Wilson Disease: Epigenetic effects of choline supplementation on phenotype and clinical course in a mouse model

Figures & data

Figure 1. Study design. Wild type or tx-j mice were fed diets with or without choline supplementation 2 weeks before mating through embryonic day 17. Fetal livers were harvested and RNA-seq was performed. Another set of tx-j offspring were weaned and continued on diets with or without choline supplementation. Subgroups of tx-j offspring on each diet were given penicillamine (PCA) treatment.

Figure 2. Principal component analysis scores plot of significantly differentially expressed genes in fetal liver. Each symbol represents a mouse. Symbols that are closer to one another are more similar. Ellipses represent 95% confidence intervals based on Hotelling's T2 statistic.

Table 1. Top pathways identified by WebGestalt Pathway Analysis in fetal livers.

Wild type vs. tx-j
Pathway	Number of genes represented in pathway	Adjusted P-value
Protein processing in endoplasmic reticulum	11% (19/169)	>0.0001
Ubiquitin mediated proteolysis	12% (17/140)	>0.0001
Lysosome	12% (15/123)	>0.0001
Huntington's disease	9% (18/197)	>0.0001
Purine metabolism	10% (16/168)	>0.0001
Alzheimer's disease	9% (16/188)	>0.0001
Oxidative phosphorylation	10% (14/147)	>0.0001
Vasopressin-regulated water reabsorption	19% (8/43)	>0.0001
Focal adhesion	8% (16/200)	>0.0001
tx-j vs. tx-j + maternal choline
Pathway	Number of genes represented in pathway	Adjusted P-value
Oxidative phosphorylation	6% (9/147)	>0.0001
Alzheimer's disease	5% (10/188)	>0.0001
p53 signaling pathway	9% (6/70)	>0.0001
Proteasome	11% (5/45)	>0.0001
Huntington's disease	4% (8/197)	0.0006
Lysosome	5% (6/123)	0.001
Focal adhesion	4% (7/200)	0.0021
GnRH signaling pathway	5% (5/99)	0.0021
Phagosome	3% (6/176)	0.0042

Table 2A. Top four pathways identified by WebGestalt Pathway Analysis in fetal livers.

