All Creatures Great and Small: New Approaches for Understanding Down Syndrome Genetics
Authors: Anna J. Moyer, Katheleen Gardiner, Roger H. Reeves
Journal: Trends in Genetics (2020)

Down syndrome (DS) is caused by an extra copy of ~500 genes.Understanding how human chromosome 21 (Hsa21) genes contribute to DS phenotypes is essential for developing effective treatment strategies.Many Hsa21 genes functionally interact, and overexpression of these genes perturbs expression of genes throughout the genome.DS research has focused on only a few Hsa21 genes.Diverse model organisms from yeast to mice are being used to study the effects of aneuploidy, to annotate functions of Hsa21 orthologs, and to model aspects of DS pathology. Human chromosome 21 (Hsa21) contains more than 500 genes, making trisomy 21 one of the most complex genetic perturbations compatible with life. The ultimate goal of Down syndrome (DS) research is to design therapies that improve quality of life for individuals with DS by understanding which subsets of Hsa21 genes contribute to DS-associated phenotypes throughout the lifetime. However, the complexity of DS pathogenesis has made developing appropriate animal models an ongoing challenge. Here, we examine lessons learned from a variety of model systems, including yeast, nematode, fruit fly, and zebrafish, and discuss emerging methods for creating murine models that better reflect the genetic basis of trisomy 21. Human chromosome 21 (Hsa21) contains more than 500 genes, making trisomy 21 one of the most complex genetic perturbations compatible with life. The ultimate goal of Down syndrome (DS) research is to design therapies that improve quality of life for individuals with DS by understanding which subsets of Hsa21 genes contribute to DS-associated phenotypes throughout the lifetime. However, the complexity of DS pathogenesis has made developing appropriate animal models an ongoing challenge. Here, we examine lessons learned from a variety of model systems, including yeast, nematode, fruit fly, and zebrafish, and discuss emerging methods for creating murine models that better reflect the genetic basis of trisomy 21. Down syndrome (DS) comprises a collection of phenotypic features that are common in individuals who inherit an extra copy of all or, rarely, part of the long arm of human chromosome 21. All individuals with DS display some level of intellectual disability and will develop the neuropathology of Alzheimer's disease (AD) at an early age. They also present with hypotonia, cerebellar hypoplasia, and midface skeletal retrusion. Additional common, but not universal, features include congenital heart defects, abnormalities in immune system function, thyroid deficiency, childhood leukemia, and early-onset dementia [1.Capone G.T. Down syndrome: advances in molecular biology and the neurosciences.J. Dev. Behav. 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Given that the incidence of DS worldwide remains at more than one in 1000 live births, it represents a significant social and medical challenge [10.Canfield M.A. et al.National estimates and race/ethnic-specific variation of selected birth defects in the United States, 1999-2001.Birth Defects Res. A Clin. Mol. Teratol. 2006; 76: 747-756Crossref PubMed Scopus (464) Google Scholar,11.Irving C. et al.Twenty-year trends in prevalence and survival of Down syndrome.Eur. J. Hum. Genet. 2008; 16: 1336-1340Crossref PubMed Scopus (93) Google Scholar]. The long-term approach to DS research has been to focus on understanding which subsets of human chromosome 21 (Homo sapiens, Hsa21) genes contribute to specific phenotypic features and to develop therapies to control these outcomes. While gene-based approaches were necessarily reductionist before the completion of the genome sequence, advances in understanding genome organization, its regulation, and its products have highlighted the extraordinarily complex challenge of DS. Hsa21 encodes >500 genes (~200 protein-coding genes, a small number of known miRNAs, and >300 genes that may represent functional RNAs, novel proteins, or transcriptional noise) [12.Gupta M. et al.Mouse models of Down syndrome: gene content and consequences.Mamm. Genome. 