Questioned validity of Gene Expression Dysregulated Domains in Down's Syndrome

Recently, in studies examining fibroblasts obtained from the tissues of one set of monozygotic twins (i.e. fetuses derived from the same egg) discordant for trisomy 21 (Down syndrome; DS), Letourneau et al., reported the presence of a defined pattern of dysregulation within specific genomic domains they referred to as Gene Expression Dysregulated Domains (GEDDs). GEDDs were described as alternating segments of increased or decreased gene expression affecting all chromosomes. Strikingly, GEDDs in fibroblasts were largely conserved in induced pluripotent cells (iPSCs) generated from the twin’s fibroblasts as well as in fibroblasts from the Ts65Dn mouse model of DS. Our recent analysis failed to find GEDDs. We reexamined the human iPSCs RNAseq data from Letourneau et al., and data from this same research group published earlier examining iPSCs from the same monozygotic twins. An independent analysis of RNAseq data from Ts65Dn fibroblasts also failed to confirm presence of GEDDs. Our analysis questions the validity of GEDDs in DS.


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The surprising fndings by Letourneau and colleagues prompted us to examine our own, as yet unpublished, Ts65Dn transcriptome data for the developing and mature hippocampus in an attempt to identify GEDDs. Our data provided no evidence for the pattern reported in Letourneau et al.'s work. We first entertained the possibility that GEDDs were not present in post mitotic cells or cells undergoing neural differentiation. However, to ensure that we fully understood the published GEDD data, we examined the entire RNAseq dataset from the Letourneau manuscript, as provided publicly via the Gene Expression Omnibus.
Principal component analysis (PCA) of RNAseq replicates from the twin's fibroblast (T1DS: twin with DS; T2N: disomic twin) revealed a great deal of variability ( Figure 1A). When the datasets from Letourneau et al., are compared, in two of four cases, a closer relationship exists between the DS and disomic twin fibroblasts than for replicates from the same individual. [The datasets from Letourneau et al. are denoted by -L]. For example, one of the 2N-hFibro-L replicates clustered more tightly with a DS-hFibro-L replicate than with any of its own replicate (2N) samples.
We next checked the variability of the twin's iPSCs RNAseq data. We found an additional three RNASeq replicates (hiPSCs-H) performed by the same research group and published earlier using fibroblasts from the same monozygotic twins 2 . PCA analysis of these data revealed that replicates of hiPSCs-H (H for Hibaoui) clustered well together; however, they did not cluster well with the data for hiPSCs-L ( Figure 1A). Altogether, PCA analysis indicated marked variability between datasets, raising the possibility that technical issues in the RNAseq samples or in their analysis compromised the Letourneau study.
To further explore the additional three hiPSCs-H RNAseq replicates, we searched for GEDDs using methods similar to those utilized by Letourneau and colleagues. Our analysis of the hiPSCs-H did not find conserved patterns indicative of GEDDs. Figure 1B shows the results for two chromosomes, as examples. The authors reported high global gene fold-change correlations between the twin's fibroblasts and derived iPSCs. Our re-analysis found a similar high correlation value of ρ = 0.82 between the hiPSCs-L and hFibro-L. However, we found the hiPSCs-H poorly correlated with the original datasets; ρ = 0.31 between hiPSCs-L and hiP-SCs-H; ρ = 0.07 between hiPSCs-H and hFibro-L ( Figure 1C and Supplementary figure 1A).
Conservation of GEDDs in Ts65Dn mouse model of DS were quite unexpected given that Ts65Dn mouse is segmentally trisomic (34Mb) for a portion of mouse chromosome 16 (MMU16); the segment contains about 88 mouse homologues to human genes on the   it also carries an extra copy of the approximately 10Mb centromeric segment of MMU17 that is not syntenic to any region on human chromosome 21 3-6 . To further explore the possibility of GEDDs, RNAseq data was obtained from three replicates each from Ts65Dn and wild type mouse embryonic fibroblasts (MEFs-D) (D for Do denotes MEFs in the current study). PCA revealed tight clustering between our replicates ( Figure 1D), but not those for the MEFs-L samples. While we found expected changes in gene expression in MEFs-D, analysis of MEFs-L and MEFs-D found a poor global correlation (ρ = -0.31) ( Figure 1E and Supplementary figure 1B); this was also the case across all mouse chromosomes (for examples see Figure 1F and Supplementary figure 1B). In summary our findings raise serious concerns regarding the validity of GEDDs. We find no evidence for such domains in the studies on DS referenced herein or in cells from the Ts65Dn mouse model of DS.

