes. The stoichiometry of subunits is usually well controlled for the normal function of the complex. Disturbance of the normal stoichiometry of subunits, owing to a super- numerary copy of the gene that encodes one subunit, might result in protein complexes of abnormal compo- sition and function. The nucleosome, for example, con- sists of a central core of eight histone proteins (two of each H2A, H2B, H3 and H4) with 146 bp of dsDNA coiled around it. The ratio of H2A and H2B over H3 and H4 is important for the fidelity of chromosomal replications and/or segregation in yeast 78 . Another example is that of nicotinic acetylcholine receptors (nAChRs), which are usually composed of 2 ?- and 3 ?-subunits. Experiments in Xenopus laevis oocytes and human embryonic kidney cells have shown that alteration of the stoichiometry of the sub- units of nAChR results in alterations of functional properties of the receptors 79,80 . Transcription regulators. Three or more copies of genes that encode transcription regulators might result in abnormal expression of downstream target genes, which in turn could have phenotypic consequences. A characteristic example is provided by the mouse BAC transgenic experiments using the clock gene, which encodes a basic helix-loop-helix-PAS domain transcrip- tion factor, a component of the circadian pacemaker system. Normal mice have mean circadian periods of 23.48 hours, whereas transgenic mice harbouring just 1?2 supernumerary but normal copies of the clock transgene on a BAC had significantly shorter circadian periods with a mean of 22.89 hours 81 . A further example is the protein product of the mouse Bmi1 locus. Bmi1 is a component of the chro- matin-associated polycomb complex and is involved in maintaining the transcriptionally repressed state of genes; it modifies chromatin in a heritable way. Transgenic mice that overexpress Bmi1 have dose- dependent skeletal anomalies, namely anterior trans- formation of vertebral identity 82 .In both examples, the phenotypic abnormalities are a consequence of the increased amount of a specific protein. FISH Fluorescent in situ hybridization is a method that uses fluorescent molecular DNA probes to visualize specific regions in chromosomes that hybridize to the probe. CHROMOSOME PAINTING Fluorescent in situ hybridization to chromosomes using a probe that represents a whole chromosome or a part of a chromosome. POLYCYTHAEMIA The increase of the red blood cell count, haemoglobin and the total red blood cell volume, accompanied by an increase in total blood volume. HAEMATOCRIT Percentage of red blood cells in blood. � 2004 Nature Publishing Group 736 | OCTOBER 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS regions might also contribute to similar phenotypes in non-trisomy 21 individuals. The extent of partial trisomy 21 and correlation with the presence of certain phenotypic features of DS needs to be re-evaluated. A complete tiling path of BAC or oligonucleotide array CGH, or other meth- ods, could be used to determine precisely the genomic trisomy 21, and therefore provide a comprehensive list of candidate genes (coding and non-coding) and conserved functional elements. Relevant miRNAs and other non-coding RNAs and their targets need to be identified, and dosage sensitiv- ity of the miRNAs and their potential involvement in certain DS phenotypes need to be evaluated. Global gene expression differences between trisomy and eusomy states need to be evaluated in human cell lines and tissues, or in those of model organisms such as the mouse. Once defined, such differences could be used for diagnostic purposes, but they will be most impor- tant for the initial determination of biological processes that are dysfunctional in full or partial trisomy 21. The problem of variability of expression owing to polymor- phisms could be avoided by studying tissues from dis- crepant identical twins (one twin with trisomy 21 and the other with a normal karyotype), or from clonal cell lines obtained from individuals with mosaic trisomy 21. All functional genomic elements of HSA21 need to be identified. The exploration of the genomic sequences is far from complete. Multi-species com- parative sequence analysis will provide initial evidence for additional protein-coding potential, ncRNAs and regulatory elements. Experimental analysis using whole chromosome approaches will hopefully provide convincing evidence for the diverse biological roles of a plethora of so far unknown functional regions. Finally, the sequencing of the short arm of HSA21, or at least the short arm of an acrocentric chromosome awaits completion. A complete contig of these sequences might be difficult or impossible to obtain owing to the presence of multiple repeats in each short arm and the ?homogenization? of the short arms of the acrocentric chromosomes. It is likely, however, that important but as yet unknown functional elements (including genes) might be present on these short arms. Therapy for trisomy 21? Our understanding of the molecular pathogenesis of DS is remarkably poor; it is therefore unlikely that an effec- tive therapeutic intervention will be found within the next decade. However, attempts towards treatment could be made in model