Down syndrome (DS) is caused by trisomy of human chromosome 21 (HSA21). It is the most common genetic cause of significant intellectual disability. The incidence of trisomy is influenced by maternal age and differs between populations (between 1 in 319 and 1 in 1000 live births are trisomic for Hsa21) (O’Nuallain et al., 2007).There is wide variability in the phenotypes associated with DS. Besides mental retardation, present in every individual with Down syndrome (DS), trisomy 21 is associated with more than 80 clinical traits including congenital heart disease, duodenal stenosis or atresia, imperforate anus, Hirschprung disease, muscle hypotonia, immune system deficiencies, increased risk of childhood leukemia and early onset Alzheimer’s disease. The mechanisms by which this aneuploidy produces the complex and variable phenotype observed in people with Down syndrome are still under discussion. The recent recognition that there are many genetically active elements that do not encode proteins makes the situation more complex. Additional complexity may exist due to possible epigenetic changes that may act differently in DS. This article is the second one discussing suggested genetic mechanisms that underlie DS.  
 

Genes on 21q: An Update 
 

The almost complete DNA sequence of the long arm (21q) of HSA21 was determined and published in Nature (Hattori et al., 2000. This represented a breakthrough for research in DS, greatly assisting in the identification of every gene and non-coding sequence of 21q. Although the precise gene catalogue has not yet been conclusively determined, Gardiner and Costa (2006) have estimated more than 400 genes and putative genes from the finished sequence of HSA21. This number of genes recognized on HSA21 is likely to continue to increase. The proteins encoded by these genes fall into several functional categories including transcription factors, regulators and modulators (18 genes); proteases and protease inhibitors (6 genes); ubiquitin pathway (4 genes); interferons and immune response (9 genes); kinases (8 genes); RNA processing (5 genes); adhesion molecules (4 genes); channels (7 genes); receptors (5 genes); and energy metabolism (4 genes). Interestingly, ~1% of the HSA21 corresponds to conserved non-genic (CNG) sequences, that is, sequences that are not “functionally” transcribed and do not correspond to protein-coding genes (Dermitzakis et al., 2002; Dermitzakis et al., 2004. The significant conservation of these sequences indicates that they are functional, although their function is unknown.  
 

The existence of a “Down Syndrome Critical Region” (DSCR), a small segment of HSA21 that contains genes responsible for many features of DS, has dominated the field of DS research for three decades. Accordingly, a number of genes contained in this ~5.4 Mb region have been extensively studied as an attempt to find out their potential contributions to DS. Two of these genes are DSCR1 andDSCR2.  
 

The DSCR1 (“Down Syndrome Critical Region 1”) protein, now renamed RCAN1 (from “Regulator of Calcineurin 1”) (Davies et al., 2007) is over-expressed in the brain and heart of Down syndrome fetuses suggesting that its overexpression may be involved in the pathogenesis of Down syndrome, particularly mental retardation and/or cardiac defects. Arron et al. (2006) reported that the genes RCAN1 and DYRK1A, both contained within the DSCR, act synergistically to prevent the nuclear occupancy of NFATc transcription factors. DYRK1A is a priming kinase that facilitates the further phosphorylation of numerous proteins by other kinases (Fig. 1). They suggested that the 1.5-fold increase in dosage of RCAN1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Down syndrome.  
 

In an independent study, increased DYRK1A gene dosage was shown to decrease the expression level of RE1-silencing transcription factor (REST) (Canzonetta et al., 2008). As REST is required both to maintain pluripotency and to facilitate neuronal differentiation, a perturbation in REST expression may alter the development of many cell types. Indeed, over-expression of DYRK1A in some animal models is associated with a number of phenotypes, including heart defects and abnormal learning and memory. However, not all animal models that over-express DYRK1A exhibit these defects, suggesting that polymorphisms or differences in the expression of other genes influence the outcome of DYRK1A trisomy (Olson et al., 2004). On the other hand, Korbel et al., (2009) excluded a necessary role for a number of genes previously suggested to be critical for DS congenital heart disease (DSCHD), including D21S55/KCNJ6, RCAN, Collagens 6A1/2 and 18, and DYRK1A. They proposed a 1.77-Mb DSCHD critical region, which contains 10 genes including the promoter and a portion of the Down syndrome cell adhesion molecule (DSCAM) gene (up to intron 11–12). Of the genes in the region, only DSCAM is known to be highly expressed in the developing heart, implicating it as a likely candidate for causing DSCHD/AVSD (atrio-ventricular septal defect). Moreover, their map ruled out an essential role for several genes in specific DS phenotypes because the phenotype is observed in the absence of trisomy of the relevant region. For example, their data do not support a necessary synergistic contribution to MR or DSCHD of the genesDSCR1 and DYRK1A that were proposed to destabilize NFATC pathways although both genes may contribute. Finally, their data were inconsistent with the hypothesis that the DSCR1 and DYRK1A genes form a critical region causing most DS features - indeed, several patients with severe DS features display segmental trisomies that do not include DSCR1. Thus, their results indicated that there was no DSCR, i.e., no single region ofHSA21 responsible for all or most severe DS features. 
 

