Down syndrome is a condition caused by having an extra copy of one of the 46 chromosomes found inside human cells. Specifically, instead of two copies, people with Down syndrome are born with three copies of chromosome 21. This results in many different effects, including learning and memory problems, heart defects and Alzheimer’s disease. Each of these different effects is caused by having a third copy of one or more of the approximately 230 genes found on chromosome 21. However, it is not known which of these genes cause any of these effects, and how an extra copy of the genes results in such changes.

Now, Lana-Elola et al. have investigated which genes on chromosome 21 cause the heart defects seen in Down syndrome, and how those heart defects come about. This involved engineering a new strain of mouse that has an extra copy of 148 mouse genes that are very similar to 148 genes found on chromosome 21 in humans. Like people with Down syndrome, this mouse strain developed heart defects when it was an embryo.

Using a series of six further mouse strains, Lana-Elola et al. then narrowed down the potential genes that, when in three copies, are needed to cause the heart defects, to a list of just 39 genes. Further experiments then showed that at least two genes within these 39 genes were required in three copies to cause the heart defects.

The next step will be to identify the specific genes that actually cause the heart defects, and then work out how a third copy of these genes causes the developmental problems.

The formation of a functional four-chambered heart is a complex process and perturbations in its development can lead to congenital heart defects (CHD). These affect almost 1% of the population and are a major cause of morbidity and infant mortality (Fahed et al., 2013). The chance of being born with CHD is drastically increased (to ~50%) in DS (Vis et al., 2009) in which a range of heart defects is seen, including ventricular septal defects (VSD) and outflow tract abnormalities such as overriding aorta (OA) and double outlet right ventricle (DORV). Notably, defects that affect the atrioventricular (AV) junction, especially atrio-ventricular septal defects (AVSD) (Freeman et al., 1998) are often seen in DS. AVSD comprise a spectrum of cardiac malformations characterized by a common AV junction, guarded by an essentially common valve, as opposed to separate AV junctions guarded by mitral and tricuspid valves. People diagnosed with AVSD can present with communication between left and right heart chambers (shunting) at the atrial level (ostium primum defect) or at the ventricular level or with shunting at both atrial and ventricular levels (Mahle et al., 2006).

The process of forming the AV junction that divides the embryonic heart into a four-chambered structure involves the growth and fusion of a number of tissues from different precursor populations (Webb et al., 1998). These include the atrial and ventricular septa (mainly of myocardial origin), the mesenchymal cap of the atrial septum, and the superior and inferior endocardial cushions (endocardial origin). In addition, the dorsal mesenchymal protrusion (DMP) first described as the ‘spina vestibuli’ by Wilhelm His the elder in 1880 (His, 1880; Mommersteeg et al., 2006) makes an important contribution to septation of the AV junction (Anderson et al., 2015; Snarr et al., 2007a; Snarr et al., 2007b; Webb et al., 1998). The DMP is a mesenchymal structure at the venous pole of the developing heart derived from the second heart field (SHF) and perturbations of its development result in AVSD (Briggs et al., 2013; Goddeeris et al., 2008; Rana et al., 2014; Tian et al., 2010; Webb et al., 1999; Xie et al., 2012). Abnormalities in the DMP have also been detected in human fetal hearts with trisomy 21, leading to the hypothesis that malformation of this tissue causes the AVSD in DS (Blom et al., 2003).

Hsa21 carries 233 protein-coding genes (genome assembly GRCh38.p5) and it is thought that DS phenotypes result from an increased dosage of one or more of the genes on Hsa21. The search for dosage-sensitive genes that when present in 3 copies cause DS phenotypes has been approached using both human and mouse genetics. In humans, rare cases of partial trisomy 21 have been used to identify critical regions that contain dosage-sensitive genes that when present in 3 copies cause DS phenotypes (Delabar et al., 1993; Korbel et al., 2009; Korenberg et al., 1994; Lyle et al., 2009). Alternatively, mouse strains have been generated to study the pathology and genetics of DS.

Hsa21 shares synteny with a large region on mouse chromosome 16 (Mmu16) and with shorter regions on Mmu10 and Mmu17. The first two DS models generated, Ts65Dn (Davisson et al., 1990) and Ts1Cje (Sago et al., 1998) have duplications of regions of mouse chromosome 16 (Mmu16) that are orthologous to Hsa21, and have been used to identify dosage-sensitive genes contributing to some DS phenotypes (Baek et al., 2009; Chakrabarti et al., 2010; Lana-Elola et al., 2011; Salehi et al., 2006; Sussan et al., 2008). However, both models also have additional aneuploidy (trisomy of 60 genes on Mmu17 in Ts65Dn and monosomy of 7 genes on Mmu12 in Ts1Cje mice), making interpretation of their phenotypes difficult (Duchon et al., 2011). Another DS model, the Tc1 mouse, carries a freely-segregating copy of Hsa21 and shows many DS phenotypes, including CHD (Dunlevy et al., 2010; O'Doherty et al., 2005), although the Hsa21 in this strain is not intact and the mice are mosaic for the human chromosome, again making interpretation difficult (Gribble et al., 2013). Thanks to recent advances in chromosome engineering, a number of mouse strains with duplications of regions of mouse chromosomes orthologous to Hsa21 have been generated, resulting in partial trisomies, including the most complete mouse model for DS to date (Dp(16)1Yey/+;Dp(17)1Yey/+;Dp(10)1Yey/+), which carries a duplication of all Hsa21-orthologous regions on Mmu16, Mmu17 and Mmu10 (Brault et al., 2015; Li et al., 2007; Liu et al., 2013; Liu et al., 2011; Olson et al., 2004; Pereira et al., 2009; Yu et al., 2010). However, a comprehensive mapping panel to finely map dosage-sensitive genes in DS has not been available until now.