Wild type vs. tx-j
Pathway		Wild type Mean RPKM	tx-j Mean RPKM	Percent Difference (Wild type relative to tx-j)
Protein processing in endoplasmic reticulum (KEGG Pathway 04141)
C = 169; O = 19; E = 3.36; R = 5.65; rawP = 1.52e-09; adjP = 4.71e-08
Tusc3	tumor suppressor candidate 3	15.1	21.5	43%
Dnaja1	DnaJ (Hsp40) homolog, subfamily A, member 1	11.6	16.4	41%
Hsph1	heat shock 105kDa/110kDa protein 1	8.5	11.5	35%
Ube2e2	ubiquitin-conjugating enzyme E2E 2	4.1	5.5	34%
Casp12	caspase 12	0.2	0.3	31%
Map3k5	mitogen-activated protein kinase kinase kinase 5	2.5	3.2	30%
Ube2d2a	ubiquitin-conjugating enzyme E2D 2A	34.0	43.9	29%
Ube2g1	ubiquitin-conjugating enzyme E2G 1	10.6	13.6	28%
Rbx1	ring-box 1	21.0	25.9	24%
Dnajc10	DnaJ (Hsp40) homolog, subfamily C, member 10	16.0	19.5	22%
Mbtps2	membrane-bound transcription factor peptidase, site 2	2.3	2.8	22%
Bcap31	B cell receptor associated protein 31	46.7	55.1	18%
Ube2e1	ubiquitin-conjugating enzyme E2E 1	14.0	16.3	17%
Sar1a	SAR1 gene homolog A (S. cerevisiae)	29.0	33.8	16%
Ern1	endoplasmic reticulum (ER) to nucleus signalling 1	6.7	5.5	−18%
Rnf5	ring finger protein 5	27.1	21.5	−20%
Sil1	endoplasmic reticulum chaperone SIL1 homolog	10.0	7.9	−20%
Atf6b	activating transcription factor 6 β	4.6	3.3	−27%
Os9	amplified in osteosarcoma	25.7	18.6	−28%
Ubiquitin mediated proteolysis (KEGG Pathway 04120)
C = 140; O = 17; E = 2.78; R = 6.10; rawP = 3.35e-09; adjP = 6.92e-08
Ube2e2	ubiquitin-conjugating enzyme E2E 2	4.1	5.5	34%
Klhl9	kelch-like 9 (Drosophila)	9.6	12.6	31%
Ube2d2a	ubiquitin-conjugating enzyme E2D 2A	34.0	43.9	29%
Birc2	baculoviral IAP repeat-containing 2	8.1	10.3	28%
Ube2g1	ubiquitin-conjugating enzyme E2G 1	10.6	13.6	28%
Rchy1	ring finger and CHY zinc finger domain containing 1	11.8	14.9	27%
Itch	itchy, E3 ubiquitin protein ligase	13.9	17.5	25%
Rbx1	ring-box 1	21.0	25.9	24%
Vhl	von Hippel-Lindau tumor suppressor	4.9	5.9	21%
Xiap	X-linked inhibitor of apoptosis	17.4	20.5	18%
Ube2e1	ubiquitin-conjugating enzyme E2E 1	14.0	16.3	17%
Klhl13	kelch-like 13 (Drosophila)	10.9	12.6	16%
Wwp2	WW domain containing E3 ubiquitin protein ligase 2	9.7	8.3	−14%
Tceb2	transcription elongation factor B (SIII), polypeptide 2	33.8	24.9	−26%
Ube2o	ubiquitin-conjugating enzyme E2O	20.3	14.9	−27%
Cdc34	cell division cycle 34	23.6	16.3	−31%
Fzr1	fizzy/cell division cycle 20 related 1 (Drosophila)	15.2	9.5	−38%
Lysosome (KEGG Pathway 04142)
C = 123; O = 15; E = 2.45; R = 6.13; rawP = 2.62e-08; adjP = 4.06e-07
Ctsk	cathepsin K	0.7	1.2	76%
Hexb	hexosaminidase B	18.3	24.1	32%
Atp6v1h	ATPase, H^+ transporting, lysosomal V1 subunit H	12.1	15.9	31%
Gla	galactosidase, α	1.8	2.4	30%
Ap4e1	adaptor-related protein complex AP-4, ε 1	2.2	2.9	29%
Ppt1	palmitoyl-protein thioesterase 1	9.6	11.9	24%
Lamp2	lysosomal-associated membrane protein 2	43.1	52.2	21%
Laptm4a	lysosomal-associated protein transmembrane 4A	56.1	67.6	21%
Cd164	CD164 antigen	66.5	79.5	20%
Asah1	N-acylsphingosine amidohydrolase 1	22.1	25.8	17%
Manba	mannosidase, beta A, lysosomal	4.5	5.2	17%
Cd63	CD63 antigen	21.0	24.4	16%
Gusb	glucuronidase, beta	10.4	12.1	16%
Napsa	napsin A aspartic peptidase	2.8	2.2	−22%
Abcb9	ATP-binding cassette, sub-family B (MDR/TAP), member 9	0.4	0.2	−47%
Huntington's disease (KEGG Pathway 05016)
C = 197; O = 18; E = 3.92; R = 4.59; rawP = 1.04e-07; adjP = 1.29e-06
Atp5g3	ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9)	172.2	237.1	38%
Ndufb6	NADH dehydrogenase 1 beta subcomplex, 6	25.9	34.2	32%
Cox5a	cytochrome c oxidase, subunit Va	67.1	88.2	32%
Tfam	transcription factor A, mitochondrial	9.0	11.8	31%
Ndufs4	NADH dehydrogenase Fe-S protein 4	22.1	28.5	29%
Vdac3	voltage-dependent anion channel 3	64.0	80.0	25%
Ndufab1	NADH dehydrogenase 1, alpha/beta subcomplex, 1	8.7	10.6	22%
Ndufb2	NADH dehydrogenase 1 beta subcomplex, 2	4.3	5.2	20%
Atp5j	ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F	86.2	102.1	18%
Ndufc1	NADH dehydrogenase 1, subcomplex unknown, 1	15.1	17.7	17%
Uqcrc2	ubiquinol cytochrome c reductase core protein 2	102.6	119.8	17%
Ndufb5	NADH dehydrogenase 1 beta subcomplex, 5	20.3	23.6	16%
Ap2a1	adaptor protein complex AP-2, alpha 1 subunit	9.6	7.5	−22%
Gpx1	glutathione peroxidase 1	474.7	331.8	−30%
Dctn1	dynactin 1	5.3	3.5	−33%
Bbc3	BCL2 binding component 3	1.9	1.2	−36%
Ndufs7	NADH dehydrogenase Fe-S protein 7	9.3	5.7	−39%
Polr2i	polymerase (RNA) II (DNA directed) polypeptide I	4.4	2.6	−42%
Number of reference genes in the category (C)
Number of differentially expressed genes in reference category (O)
Expected number in the category (E)
Ratio of enrichment (F)
P-value from hypergeometric test (rawP)
P-value adjusted by the multiple test adjustment (adjP)
RPKM: Reads per kilobase of transcript per million mapped reads
*Percent difference = [(tx-j - wild type)/wild type]*100

