Associative DNA Methylation Changes in Children with Prenatal Alcohol Exposure

Figures & data

Figure 1. Methylation signals from fetal alcohol exposed mice and children diagnosed with fetal alcohol spectrum disorders.

(A) Heatmap of top significant (p < 0.01) contigs from the adult mouse prenatally exposed to alcohol. (B) Heatmap of significantly (p < 0.005) differentially methylated targeted CpG sites generated using hierarchical clustering of buccal epithelial DNA obtained via swab from fetal alcohol spectrum disorders (n = 6) and matched control (n = 5) children from the discovery cohort. (C) Heatmap of significantly (p < 0.005) differentially methylated targeted CpG sites generated using hierarchical clustering of buccal epithelial DNA obtained via swab from fetal alcohol spectrum disorders (n = 6) and matched control (n = 6) children from the replication cohort. (D) Manhattan plot for human chromosome 5 from the discovery sample. Genomic location is plotted on the x-axis and –log₁₀ (p-value) of differential CpG methylation from prenatal alcohol exposure is on the y-axis. Each dot represents a single CpG site. A red dot indicates an increase in methylation and a blue dot indicates a decrease in methylation. The black arrow identifies clustered protocadherin genes on 5q31.

Figure 2. GeneMANIA network analysis using differentially methylated CpGs (p < 0.005) from cheek swabs of the discovery sample.

The network highlights preferential and synchronous alteration involving Protocadherin genes in children with fetal alcohol spectrum disorders.

Figure 3. CpG methylation of the Protocadherins in children with fetal alcohol spectrum disorders and matched controls.

(A) Representative methylation patterns in 14 CpGs in the gamma cluster in six fetal alcohol spectrum disorders children and their five matched controls. (B) Significantly (p < 0.005) increased methylation in the clustered protocadherin region of patients (red dots), known CpG island (green dots) and potential transcripts (blue lines) along with their similarity to the mouse model (p < 0.01). Figure is not to scale as non-informative regions have been removed for clarity.

Figure 4.  Top canonical (KEGG) pathways affected by altered CpG methylation in children with fetal alcohol spectrum disorders.

Genes were identified and then assembled as protein complexes. (A) Hippo signaling; (B) glutamatergic synapse pathway. Green marks a decrease in methylation and potentially increased gene expression while red indicates a increase in methylation and potentially decreased gene expression.

Table 1. Clinical features of patients from the discovery and replication cohorts with the diagnosis of fetal alcohol spectrum disorders.

(A) Discovery cohort
Patient ID	D1	D2	D3	D4	D5	D6
Patient (age, years) and sex	(6) M	(6) M	(5) M	(4) M	(3) M	(3) M
Clinical features
Developmental delay	x	x	x	x	x	x
Hyperactivity, poor impulse control	x	x	x	x	x	x
Learning disorders	x	x	x
Short attention span and inattention	x	x		x
Conduct disorder	x	x	x		x
Oppositional defiant disorder	x			x	x
Social difficulties	x			x	x
Nervousness and anxiety		x	x	x
Mood disorder	x			x
Macrocephaly/macrotia		x	x
Microcephaly/microtia				x	x
Distinctive facial features	x			x	x	x
Stimulant/psychotrophic medication	No	No	No	No	No	No
(B) Replication cohort
Patient ID	R1	R2	R3	R4	R5	R6
Patient (age, years) and sex	(7) F	(6) M	(6) M	(9) F	(6) M	(10) M
Clinical features
Developmental delay	x	x	x		x	x
Hyperactivity, poor impulse control	x	x	x	x	x	x
Learning disorders	x		x		x	x
Short attention span and inattention	x	x	x	x	x	x
Conduct disorder	x	x	x	x	x	x
Oppositional defiant disorder	x	x	x	x
Social difficulties	x	x	x
Nervousness and anxiety	x	x	x			x
Mood disorder			x	x		x
Macrocephaly/macrotia		x
Microcephaly/microtia						x
Distinctive facial features	x		x			x
Stimulant/psychotrophic medication	Yes	No	No	Yes	No	No

Table 2. Nucleotide-specific analysis of CpGs in the protocadherin gene family clusters showing significant (p < 0.005) alterations to CpG methylation in human buccal epithelial DNA from children of the discovery sample (with fetal alcohol spectrum disorders compared to matched controls).