To identify dosage-sensitive critical regions and candidate genes, we now report the generation of a fine mapping panel of 7 partial trisomies of Mmu16 that can be used to identify the genetic basis of DS phenotypes where such genes reside in the region of Mmu16 orthologous to Hsa21. We use this panel to investigate the cardiac defects in DS, employing high-resolution episcopic microscopy (HREM) to analyze the precise three-dimensional (3D) morphology of the developing hearts. This combined approach enabled us to narrow down the critical region for CHD in DS to a minimal region containing 39 protein-coding genes and to show that this region contains at least two dosage-sensitive loci required in three copies to cause CHD. Furthermore using dual-wavelength HREM we show that the DMP develops normally in these DS models, and conclude that CHD in these DS models are not caused by failure in formation or growth of the DMP.

Cardiac abnormalities are very common in DS. Approximately half of all babies born with DS have a heart defect, many of which are serious and need to be surgically repaired. In order to understand the genetic and molecular mechanisms that lead to CHD in DS it is essential to establish mouse models for DS that accurately recapitulate them. Here we report the generation of a new mouse strain Dp1Tyb with a 23 Mb duplication of the entire region of Mmu16 orthologous to Hsa21, and show that it recapitulates the main types of CHD seen in DS. The Dp(16)1Yey mouse strain carries a similar duplication to Dp1Tyb encompassing the same set of genes and was also reported to have heart defects during embryogenesis (Li et al., 2007). Interestingly, the cardiac phenotypes in Dp1Tyb are very similar to the ones we previously observed in the transchromosomic strain Tc1 (Dunlevy et al., 2010; O'Doherty et al., 2005), despite differences in the Hsa21 orthologous gene content in the two strains and the different species origin for the duplicated genes: mouse in Dp1Tyb mice, human in Tc1 mice. This phenotypic similarity supports the conclusion that the CHD seen in these strains are caused by the same pathological mechanisms as those in people with DS. As with CHD in humans with DS we observed incomplete penetrance of the phenotype in Dp1Tyb mice. In humans this has been ascribed to genetic modifiers (Li et al., 2012). However the Dp1Tyb mouse strain was analyzed on an inbred background, suggesting that incomplete penetrance may be caused by stochastic effects during embryonic cardiogenesis. While the recovery of E14.5 Dp1Tyb embryos was consistent with expected Mendelian numbers (not shown), the recovery of Dp1Tyb mice at weaning was reduced to 50% of the expected numbers (Table 1). Some of this loss could be due to the most severe CHDs, since AVSDs were found in ~25% of E14.5 Dp1Tyb embryos, but it is likely that there are other unknown causes for this perinatal lethality. The other strain to show AVSDs, Dp3Tyb, showed a 25% reduction in recovery of mice at weaning, similar to the observed frequency of AVSDs at E14.5, making this a likely cause of the reduced yield of mutant mice.

Together with the Dp1Tyb strain we generated a collection of 7 new mouse strains with segmental duplications ranging from 1.5 Mb to 23 Mb that provide a genetic resource for mapping dosage-sensitive genes required in 3 copies to cause DS phenotypes. We used this mapping panel to identify a 4.9 Mb genomic region (from Mir802 to Zbtb21) that when present in 3 copies is sufficient to cause CHD. Furthermore, from analysis of shorter duplications, we show that the phenotype is caused by at least two distinct dosage-sensitive loci. By combining this data with analysis of the Ts1Rhr strain, we have determined that, minimally, one of the two loci lies either in an interval at the centromeric end of the duplication in Dp3Tyb (from Mir802 to Setd4) or at the telomeric end (from Mx2 to Zbtb21) (Figure 6a). These two segments between them contain only 7 coding genes (Setd4, Mx2, Tmprss2, Ripk4, Prdm15, C2cd2 and Zbtb21) and one microRNA gene (Mir802), none of which had been previously implicated in causing CHD in DS. A previous study proposed that a 3.7 Mb genomic region of Mmu16 (Ifnar1 – Kcnj6) in Dp(16)4Yey was sufficient to cause CHD (Liu et al., 2013); this region overlaps the centromeric end of the duplicated region of Dp3Tyb by 1.9 Mb from Mir802 to Kcnj6, which may help to further narrow down the search for causative genes (Figure 6b). We note that while the Dp5Tyb and Ts1Rhr strains did not show a statistically significant increase in CHDs compared to their wild-type littermate controls, in both strains we detected 2 mutant embryos with AVSDs (out of 20 or 21 embryos analyzed respectively). Thus it is possible that these strains have a weak phenotype – a larger number of embryos from these strains would need to be analyzed to establish whether this is significant. If true, it would suggest that one or more of the causative genes may reside within the Dp5Tyb interval (which is entirely contained within the Ts1Rhr interval).