2016; 27: 538-555Crossref PubMed Scopus (64) Google Scholar]. The functions of most Hsa21 genes, even those encoding proteins, are unknown. Overexpression of these genes due to trisomy (see Glossary) causes widespread disruptions in the transcriptome and proteome, affecting development and function of most organs and organ systems. Model organisms are essential for determining gene functions and for creating models of trisomy. While mouse has long been the model organism of choice for many good reasons, it has its own limitations and challenges. Increasingly, other less complex organisms are being exploited, among them zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), nematode (Caenorhabditis elegans), and yeast (Saccharomyces cerevisiae). To maximize the potentials of these systems, it is necessary to understand the unique advantages and limitations of each. Here, we discuss conservation of Hsa21 genes among these organisms and review recent studies that have shed light on the functions of individual Hsa21 genes and how their collective misexpression may work in concert to create the features of DS. Hsa21 is the smallest of human chromosomes. It was the second human chromosome to be completely sequenced [13.Hattori M. et al.The DNA sequence of human chromosome 21.Tanpakushitsu Kakusan Koso. 2001; 46: 2254-2261PubMed Google Scholar] and, since then, it has been extensively annotated [12.Gupta M. et al.Mouse models of Down syndrome: gene content and consequences.Mamm. Genome. 2016; 27: 538-555Crossref PubMed Scopus (64) Google Scholar,14.Harrow J.L. et al.The Vertebrate Genome Annotation browser 10 years on.Nucleic Acids Res. 2014; 42: D771-D779Crossref PubMed Scopus (36) Google Scholar]. Given that the short arm, Hsa21p, encodes largely repetitive DNA, it likely makes little contribution to the phenotypes of DS. The most recent manual annotation of the long arm, Hsa21q, which spans ~35 Mb, identified ~500 genes (this number excludes pseudogenes because they are nonfunctional). These included 164 classical protein-coding genes, 49 members of the gene family of keratin-associated proteins (KRTAPs), and ~300 additional transcripts that encode ambiguous open reading frames (ORFs; short ORFs, multiple ORFs, or no ORFs), which may or may not be functional. Given that expression of KRTAP genes is restricted to skin and because the family may include many pseudogenes, these genes are not considered here. Among the non-KRTAP protein-coding genes are many transcription factors/modulators, components of the ubiquitin/proteasome and RNA processing pathways, several with known sex differences in functional properties, and many others relevant to AD, including the amyloid precursor protein (APP) and proteins that modulate APP processing or trafficking [12.Gupta M. et al.Mouse models of Down syndrome: gene content and consequences.Mamm. Genome. 2016; 27: 538-555Crossref PubMed Scopus (64) Google Scholar]. Most of these functional properties are merely predicted from sequence similarities and, similar to genes throughout the genome, few Hsa21 proteins have been experimentally assessed. Fewer still have been studied for consequences when trisomic and overexpressed individually or in the context of trisomy 21. Reliable identification of orthologous genes in model organisms requires fulfillment of several criteria, the first being a statistically significant sequence similarity (Box 1). Given their high sequence conservation, orthologs of Hsa21 protein-coding genes can be identified with high confidence in mammals when a comprehensive transcriptome and/or genomic sequence is available. In chimpanzee, Ptr21q is essentially conserved with Hsa21q, with no species-specific genes (Table 1). In mouse, Hsa21q genes are found within three chromosomal regions: 105 protein coding genes in the ~28 Mb of the telomeric region of mouse chromosome (Mmu) 16; ~20 in an internal ~1.5-Mb segment of Mmu17; and ~40 genes in an internal ~3-Mb segment of Mmu10 [15.Davisson M.T. et al.Evolutionary breakpoints on human chromosome 21.Genomics. 2001; 78: 99-106Crossref PubMed Scopus (28) Google Scholar]. In rat, the segment syntenic with Mmu16 is found on Rattus norvegicus chromosome (Rnor) 11, while the regions syntenic with Mmu17 and Mmu10 are contiguous on Rnor20. Four Hsa21q protein-coding genes are specific to primates (Table 2); all four show highly restricted expression patterns, largely confined to testes, prostate, placenta, and/or ovary.Box 1Considerations for Hsa21 Ortholog IdentificationStatistical Considerations Concerning Alignment with Hsa21 Protein(i) Minimum Expect (E) value: the number of matches expected by chance.(ii) Percentage of amino acids that are identical and similar (in physiochemical properties) to Hsa21 protein: criteria that are too strict will miss Hsa21 orthologs, while those that are too relaxed will identify non-orthologs.Functional Considerations(i) Gaps and coverage: differences in protein lengths suggest differences in function(s).