Prenatal Neun+ Neurons of Down Syndrome Display Aberrant Integrative DNA Methylation and Gene Expression Profiles

Figures & data

Figure 1. Fluorescent activated sorting of NeuN+ nuclei.

(A) Right: fluorescent activated sorting of NeuN+ and NeuN- nuclei based on scatter properties and verified with cytospin staining. In short, DAPI signal was removed using forward and side scatter (data not shown), leaving only nuclei (left panel). Selected areas represent selection thresholds for nuclei selection, blue annotated nuclei represent NeuN+ nuclei and orange annotated nuclei represent NeuN- nuclei. The x-axis represents NeuN absorption (nm) spectrum and the y-axis represents Hoechst absorption (nm). The panel on the right side represents the NeuN counts, where the first peak is NeuN- (56%) and the second is Neun+ (37%). (B) Cytospin image confirmation of fluorescent activated sorting procedure of nuclei. Left: NeuN staining, middle: DAPI staining and right: overlay of NeuN and DAPI staining.

Table 1. Characteristics of cerebrum specimens that passed quality control for both DNA methylation and gene expression data.

Group	Sex	GA (weeks)	PMI (h)	QC meth/GE
DS	F	20	2	pass / pass
DS	F	19	1	pass / pass
DS	F	23	2	pass / pass
DS	F	16	1	pass / pass
DS	M	21	2	pass / pass
DS	M	22	3	pass / pass
DS	M	19	6	pass / pass
DS	M	22	3	pass / pass
DS	F	20	2	pass / pass
Control	F	19	1	pass / pass
Control	F	19	2	pass / pass
Control	F	18	2	pass / pass
Control	F	16	3	pass / pass
Control	F	19	1	pass / pass
Control	F	19	1	pass / pass
Control	M	19	1	pass / pass
Control	M	19	2	pass / pass
Control	M	19	4	pass / pass

DS: Down syndrome; F: Female; GA: Gestational age; GE: Gene expression profile; M: Male; PMI: Postmortem interval until tissue preservation; QC meth: Quality control DNA methylation.

Figure 2. Differentially methylated positions.

(A) Explorative principal component analysis of normalized DNA methylation profiles annotated by group. The x-axis represents the first principal component and the y-axis represents the second principal component. (B) Correlation analysis of the first eight principal components of normalized DNA methylation profiles (x-axis) with the following metadata: group, sex, section of the region, age, cerebrum region, postmortem interval, ethnicity, array and slide position. The y-axis represents the correlation coefficient. (C) Manhattan plot showing the relative percentage of DMPs at a FDR < 0.05. The x-axis represents chromosome number and the y-axis represents relative percentage of DMPs (with respect to the total per chromosome) and separated for direction of effect. (D) Volcano plot DMPs. The x-axis represents delta mean β difference (DS vs controls). The y-axis represents -log10 (FDR). Green dots represent significant DMPs at a delta difference > 0.4. Blue dots represent significant DMPs with a FDR < 0.05. Orange dots represent non-significant DMPs.

DMP: Differentially methylated position.

Table 2. Overview of top significant differential associations between Down syndrome and controls.