Figure 6

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In contrast, analysis of humans with partial trisomies of Hsa21 identified distinct regions that contribute to cardiac abnormalities (Barlow et al., 2001; Korbel et al., 2009). An individual (PM) trisomic for the 7.7 Mb region from PSMG1 to PRMT2 had a VSD, whereas the shortest partial trisomy (individual BA) that gave rise to AVSD extended 10.0Mb from HLCS to PRMT2 (Figure 6b). We note that that while both of these intervals overlap substantially with the minimal Dp3Tyb region identified in our studies, the duplicated region in individual PM does not overlap at all with the Ifnar1 – Kcnj6 region identified by Liu et al as being sufficient to cause CHDs (Liu et al., 2013). In particular the human studies suggested that increased dosage of the DSCAM gene may be responsible for the CHDs (Korbel et al., 2009). In contrast the mouse studies show that duplication of Dscam is neither necessary or sufficient to cause CHDs, since the gene is not within the minimal Ifnar1 – Kcnj6 region identified by Liu et al as being sufficient for CHDs (Liu et al., 2013) but is duplicated in the Dp6Tyb strain which we show here does not have CHDs. Taking the human and mouse data together, this suggests that several dosage-sensitive genes may contribute to CHDs, with potentially no single gene being absolutely required.

Dp1Tyb mice show AVSD with ventricular but not atrial shunting. Analysis of AVSD in people with DS showed that the most common subtype was a complete AVSD with both atrial and ventricular shunting and the next most common was AVSD with exclusive ventricular shunting similar to that seen in the Dp1Tyb mouse (Freeman et al., 2008). Thus, although Dp1Tyb mice show AVSD, they only model one subtype of AVSD seen in people with DS – AVSD with ventricular shunting. It is not known why some cases of DS have complete AVSD whereas others have subtypes with only atrial or ventricular shunting, but it is tempting to speculate that there are distinct mechanisms driving defects in the atrial and ventricular septa.

AVSD have been shown to result from defects in development of the DMP (Briggs et al., 2013; Goddeeris et al., 2008; Rana et al., 2014; Tian et al., 2010; Webb et al., 1999; Xie et al., 2012). Importantly, a variety of different genetic manipulations that affect the DMP all resulted in AVSD with exclusive atrial shunting, demonstrating the importance of the DMP for the correct formation of the atrial side of the AV junction (Briggs et al., 2012). In contrast, in Dp1Tyb mice we found normal DMP development and no AVSD with atrial shunting, but instead saw exclusively AVSD with ventricular shunting (Figure 6c). Thus we propose that defects in the atrial septum causing the ostium primum type of AVSD are caused by malformation of the DMP, whereas ventricular defects are due to perturbations in other tissues, such as endocardial cushions or myocardium. Interestingly, Gata4-deficient mice display AVSD and have a normal DMP, but show perturbations in myocardial signaling to the adjacent atrioventricular cushion mesenchyme, suggesting that defects in myocardial signaling could lead to AVSD (Misra et al., 2014; Rajagopal et al., 2007). To our knowledge the Dp1Tyb and Dp3Tyb mouse strains are the first to have been shown to have AVSD with ventricular shunting and thus will allow direct studies of the pathological mechanisms underlying this subtype of AVSD.

In conclusion, we have generated a mapping panel of 7 mouse strains that can be used to identify dosage-sensitive genes underlying the broad range of DS phenotypes, and we have exploited this panel to map the location of genes causing CHD in DS.

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Author details

1. Eva Lana-Elola

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared

2. Sheona Watson-Scales

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared

3. Amy Slender

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared

4. Dorota Gibbins

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared

5. Alexandrine Martineau

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared

6. Charlotte Douglas

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared

7. Timothy Mohun

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared

8. Elizabeth MC Fisher

   Department of Neurodegenerative Disease, UCL Institute of Neurology, London, United Kingdom

   Contribution

   Conception and design, Drafting or revising the article

   Competing interests

   No competing interests declared

9. Victor LJ Tybulewicz

   1. The Francis Crick Institute, London, United Kingdom
   2. Imperial College London, London, United Kingdom

   Contribution

   Conception and design, Drafting or revising the article

   For correspondence

   Victor.T@crick.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2439-0798

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