organisms or in cultured cells. For example, reducing the total transcript levels of the dosage sensitive genes (perhaps by small interfering RNA (siRNA)) might alter cellular or whole organism phenotypes. Another option could involve pharmaco- logical interference with the dysregulated metabolic pathways or key target molecules that are crucial for phenotypic characteristics. Ultimately, the elucidation of the phenotypic conse- quences of gene dosage imbalance in DS might provide new opportunities for therapeutic interventions. provide new opportunities for understanding the func- tion of the HSA21-encoded genes, and for explaining the molecular pathogenesis of different phenotypic manifestations of DS. There are many important future research goals, which are listed below, but are by no means complete. Functional analysis of all HSA21 genes is a priority, par- ticularly in the context of development (timing and cel- lular specificity of expression). Studying HSA21 ortho- logues in model organisms would facilitate such analysis. The results might lead to the identification of candidate genes for specific DS phenotypes and for gene dosage sensitivity. Determining the variation of gene expression in dif- ferent tissues is crucial. Genes with considerable allelic variation of expression in the population will probably be identified. These genes are probable candidates for allele-specific contribution to specific DS phenotypes. At the same time, genes without polymorphic variabil- ity of expression are probably those that contribute in an allele-independent manner to DS phenotypes. Furthermore, genes that show considerable overlap of the RNA expression output between normal and tri- somy tissues or cells could be excluded as candidates. However, alterations in gene expression measured at the transcript level might not always accurately reflect alter- ations in protein levels 88 .It is therefore necessary to either validate the differences at the protein level and/or study differences in the proteome. Genomic variation in cis that is responsible for gene expression variation will need to be uncovered. This variation could then be used in association stud- ies to detect genes that are responsible for certain DS phenotypes. Initial association studies have been published for the heart defects that are associated with DS (REF. 89). Mouse models with trisomy of all HSA21 syntenic regions (that is, the MMU segments of chromosomes 16, 17 and 10) should be created and their phenotypes characterized in detail. Mice that are trisomic for single genes should also be generated as they provide valuable information on certain candidate genes. Another infor- mative approach involves ?subtracting? the third allele of a gene from the trisomy mouse using appropriate crosses with heterozygous or homozygous mice that carry a deletion of that gene. The function of the numerous CNGs on HSA21 will need to be determined. Identification of CNGs with potential gene regulatory function will reveal candidate genomic elements for dosage imbalance. Trisomy for some of them or their genomic variation might be related to certain DS phenotypes. CNGs that are not regulatory might also be involved in trisomy 21-linked phenotypes, owing to as yet unknown mechanisms. Determination of the non-HSA21 (trans-acting) genes or non-coding sequences that predispose to DS phenotypes will also be important. 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E., Lyle, R., Deutsch, S. & Reymond, A. Chromosome 21: a small land of fascinating disorders with unknown pathophysiology. Int. J. Dev. Biol. 46, 89?96 (2002). 98. Olson, L. et al. Down syndrome mouse models Ts65Dn, Ts1Cje, and Ms1Cje/Ts65Dn exhibit variable severity of cerebellar phenotypes. Dev. Dyn. 230, 581-589 (2004). Acknowledgements We thank H. Attar, E. Hafen, U. Schibler, K. Basler, A. Estreicher, M. Friedli, K. Casada, L. Curtis, D. Sutter, C. Borel, and C. Attanasio for their contributions in the preparation of the manuscript; S. Dahoun and D. Marelli for figures 4a,b, and F. Bena for figure 4c. We also thank R. Reeves and three anonymous reviewers for criti- cal reading of the manuscript and numerous insightful suggestions; all members (past and present) of the Antonarakis laboratory for discussions, debates and experimental data; the Swiss National Science Foundation, NCCR ?Frontiers in Genetics?, European Union/Swiss OFES, Lejeune and ?Childcare? Foundations for sup- port. We also thank the patients and their families for the donation of their samples and the continuous inspiration for research. Competing interests statement The authors declare no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene PLP1 | PMP22 | clock OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM Down syndrome | CMT1A | SHFM3 | Hirschsprung disease Swiss-Prot: http://www.expasy.ch/ FURTHER INFORMATION Interpro: http://www.ebi.ac.uk/interpro/ Gene Ontology Annotation: http://www.ebi.ac.uk/GOA/ Single Nucleotide Polymorphism: http://www.ncbi.nlm.nih.gov/SNP/ SUPPLEMENTARY INFORMATION See online article: S1 (table) | S2 (table) Access to this links box is available online � 2004 Nature Publishing Group "
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