Table 2B. Top four pathways identified by WebGestalt Pathway Analysis.

tx-j vs tx-j + maternal choline supplementation
Pathway		tx-j Mean RPKM	tx-j + maternal choline Mean RPKM	Percent Difference (tx-j relative to tx-j + maternal choline)
Oxidative phosphorylation (KEGG Pathway 00190)
C = 147; O = 9; E = 1.15; R = 7.85; rawP = 2.61e-06; adjP = 1.48e-05
Atp6v1a	ATPase, H+ transporting, lysosomal V1 subunit A	18.5	16.1	−13%
Uqcrfs1	ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1	77.4	66.3	−14%
Ndufb6	NADH dehydrogenase (ubiquinone) 1 β subcomplex, 6	34.2	28.6	−16%
Atp6v1b2	ATPase, H^+ transporting, lysosomal V1 subunit B2	35.2	29.4	−17%
Cox17	cytochrome c oxidase, subunit XVII assembly protein homolog (yeast)	5.0	4.2	−17%
Atp6v1h	ATPase, H+ transporting, lysosomal V1 subunit H	15.9	13.1	−18%
Atp5g3	ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9)	237.1	189.9	−20%
Cox5a	cytochrome c oxidase, subunit Va	88.2	69.8	−21%
Cox11	COX11 homolog, cytochrome c oxidase assembly protein (yeast)	5.5	3.8	−30%
Alzheimer's disease (KEGG Pathway 05010)
C = 188; O = 10; E = 1.47; R = 6.82; rawP = 2.60e-06; adjP = 1.48e-05
Ern1	endoplasmic reticulum (ER) to nucleus signalling 1	5.5	6.8	24%
Uqcrfs1	ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1	77.4	66.3	−14%
Casp9	caspase 9	6.4	5.4	−15%
Ndufb6	NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6	34.2	28.6	−16%
Itpr1	inositol 1,4,5-trisphosphate receptor 1	3.7	3.1	−17%
Aph1a	anterior pharynx defective 1a homolog (C. elegans)	13.6	11.0	−19%
Bace2	beta-site APP-cleaving enzyme 2	1.2	1.0	−19%
Atp5g3	ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9)	237.1	189.9	−20%
Casp7	caspase 7	6.6	5.2	−21%
Cox5a	cytochrome c oxidase, subunit Va	88.2	69.8	−21%
p53 signaling pathway    ID:04115
C = 70; O = 6; E = 0.55; R = 11.00; rawP = 1.87e-05; adjP = 7.95e-05
Steap3	STEAP family member 3	13.4	11.5	−14%
Casp9	caspase 9	6.4	5.4	−15%
Rchy1	ring finger and CHY zinc finger domain containing 1	14.9	12.0	−20%
Perp	PERP, TP53 apoptosis effector	15.8	12.5	−21%
Igf1	insulin-like growth factor 1	3.0	2.3	−24%
Sesn3	sestrin 3	3.2	2.4	−25%
Proteasome (KEGG Pathway 03050)
C = 45; O = 5; E = 0.35; R = 14.25; rawP = 2.66e-05; adjP = 9.04e-05
Pomp	proteasome maturation protein	74.6	63.6	−15%
Psmf1	proteasome inhibitor subunit 1	7.2	6.1	−15%
Psmc5	protease 26S subunit, ATPase 5	33.3	26.7	−20%
Psmd13	proteasome 26S subunit, non-ATPase, 13	19.6	15.5	−21%
Psmd2	proteasome 26S subunit, non-ATPase, 2	49.3	38.5	−22%
Number of reference genes in the category (C)
Number of differentially expressed genes in reference category (O)
Expected number in the category (E)
Ratio of enrichment (F)
P-value from hypergeometric test (rawP)
P-value adjusted by the multiple test adjustment (adjP)
*Percent difference = [(tx-j - tx-j+maternal choline)/tx-j]*100

RPKM: Reads per kilobase of transcript per million mapped reads

Table 3. Fetal RNA-seq transcript levels for select oxidative phosphorylation genes.