(ii) Domain composition: differences presumably affect function(s).(iii) Alternative splice variants: for example, Hsa21-encoded SYNJ1 and ITSN1 are produced in two major variants (L and S) that differ in tissue specificity of expression, suggesting tissue-specific differences in functions; only one major form is found in non-vertebrates.(iv) Gene copy number: members of a gene family may have evolved different functions; this is relevant when a Hsa21 gene is a member of a gene family versus a single gene in non-vertebrates, and because whole-genome duplication in teleost fish has resulted in some Hsa21 genes with two co-orthologs in zebrafish.Technical Considerations and Relevance to DS(i) Expression level: in DS, trisomy most often results in a modest ~50% increase in mRNA and protein level versus a knockout, mutation, or many-fold elevation in expression with cDNAs.(ii) Genomic context: in mouse models of DS, the number and nature of trisomic genes influences phenotype. Examples include: trisomy of Hsa21-encoded DYRK1A alone or in concert with KCNJ6 versus one of several genes trisomic in the Ts1Rhr or the Ts65Dn models has different phenotypic consequences, and the phenotype of the mouse trisomic for the Mmu16 Hsa21 orthologous segment is altered by addition of trisomy of the Mmu17 orthologous segment.(iii) Stoichiometry of pathway/complex components, substrates, or interaction partners: a different phenotype may be produced with trisomy of only one Hsa21 component versus complete trisomy. Examples include: the Hsa21 proteins ITSN1, APP, and SYNJ1 are substrates of the Hsa21 kinase DYRK1A, and the Hsa21-encoded PDXK phosphorylates Vitamin B6 to pyridoxal-5-phosphate, which is required for Hsa21-encoded CBS protein activity.(iv) Genomic background/strain: phenotype may be altered, exacerbated, or masked, dependent on strain.(v) Sex dependence: some phenotypes in DS differ in penetrance or expressivity between males and females. In animal models, separate cohorts of males and females should be analyzed.Table 1Orthologs of Hsa21q Protein Coding GenesSpeciesConservedSyntenic regionsGenetic manipulations availableImplied hypothesis of DS pathogenesisExample phenotypesChimpanzee (Pan troglodytes)164Ptr21Naturally occurring trisomy of Ptr21Same as human DSnot applicable, except observations of captive-born animals [94.Hirata S. et al.Chimpanzee Down syndrome: a case study of trisomy 22 in a captive chimpanzee.Primates. 2017; 58: 267-273Crossref PubMed Scopus (15) Google Scholar]Rat (Rattus norvegicus)158Rnor11,20Individual gene targeting with CRISPR/Cas9, direct subchromosome duplication, introduction of human chromosomesSimultaneous overexpression of many genes causes net phenotypeRefined behavior, preferred model for pharmacology, larger animal allows some surgical interventions that are more difficult in miceMouse (Mus musculus)158Mmu16,17,10Segmental duplications of regions with conserved synteny, overexpression or knockout of single genes, introduction of human chromosomesOverexpression of many genes in segmental duplications or overexpression of single genes causes phenotypesDiverse phenotypes in many organ systems, diverse behavioral assaysZebrafish (Danio rerio)137Addition and knockdown/knockout of genes, many transgenic fish marking key developmental pathways availableOverexpression of one or a few genes is sufficient to cause a phenotype; assumes genes and pathways are well conserved and have conserved roles in developmentDevelopmental phenotypes caused by perturbation of major growth factor pathways (Shh and Wnt), behavioral assaysFruit fly (Drosophila melanogaster)59Tissue-specific knockdown and overexpression, temporal control, mutations in most genes available, mosaic analysisOverexpression of one or a few genes is sufficient to cause a phenotype; assumes genes and pathways are well conserved and have conserved roles in developmentLarval crawling, negative geotaxis, phototaxis, flight, activity, rhythmicity, courtship conditioning, habituation, associative learning, cellular morphology and physiologyNematode (Caenorhabditis elegans)42Tissue-specific knockdown and overexpression, temporal control, efficient transgenesis, RNAiOverexpression of one or a few genes is sufficient to cause a phenotype; assumes genes and pathways are well conserved and have conserved roles in developmentMechanosensation, chemosensation, habituation, thermotaxis, locomotion, feeding, reproduction, cellular morphology and physiologyYeast (Saccharomyces cerevisiae)26Modify expression of single genes, create defined aneuploidies, population selectionGeneral effects of