(I) Top five significant DMPs
Probe	FDR	Delta	Position (hg19)	Gene (feature)
cg16204717	1.798444e-05	0,34	chr12:65894458	MSRB3 (3′downstream)
cg15297841	1.133111e-04	0,17	chr1: 54881909	SSBP3 (5′upstream)
cg05038268	3.180800e-04	0,35	chr11:66885281	KDM2A (TSS)
cg21557180	3.180800e-04	0,15	chr7: 30323717	ZNRF2 (TSS)
cg25552887	3.180800e-04	-0,17	chr16:66751144	DYNC1LI2 (5′upstream)
(II) Top five significant DMRs
Position (hg19)	Stouffer C	Delta	CpGs	Gene (feature)
Chr22: 51016386–51017723	2.309020e-22	0,198	17	CPT1B, CHKB (Body, TSS)
Chr10: 94819559–94822747	9.484489e-20	-0,20	17	CYP26C1 (TSS)
Chr5: 92922043–92930052	3.181663e-18	-0,35	19	NR2F1 (Body)
Chr18: 77904435–77905947	1.104188e-13	-0,206	10	ADNP2 (TSS)
Chr5: 92905692–92908988	5.684382e-13	0,171	11	NR2F1 (TSS)
(III) Top five significant DEGs
Gene	Position (GRCh38/hg38)	-log2FC	FDR
GAL3ST2	chr2: 241776825–241804208	-47.1	2.021976e-54
HMCN2	chr9: 130265882–130434123	-43.8	3.323494e-52
HAR1A	chr20: 63102205–63104386	-39.8	3.681543e-51
EXPH5	chr11: 108505435–108593768	40.2	1.947846e-48
TRBV26OR9-2	chr9: 33695767–33696059	-36.3	1.624162e-38
(IV) Top five overlap DMRs and DEGs
Gene	Feature, delta	Stouffer C	Position (hg19)	-log2FC	FDR
EXPH5	Body, -0,145	1,08E-03	chr11: 108408907–108409825	-40.2	4.216801e-52
ADAMTS18	TSS, -0,198	3,17E-07	chr16: 77465215–77467409	22.3	2.565373e-12
LHX2	TSS, -0,166	2,82E-04	chr9: 126772210–126773095	-1.58	2.072478e-11
HSPA12A	TSS, 0,100	3,11E-02	chr10: 118502872–118503075	1.89	1.715203e-10
ITPR2	Body, -0,142	8,18E-04	chr12: 26783594–26783717	10.2	5.649025e-10
(V) Top five significant eQTMs
Gene	Chr (N probes)	Feature, direction	-log2FC	PCC	95% CI	p-value
SCUBE1	22 (6)	Body, 0,059	0.7	-0,8	-0,93 / -0.45	1,30E-04
NR2F1	5 (19)	Body, -0,350	-1.36	-0,83	-0,90 / -0.64	2,30E-04
SDK1	7 (7)	TSS, 0,112	2.29	-0,89	-0,95 / -0.77	3.00E-04
SH2D4B	10 (10)	TSS, 0,117	1.00	-0,76	-0,90 / -0.38	4.10E-04
CAMTA2	17 (2)	TSS, 0,058	2.96	-0,72	-0,85 / -0.47	9.00E-09

Full lists are described in Supplementary Table 1.

Delta: Delta mean beta-value difference; DEG: Differentially expressed gene; DMP: Differentially methylated position; DMR: Differentially methylated region; eQTM: Expression quantitative trait methylation; FDR: False discovery rate; IGR: Intergenic region; -log2FC: Minus natural log fold change; PCC: Pearson’s correlation coefficient; Stouffer C: Stouffer coefficient; TSS: Transcription start site, 1500 bp.

Figure 3. Differentially expressed genes.

(A) Explorative principal component analysis of gene expression (count) profiles annotated by group. The x-axis represents the first principal component and the y-axis represents the second principal component. (B) Manhattan plot showing the relative percentage of DEGs at a FDR < 0.05. The x-axis represents chromosome number and the y-axis represents relative (with respect to the total per chromosome) percentage of DEGs, annotated for direction of effect. (C) Volcano plot representing DEGs. The x-axis represents -log2 fold change counts and the y-axis represents -log10 (FDR). Green dots represents significant DEGs with mean effect size change >10. Blue dots represent significant DEGs with -log10 (FDR) < 0.05. Orange dots represent non-significant DEGs.

DEG: Differentially expressed gene; FDR: False discovery rate.

Figure 4. Integrative analyses of differentially DNA methylation and gene expression profiles.

(A) Genomic overlap between Down syndrome-associated DMRs and DEGs. The x-axis represents -log10 Stouffer coefficient of DMRs and the y-axis represents -log10 (FDR) of DEGs. Purple dots represent significant DEG/DMR overlap, blue dots represent significant DEGs, green dots represent significant DMRs and red dots represent non-significant DEG/DMRs. (B) Expression quantitative trait methylation analysis. The x-axis represents the Pearson correlation coefficient between DMRs (median percentage difference of the β-value) and DEGs (-log2 fold change). the y-axis represents -log10 p-value of the Pearson correlation coefficient.

DEG: Differentially expressed gene; DMR: Differentially methylated region; FDR: False discovery rate.

Figure 5. Top five Down syndrome-associated genes representing significant expression quantitative trait methylations (eQTMs).

The x-axis represents gene expression count data, and the y-axis represents median percentage difference of the β-value. The blue line represents correlation coefficient. r² represents the absolute Pearson correlation coefficient. The red dots represent Down syndrome and the blue dots represent controls. (A) eQTM observed for the SCUBE1 gene. (B) eQTM observed for the NR2F1 gene. (C) eQTM observed for the SDK1 gene. (D) eQTM observed for the SH2D4B gene. (E) eQTM observed for the INCA1 gene.