Gene	Wild type	Wild type + maternal choline	tx-j	tx-j + maternal choline
Nuclear encoded subunit of NADH dehydrogenase (Complex I)
Ndufa4	205.34	205.27	232.51	224.94
Ndufa9	54.45	59.13	60.06	54.44
Ndufab1	8.68^a	8.89^a,b	10.60^b	9.31^a,b
Ndufb2	4.35^a	4.28^a	5.20^b	4.66^a,b
Ndufb5	20.28^a	21.10^a	23.59^b	21.58^a,b
Ndufb6	25.90^a	25.59^a	34.22^b	28.58^a
Ndufc1	15.08	15.59	17.68	16.87
Ndufc2	31.24	32.61	35.81	31.72
Ndufs4	22.05^a	23.55^a	28.46^b	26.15^b
Ndufs7	9.31	9.67	5.68	7.30
Nuclear encoded subunit of succinate dehydrogenase (Complex II)
Sdha	74.36^a,b	76.68^a	77.06^a	67.80^b
Nuclear encoded subunits of ubiquinol-cytochrome c reductase (Complex III)
Uqcrc2	102.64	109.76	119.80	112.96
Uqcrfs1	68.13^a,b	65.85^a	77.45^b	66.28^a
Nuclear encoded subunits of cytochrome c oxidase (Complex IV)
Cox17	3.80^a	4.50^a,b	5.02^b	4.17^a
Cox5a	67.06^a	64.76^a	88.23^b	69.81^a
Nuclear encoded subunits of mitochondrial ATP synthase (Complex V)
Atp5f1	139.19	140.28	152.81	146.87
Atp5g3	172.22^a	187.34^a	237.14^b	189.94^a
Atp5j	86.24^a	84.33^b	102.15^b	89.90^a,b
Atp5l	135.50	131.52	149.37	135.43
Atp6v1a	15.31^a	17.00^a,b	18.52^b	16.07^a,b
Atp6v1h	12.12^a	13.85^a,b	15.93^b	13.07^a,b

Values are expressed as average reads per kilobase of exon per million reads mapped (RPKM). Between group differences were analyzed by 1-way ANOVA; values with different letter symbols are significantly different (P < 0.05) from each other.

Figure 3. Liver histology from tx-j mice at 24 weeks of age. Hematoxylin and eosin, 100 X. Untreated tx-j mice presented mild inflammation and steatosis with more inflammation occurring in males compared to females in the same diet group. Steatosis improved in female tx-j mice after penicillamine (PCA) treatment. Whereas choline did not change histology scores, the combination of PCA and choline was associated with higher grade of steatosis compared to all other groups. Results are expressed as mean ± standard deviation. Between group differences were analyzed by 1-way ANOVA; values with different letter symbols are significantly different (P < 0.05) from each other. Differences between sexes in the same treatment group were analyzed by Student's t-test; values with an asterisk (*) are significantly different (P < 0.05) between sexes.

Figure 4. Hepatic copper, iron zinc concentrations, plasma triglycerides and ALT, and global hepatic DNA methylation. Results are expressed as mean ± standard deviation. Between group differences were analyzed by 1-way ANOVA; values with different letter symbols are significantly different (P < 0.05) from each other. Differences between sexes in the same treatment group were analyzed by Student's t-test; values with an asterisk (*) are significantly different (P < 0.05) between sexes.

Figure 5. Transcript levels of genes representative of oxidative phosphorylation (Ndufab1, Ndufb5, Sdha, Cox5a, Atp5j). Transcript expression data are normalized to Gapdh (10–22 mice/group). Protein relative expression (R.I.) according to Western blots analyses for SDHA are reported; lane 1: tx-j; lane 2: tx-j + PCA; lane 3: tx-j + choline; lane 4: tx-j + choline + PCA. Results are expressed as mean ± standard deviation. Between group differences were analyzed by 1-way ANOVA; values with different letter symbols are significantly different (P< 0.05) from each other. Differences between sexes in the same treatment group were analyzed by Student's t-test; values with an asterisk (*) are significantly different (P < 0.05) between sexes.

Figure 6. Plasma acylcarnitines and plasma triglycerides. Results are expressed as mean ± standard deviation. Plasma Acylcarnitines: Significant differences between 2 groups were assessed by Student's t-test. Values with different letter symbols are significantly different (P< 0.05) from each other. Plasma Triglycerides: Between group differences were analyzed by 1-way ANOVA; values with different letter symbols are significantly different (P < 0.05) from each other.

Figure 7. Transcript levels of genes representative of methionine metabolism, DNA methylation (Sahh, Dnmt1, Dnmt3a, Dnmt3b), and lipid metabolism (Srebf1). Transcript expression data are normalized to Gapdh (10–14 mice/group). Protein relative expression (R.I.) according to Western blot analyses for SAHH and SREBP1 are reported; lane 1: tx-j; lane 2: tx-j + PCA; lane 3: tx-j + choline; lane 4: tx-j + choline + PCA. Results are expressed as mean ± standard deviation. Between group differences were analyzed by 1-way ANOVA; values with different letter symbols are significantly different (P < 0.05) from each other. Differences between sexes in the same treatment group were analyzed by Student's t-test; values with an asterisk (*) are significantly different (P < 0.05) between sexes. PCA, penicillamine.