aneuploidy on cell fitness, stress response pathways, etc.Cell growth, selection under different metabolic conditions, perturbations to overall protein/RNA levels Open table in a new tab Table 2Diversity in Evolutionary Conservation (from Protein Sequence Alignment) of Hsa21 Protein-Coding GenesaGene descriptions, functional and expression information, and amino acid (AA) length are for Hsa21 orthologs., bAbbreviations: Coverage, AA range of model organism protein included in alignment; E (Expect) value, the number of sequence hits in the database expected by chance; ID, identical amino acids; LTD, long-term depression; MWM, Morris Water Maze; NC, not conserved; Sim, amino acids with similar physiochemical properties; gaps, length of amino acids introduced to maximize alignment.SpeciesE value% ID% Sim% GapsCoverage (AA)Ortholog notesCCT8: θ subunit of the T-complex 1 chaperonin; may be involved in the transport and assembly of newly synthesized proteins; 548 AASaccharomyces cerevisiaee-17046%65%3%1–568/568Caenorhabditis eleganse-15851%67%6%1–578/581Increased expression in somatic tissues extends C. elegans lifespanDrosophila0.058%75%0%1–535/546Danio rerio0.082%89%0%1–546/548Overexpression causes pigment abnormalitiesMus musculus0.097%98%0%1–548/548ITSN1: multidomain, cytoplasmic membrane-associated protein involved in endocytosis, synaptic vesicle recycling. ITSN1-S, 2 EH, 5 SH3 domains,1220 AA; ITSN1-L, 2 EH, 5 SH3, PH, Rho-GEF domains; 1721 AAS. cerevisiae3e-2126%43%19%124–360/1381Not a likely ortholog; similarity only over two EH domains; lacks SH3 domainsC. elegans5e-61, 2e-4529%47%11%5–728, 786–1085/1087Two EH and five SH3 domains presentDrosophila3e-16933%59%12%1–1085/1190Two EH and five SH3 domains present; ITSN1-S onlyD. rerio0.076%83%5%1–1751/1751ITSN1-S and ITSN1-LM. musculus0.095%97%0%1–1719/1719ITSN1-L and ITSN1-SDONSON: DNA replication fork stabilization factor; mutations cause microcephalic dwarfism; ubiquitous expression in adult; 566 AAS. cerevisiaeNCC. elegans2e-1421%38%21%139–565/596Expression restricted to embryo; low similarity; possibly not an orthologDrosophila3e-5835%50%10%145–567/568Humpty Dumpty; required for cell proliferation in developmentD. rerio2e-3038%50%19%7–217/230Shorter protein length could be due to incomplete genomic annotationM. musculus0.077%85%1%1–56/560Ubiquitous expression in embryo and adultRWDD2B: arginine, tryptophan, aspartate (RWD) domain protein 2B; also contains domain of unknown function (DUF) 1115 domain; 319 AAS. cerevisiaeNCC. elegansNCDrosophila2e-2830%48%8%83–287/288DUF1115; no RDW domain; cannot be considered an orthologD. rerioe-10852%70%2%5-289/289DUF1115, RWD domains; overexpression causes U-shaped somitesM. musculus0.082%88%0%1-290/290DUF1115, RWD domainsPCP4: Purkinje cell protein 4; contains an IQ-motif [A-x(3)-I-Q-x(2)-F-R-x(4)-K-K]; modulates calmodulin signaling; 62 AAS. cerevisiaeNCC. elegansNCDrosophilaNCD. rerio5e-1678%82%1%1–58/63M. musculus1e-3797%98%0%1–62/62Involved in a cilia-beating deficit resulting in ventriculomegaly; overexpression results in elevated cerebellar LTD and a MWM deficitTFF3: three trefoil genes are clustered on Hsa21q22.3; contains a 40-AA trefoil motif with three conserved disulfides; expressed in gastrointestinal mucosa, immune system, and brain; 91 AAS. cerevisiaeNCC. elegansNCDrosophilaNCD. rerioNCM. musculus6e-3175%81%0%31–81/81Knockout causes hearing lossPrimate specificTCP10LT-complex 10-like; 210 AA; expressed in liver and testisDSCR4118 AA ORF; classed as noncoding RNA gene; expressed in placenta, prostate, testis, and small intestinePOTEDProstate, ovary, testis-expressed (POTE) ankyrin domain family member D; 584 AAPLAC4Placenta enriched protein 4; 10 kb RNA; longest ORF 150 AA; 13 ORFs >60 AA; classed as noncoding RNAa Gene descriptions, functional and expression information, and amino acid (AA) length are for Hsa21 orthologs.b Abbreviations: Coverage, AA range of model organism protein included in alignment; E (Expect) value, the number of sequence hits in the database expected by chance; ID, identical amino acids; LTD, long-term depression; MWM, Morris Water Maze; NC, not conserved; Sim, amino acids with similar physiochemical properties; gaps, length of amino acids introduced to maximize alignment. Open table in a new tab Statistical Considerations Concerning Alignment with Hsa21 Protein (i) Minimum Expect (E) value: the number of matches expected by chance. (ii) Percentage of amino acids that are identical and similar (in physiochemical properties) to Hsa21 protein: criteria that are too strict will miss Hsa21 orthologs, while those that are too