The gene DSCR2 (“Down Syndrome Critical Region 2”) is highly expressed in all proliferating tissues and cells, such as fetal tissues, adult testis and cancer cell lines (Vidal-Taboada et al., 2000). Hirano et al. (2005) have recently designated DSCR2 as “Proteasome Assembling Chaperone-1” (PAC1). PAC1 and PAC2 are chaperones that function as heterodimers in the maturation of mammalian 20S proteasomes. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes. Thus, the product of the gene DSCR2 is involved in the correct assembly of 20S proteasomes. 
 

There are eighteen genes located on HSA21 that encode transcription factors and co-regulators/modulators of transcription. These proteins are directly and indirectly involved in transcription regulation and alterations in their expression levels could impact the expression of downstream targets. A recent study provided the evidence that trisomy for the DSCR is necessary but not sufficient for the brain phenotypes observed in trisomic mice (Olson et al., 2007). Thus, although HSA21 genes are likely to contribute to DS, the abnormalities seen in the patients are multifactorial conditions and are the result of genetic, environmental and stochastic influences (Reeves et al., 2001). Besides the complete characterization of HSA21 genes, we need to understand the transcriptional effects caused by trisomy 21. 
 

Infleunce of HSA21 Gene Expressions on DS

The imbalance in expression of HSA21 and non-HSA21 genes is hypothesized to result in the many phenotypes that characterize DS. However, only some of the HSA21 genes are likely to be dosage-sensitive, such that the phenotype they confer is altered by gene-copy number. Thus to understand DS, it is crucial both to understand the genomic content of HSA21 and to evaluate how the expression levels of these genes are altered by the presence of a third copy of HSA21 (Wiseman et al., 2009). A number of fusion transcripts that are encoded by two or more genes previously considered to be separate have been reported, such as the transcript encoded by exons from the HSA21, DONSON and ATP50 genes (Birney et al., 2007). The impact of this gene dosage effect on the whole transcriptome is still debated. The primary gene-dosage effects as well as the trans-acting gene-dosage effects will produce a phenotypic effect. The presence of CNG sequences on HSA21 indicates that they may also have a role in the generation of DS phenotypes although this has yet to be confirmed. Interestingly, studies performed on human trisomic tissues indicate that only a subset of HSA21 genes is over-expressed relative to euploid controls and that the increase in expression may be different from the expected ~1.5-fold (Mao et al., 2005). Also, the set of over-expressed HSA21 genes differs across the trisomic cell types (Li et al., 2006). These findings indicate that the presence of three copies of a gene does not necessarily result in overexpression and that other factors (e.g. developmental stage, tissue-specific differences) also affect gene expression. The extensive variation in the expression of HSA21 genes observed among unaffected individuals (Deutsch et al., 2005) might underlie some of the phenotypic variability seen in the patients. Furthermore, a recent report indicates that many HSA21 genes are likely to be compensated in DS and some of them are highly variable among individuals (Aït Yahya-Graison et al., 2007). The increase in expression of some HSA21 genes would induce changes in the global gene expression pattern that ultimately contribute to the DS phenotypic features. Different sets of non-HSA21 genes show up- or down regulation as a consequence of chromosomal imbalance. It is likely that some (if not all) the DS phenotypic features are not directly attributable to single gene(s) but are at least in part the result of a more generalized gene dysregulation caused by the triplicated chromosome (Sommer et al., 2008).  
 

Future Prspects

DS was once thought to be an intractable condition because of the genetic complexity underlying it. Here, we have described recently reported breakthroughs in the understanding of HSA21 trisomy, illustrating that research efforts in this field are making significant strides to understand for the debilitating aspects of the syndrome. Despite this more research is needed before we can elucidate the numerous pathogenic mechanisms associated with this complex disorder. Many issues vital to the health and well-being of people with DS remain to be studied, making this an important and exciting time for HSA21 trisomy research.

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