Methods
Total RNA was collected using TRIzol reagent and further purified using RNeasy mini Kit, (Qiagen) from primary mouse embryonic fibroblasts (MEFs) derived from 18.5-day-old Ts65Dn and 2N mouse using manufacturer's instructions. RNA quality was checked using Tapestation 2200 (Agilent technologies) and quantified using    Author contributions LD contributed in writing the manuscripts and all the scripts used to perform analysis of the data. NS contributed in writing the manuscript and performed all the experiments for sequencing. WM contributed in writing the manuscript. All authors have seen and agreed to the final content of the manuscript.

Competing interests
The authors declare no competing financial interests.    The comments by Pierce-Shimomura and Nordquist provide an excellent summary of several statistical issues in the analyses. They suggest possible clarifications to the Do study and conclude, with Do, that the existence of GEDDs is not proven. I would note only two additional two points.

Supplemental figure S1. A) Comparison of the gene expression fold-change profiles between T1DS and T2N in human fibroblasts
The However, it is immediately obvious that Letourneau et al. have no basis for many of the statistical comparisons used to identify GEDDs -a phenomenon -as they have no biological replicates, biological only technical ones. It was not clear to this reviewer whether Letourneau established four fibroblast clones and/or iPS lines from each twin (and if so, in how many independent transformation experiments) but this would only be a technical replicate for the artifact of that transformation process, not for the biology of GEDDs in trisomy, and therefore it is not the appropriate basis for the statistically-based conclusions that they make about the of the effects of trisomy 21 on transcription. For biological conclusions, there biology is an N of one euploid and one trisomic individual -statistical assessments are not possible with a single comparison.
Letourneau et al. argue that genetic variability affecting gene expression levels does not allow detection of GEDDs in any but co-isogenic conditions. However, if GEDDs exist and are so highly conserved in evolution that the same genes are mis-regulated in the same way in outbred* trisomic mice, it is difficult to understand why they are not evident in any comparison of humans with two vs. three copies of Hsa21; if GEDDs only occur in vanishingly rare, coisogenic monozygotic human twin sets discordant for a specific trisomy, the phenomenon would hardly seem worthy of the attention it has received. An explanation for the conservation of this phenomenon in outbred individuals of another species but not between human pairs would strengthen the understanding of this phenomenon. An adequately described experiment comparing multiple individuals with two vs. three copies of Hsa21 including euploid and trisomic sib pairs, in addition to the statistical clarifications suggested by Pierce-Shimomura, would help to establish the existence or not of GEDDs and provide some indication of their relevance to the goal of ameliorating effects of gene dosage in DS.
*Ts65Dn mice are maintained as an advanced intercross between any of several C57BL6 and C3H strains. Thus, individual mice and their sibs are not genetically identical and are heterozygous at ~50% of all loci -a different 50 % for each individual. SNPs occur about every 3000 bp between B6 and C3H, while SNPs for a given segment of a human chromosome pair might be on the order of one per 1000 bp. It might be important to consider variability in chr21 alleles in each twin. Assuming the original conceptus was trisomic, if trisomy resulted from a meiosis I error, the trisomic line will carry three sets of Hsa21 alleles while the euploid twin will lack one of the three sets. If the trisomy resulted from a meiosis II error, the euploid twin could be isodisomic for Chr21 and carry only a single set of alleles. One could speculate about the possible impact of isodisomy on genome-wide expression patterns.
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