relaxed will identify non-orthologs. Functional Considerations (i) Gaps and coverage: differences in protein lengths suggest differences in function(s). (ii) Domain composition: differences presumably affect function(s). (iii) Alternative splice variants: for example, Hsa21-encoded SYNJ1 and ITSN1 are produced in two major variants (L and S) that differ in tissue specificity of expression, suggesting tissue-specific differences in functions; only one major form is found in non-vertebrates. (iv) Gene copy number: members of a gene family may have evolved different functions; this is relevant when a Hsa21 gene is a member of a gene family versus a single gene in non-vertebrates, and because whole-genome duplication in teleost fish has resulted in some Hsa21 genes with two co-orthologs in zebrafish. Technical Considerations and Relevance to DS (i) Expression level: in DS, trisomy most often results in a modest ~50% increase in mRNA and protein level versus a knockout, mutation, or many-fold elevation in expression with cDNAs. (ii) Genomic context: in mouse models of DS, the number and nature of trisomic genes influences phenotype. Examples include: trisomy of Hsa21-encoded DYRK1A alone or in concert with KCNJ6 versus one of several genes trisomic in the Ts1Rhr or the Ts65Dn models has different phenotypic consequences, and the phenotype of the mouse trisomic for the Mmu16 Hsa21 orthologous segment is altered by addition of trisomy of the Mmu17 orthologous segment. (iii) Stoichiometry of pathway/complex components, substrates, or interaction partners: a different phenotype may be produced with trisomy of only one Hsa21 component versus complete trisomy. Examples include: the Hsa21 proteins ITSN1, APP, and SYNJ1 are substrates of the Hsa21 kinase DYRK1A, and the Hsa21-encoded PDXK phosphorylates Vitamin B6 to pyridoxal-5-phosphate, which is required for Hsa21-encoded CBS protein activity. (iv) Genomic background/strain: phenotype may be altered, exacerbated, or masked, dependent on strain. (v) Sex dependence: some phenotypes in DS differ in penetrance or expressivity between males and females. In animal models, separate cohorts of males and females should be analyzed. For non-mammalian model organisms, reliably identifying orthologs requires not only manual examination of significant sequence matches, but also functional information (Box 1). If a significant alignment involves only a limited segment of the total protein sequence, functional equivalence cannot be assumed; this is especially important if the model protein lacks functional domains seen in the Hsa21 protein. A similar problem exists if the ortholog lacks splice variants with specific domain features seen in the Hsa21 gene. Another criterion is expression pattern: highly tissue or developmentally specific expression in one organism and ubiquitous expression in the second must have consequences for functional similarity. Last is the challenging issue of gene families. For example, when a single gene in non-vertebrates produces equally significant alignments with a Hsa21 protein and multiple other human proteins, it cannot be assumed that the function of the non-vertebrate protein specifically reflects that of the Hsa21 protein. Six Hsa21 proteins selected to illustrate a range of the statistical and functional properties used to identify orthologs are shown in Table 2. For effective DS studies, in any model system, consideration of some specific technical issues is required. Commonly, experiments target single genes, often using cDNA constructs where normal regulation is absent and expression levels typically are higher than the modest increases of ~50% seen in DS. Thus, results of such experiments may present a gross exaggeration, lacking tissue specificity, of a very subtle perturbation. Alternatively, knockouts and/or knockdowns are used. Such experiments are useful for uncovering gene functions, but interpretation of results and claims of relevance to DS must be done with circumspection. Results do not reflect trisomic gene consequences in DS, but they can reveal the potential contribution of a pathway or cellular process worthy of investigation in a system that reflects the DS perturbation. Single gene experiments also miss the important effects of gene interactions and the stoichiometry of pathway components (Box 2). This principle has been well illustrated by the divergent phenotypes of mouse models trisomic for the Hsa21-encoded DYRK1A protein kinase (see also Table S1 in the supplemental information online for gene descriptions) in the presence of varying numbers and identities of additional Hsa21 orthologs [16.Altafaj X. et al.Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A (minibrain), a murine model of Down's syndrome.Hum. Mol. Genet. 2001; 10: 1915-1923Crossref PubMed Scopus (312) Google Scholar, 17.Olson L.E. et al.Trisomy for the Down syndrome 'critical region' is necessary but not sufficient for brain phenotypes of trisomic mice.Hum. Mol. Genet. 2007; 16: 774-782Crossref PubMed Scopus (132) Google Scholar, 18.Jiang X. et al.Genetic dissection of the Down syndrome critical region.Hum. Mol. Genet. 2015; 24: 6540-6551Crossref PubMed Scopus (39) Google Scholar]. Another consideration is that several targets of the DYRK1A kinase are encoded by Hsa21 [19.Adayev T. et al.MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins.Biochem. Biophys. Res. Commun. 2006; 351: 1060-1065Crossref PubMed Scopus (43) Google Scholar,20.Ryoo S.R. et al.Dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A-mediated phosphorylation of amyloid precursor protein: evidence for a functional link between Down syndrome and Alzheimer's disease.J. Neurochem. 2008; 104: 1333-1344Crossref PubMed Scopus (135) Google Scholar]. The consequences of elevated expression of a kinase (or protease, or other protein modifier, of which there are many encoded by Hsa21) without elevated expression of its targets may well be different from co-overexpression with its targets (as would occur in DS). The more ideal, segmentally trisomic models are particularly challenging to create in organisms other than mammals because syntenic regions tend to be short and difficult to splice together (Figure 1). A last important fact regarding Hsa21 genes is that no region of Hsa21 can be excluded from containing genes that contribute to DS features. The oft-cited 'DS Critical or Chromosomal Region' (DSCR) was clearly shown not to exist at the time it was first proposed and since [21.Korenberg J.R. et al.Down syndrome phenotypes: the consequences of chromosomal imbalance.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4997-5001Crossref PubMed Scopus (575) Google Scholar,22.Korbel J.O. et al.The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 12031-12036Crossref PubMed Scopus (273) Google Scholar]. Specifically, the diagnosis of DS, including the presence of intellectual disability, has been made in several cases of partial trisomy Hsa21 where the trisomic segments exclude the DSCR [22.Korbel J.O. et al.The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 12031-12036Crossref PubMed Scopus (273) Google Scholar]. More recently, another effort named a 34-kb segment of Hsa21 the high-resolution DSCR, again using a minimal overlap region among DS cases due to partial trisomy [23.Pelleri M.C. et al.Systematic reanalysis of partial trisomy 21 cases with or without Down syndrome suggests a small region on 21q22.13 as critical to the phenotype.Hum. Mol. Genet. 2016; 25: 2525-2538PubMed Google Scholar,24.Pelleri M.C. et al.Partial trisomy 21 map: ten cases further supporting the highly restricted Down syndrome critical region (HR–DSCR) on human chromosome 21.Mol. Genet Genomic Med. 2019; 7e797Crossref PubMed Scopus (12) Google Scholar]. No functional genetic elements are annotated in the segment and no individual trisomic for only this segment has been identified. The high-resolution DSCR has no stronger intellectual or phenotypic support than the original DSCR, for all the reasons clearly explained previously [22.Korbel J.O. et al.The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 12031-12036Crossref PubMed Scopus (273) Google Scholar]. Thus, all Hsa21 genes remain as candidates.Box 2Thinking about the Genetic Basis of DSDS-associated phenotypes are caused by triplication of Hsa21, which contains hundreds of genes that are overexpressed, often with tissue and developmental timing specificities, at modest levels. While DS phenotypes are generally easily diagnosed, they display both incomplete penetrance (occurring in only some people with DS) and variable expressivity (differing in severity among people with DS).Understanding how trisomy 21 causes the features of DS requires rigorous attention to development of DS models. Genomic context, or the number and nature of trisomic genes and genetic background, is critical. For example, relying on a 'model' that overexpresses a single Hsa21 gene implies that overexpression of the gene is 'sufficient' to cause a detectable phenotype in the absence of other trisomic genes. Modeling the additive effects of many genes requires a model system where multiple Hsa21 genes are overexpressed simultaneously. If returning a sin