Family	p-value	CpG ID	CpG coordinate	Fold change	Average β
α	2.24E-03	cg03318614	140154796	1.1	0.55
α	4.90E-03	cg16234335	140168303	1.11	0.6
α	4.07E-03	cg25225155	140202437	1.23	0.64
α	3.79E-03	cg13619597	140237001	1.54	0.68
α	4.42E-04	cg15122993	140236606	1.17	0.49
β	1.99E-03	cg05941060	140594004	1.43	0.46
β	1.90E-03	cg27086874	140549040	1.14	0.19
γ	8.85E-04	cg25657261	140705331	1.34	0.33
γ	1.37E-03	cg01561869	140704042	1.22	0.29
γ	4.86E-03	cg26856475	140734108	1.38	0.42
γ	1.08E-03	cg22087053	140734498	1.27	0.35
γ	6.14E-05	cg24922090	140720687	1.24	0.37
γ	6.18E-04	cg18705909	140754610	1.58	0.45
γ	4.98E-03	cg24633027	140752470	1.32	0.55
γ	8.56E-04	cg03831054	140769892	1.53	0.48
γ	4.36E-04	cg21117330	140769634	1.2	0.28
γ	8.64E-04	cg21915313	140779155	1.5	0.39
γ	3.24E-03	cg02074191	140731690	1.6	0.46
γ	1.36E-04	cg06757405	140731792	1.35	0.35
γ	4.41E-03	cg08854987	140759645	1.26	0.34
γ	8.46E-04	cg18297751	140749183	1.31	0.39

Table 3. Tiling-based analysis of protocadherin gene clusters showing significant (p < 0.01) increases in methylation in adult brain tissue from a mouse model of fetal alcohol spectrum disorders.

Family	Peak start	Peak end	Peak Length
α	37090887	37091731	844
α	37099823	37099982	159
α	37100493	37101082	589
α	37120628	37121169	541
α	37121813	37122462	649
α	37128647	37129396	749
α	37148470	37149114	644
α	37153966	37154515	549
α	37159353	37160304	951
α	37166096	37167215	1119
α	37181780	37182430	650
β	37456114	37456763	649
β	37603162	37603321	159
β	37645176	37646223	1047
β	37657621	37657881	260
β	37674825	37674994	169
β	37678546	37679275	729
γ	37828934	37831185	2251
γ	37835333	37836297	964
γ	37840948	37841597	649
γ	37850740	37850974	234
γ	37854441	37854897	456
γ	37868606	37868955	349
γ	37886555	37887889	1334
γ	37892011	37893005	994
γ	37922706	37922835	129

Results are from methylated DNA immunoprecipitation (MeDIP) CpG Island tilling arrays and peaks are represented as unique stretches showing significant alterations to DNA methylation in prenatal alcohol exposed mice compared with matched controls.

Table 4. Gene ontology and KEGG-based canonical pathway analysis of significant differences in CpG methylation from the discovery cohort.

Ontologies and pathways	p-value
Biological processes
Nervous system development (GO:0007399)	0.00000001
Anatomical structure development (GO:0048856)	0.00000008
Cell adhesion (GO:0007155)	0.00002176
Calcium-dependent cell–cell adhesion (GO:0016339)	0.00073511
Skeletal system development (GO:0001501)	0.00562206
Cell projection assembly (GO:0030031)	0.01521803
Cell–cell adhesion (GO:0016337)	0.01396605
Molecular functions
Neuropeptide binding (GO:0042923)	0.024
Neuropeptide receptor activity (GO:0008188)	0.022
Sodium channel activity (GO:0005272)	0.016
Protein binding (GO:0005515)	0.007
Voltage-gated sodium channel activity (GO:0005248)	0.009
RNA polymerase II transcription factor activity (GO:0003702)	0.026
Cation channel activity (GO:0005261)	0.043
Canonical pathways (number of genes affected)
Hippo signaling pathway (25 genes)	0.0002
Glutamatergic synapse (18 genes)	0.0011
Calcium signaling pathway (23 genes)	0.0051
Retrograde endocannabinoid signaling (16 genes)	0.0062
Serotonergic synapse (16 genes)	0.0092
Axon guidance (17 genes)	0.0118
Cell adhesion molecules (18 genes)	0.0132
Long-term depression (8 genes)	0.0349
Focal adhesion (21 genes)	0.0377
Dorso–ventral axis formation (5 genes)	0.0467
Cholinergic synapse (14 genes)	0.0546
Apoptosis (10 genes)	0.0608

Table 5. Pyrosequencing results for COLEC11 and HTT.

Individual ID	Colec11 panel		HTT panel
	SNP T>G rs182514706	cg15730644	cg26128129 SNP C>T rs362313	SNP A>G rs147422679
E1	T/T	91.5	89.30	A/A
E2	T/T	96.0	86.62	A/A
E3	T/T	92.0	90.84	A/A
E4	T/T	96.9	86.63	A/A
E5	T/T	94.0	84.61	A/A
E6	T/T	94.5	87.82	A/A
C1	T/T	63.8	37.37	A/A
C2	T/T	81.9	44.32	A/A
C3	T/T	84.2	84.18	A/A
C4	T/T	82.3	44.82	A/A
C5	T/T	82.2	42.57	A/A

Sample ID represents the individuals from the discovery cohort and the metrics represent percent methylation of the CpG from the sample.