Spontaneous and ART-induced large offspring syndrome: similarities and differences in DNA methylome

Figures & data

Figure 1. Example of phenotypic abnormalities of SLOS and ART-LOS calves. (a) Abdominal wall defect of US_SLOS_#5 (Angus breed). This spontaneous LOS calf was born alive and had to be euthanized due to the body wall malformation. (b and c) Macrosomia and macroglossia of US_SLOS_#6 (Charolais breed). This stillborn calf was ~77 Kg at birth. The average weight for calves of this breed is ~ 36 Kg. (d and e) Macroglossia of ES_ART_#2 and of stillborn ES_RF_necropsy_#1 (Asturian Valley x Limousin crossbred), respectively.

Table 1. Information of calves in this study. Sex with * = predicted sex based on WGBS reads alignment to chromosome Y (lack of sex information from original owners/sample providers). BW = birth weight. Used ID = animal ID used in previous publications.

Origin	Group	Animal	Breed	Sex	BW (Kg)	Tissue	Clinical diagnosis	Used ID
United States	US_Control	US_Control_#1	Holstein	Male	-	Blood, muscle	None	-
		US_Control_#2	Holstein	Male	-	Blood, muscle	None	-
		US_Control_#3	Holstein	Male	-	Blood, muscle	None	-
	US_SLOS	US_SLOS_#1	Angus	Male	58	Ear	Macrosomia	-
		US_SLOS_#2	-	Male*	-	Muscle	N/A	-
		US_SLOS_#3	Simmental	Male*	54	Tongue	Macrosomia	-
		US_SLOS_#4	Hereford	Female*	61	Muscle	Macrosomia	-
		US_SLOS_#5	Angus	Female	-	Muscle	Ventral abdominal wall: cloacal exstrophy, omphalocele, diastasis recti; heart: patent ductus arteriosus, patent foramen ovale	-
		US_SLOS_#6	Charolais	Male	77	Muscle, tongue	Macroglossia, macrosomia	-
		US_SLOS_#7	Shorthorn crossbred	Female	44	Muscle, tongue	Dexamethasone and prostaglandin inducecd delivery 16 days preterm	-
		US_SLOS_#8	Shorthorn crossbred	Male	58	Muscle, tongue	Dexamethasone and prostaglandin inducecd delivery 16 days preterm, macroglossia, macrosomia, microtia, hardened umbilicus	-
	US_SLOS_dam	US_SLOS_#6_dam	Charolais	Female	37	Blood	None	-
	US_SLOS_sibling	US_SLOS_#6_sibling	Charolais	Male	41	Blood	None	-
	US_SLOS_sire	US_SLOS_#6_sire	Charolais	Male	-	Blood	None	-
Spain	ES_Control	ES_Control_#1	Asturian Valley x Holstein (AxH)	Male	32	Blood, muscle	None	AI 1
		ES_Control_#2	AxH	Male	30	Blood, muscle	None	AI 4
		ES_Control_#3	AxH	Male	45	Blood, muscle	None	AI 5
	ES_ART	ES_ART_#1	Asturian Valley x Limousin crossbred (AxL)	Male	35	Muscle	None	BSA 1
		ES_ART_#2	AxL	Female	46	Blood	Macroglossia, hypersensitivity to needles	BSA 3
		ES_ART_#3	AxL	Male	33	Muscle	None	BSA 4
		ES_ART_#4	AxL	Male	33	Muscle	None	BSA 8
	ES_RF	ES_RF_#1	AxL	Male	29	Blood, muscle	Heart murmur, trouble standing for the first three days	RF 5
		ES_RF_#2	AxL	Male	49	Blood, muscle	None	RF 7
		ES_RF_#3	AxL	Male	33	Blood, muscle	None	RF 8
	ES_RF_necropsy	ES_RF_necropsy_#1	AxL	Male	56	Muscle	Stillbirth, dystocia, macrosomia, enlarged head, limb hyperflexion, cardiomegaly	RF 2
		ES_RF_necropsy_#2	AxL	Female	56	Muscle	Died after birth due to spine fracture, dystocia, macrosomia, limb hyperflexion, cardiomegaly	RF 6

Figure 2. Distribution of LOS associated differentially methylated regions (DMRs) across various genomic contexts. (a-c) Muscle US_SLOS vs. US_Control DMRs. (d-f) Muscle ES_RF_necropsy vs. ES_Control DMRs. (a-b and d-e) Each figure shows the total number of DMRs in the comparison and the number and percent of the hypermethylated (hyper; a and d) and hypomethylated (hypo; b and e) DMRs over each genomic context. In addition, the figures include the number and percent of DMRs that overlap with two previous studies (Li [25] and Chen [19]) for comparison purposes. (c and f) Percent of the genomic context that overlaps with DMRs. Obs = observed frequencies. Exp = expected frequencies (mean ± standard deviation; obtained from randomly shuffling DMRs across genome 10,000 times). The p values were calculated as p = n(|Exp – mean(Exp)| ≥ |Obs – mean(Exp)|)/10,000.

Figure 3. Example of LOS-associated vulnerable loci. Hypo = hypomethylation. Hyper = hypermethylation. Differentially methylated regions (DMR) identifiers are the positions in the bovine genome assembly ARS-UCD1.2. For complete information please refer to Table S2.

Figure 4. LOS-vulnerable loci around promoter regions. This figure shows DNA methylation level of vulnerable loci 7_2579941_2581100 (a), 8_43514181_43518080 (b), and 9_64659781_64660620 (c) in four LOS experiments. The aforementioned numbers refer to the chromosomes and genomic position in bovine genome assembly ARS-UCD1.2. Met% = group mean CpG methylation level in percent. Cov = group mean CpG read coverage. DMR = differentially methylated regions. Hyper = hypermethylation (red). Hypo = hypomethylation (yellow).

Figure 5. LOS-vulnerable loci overlapping CpG Islands in body of imprinted genes. This figure shows DNA methylation level of vulnerable loci 6_94882141_94883160 (a), 9_96225361_96226140 (b), and 13_66465461_66466640 (c) in four LOS experiments. The aforementioned numbers refer to the chromosomes and genomic position in bovine genome assembly ARS-UCD1.2. Met% = group mean CpG methylation level in percent. Cov = group mean CpG read coverage. DMR = differentially methylated regions. Hyper = hypermethylation (red). Hypo = hypomethylation (yellow).

Figure 6. DNA methylation at the 25 highly vulnerable loci of LOS (≥ 3 experiments) in US_SLOS_#6 calf, its sire, dam, and full-sibling. (a) Violin plots with dots showing the difference of DNA methylation between examined individual/group (mean) and mean of US_Control for highly vulnerable loci in LOS (found in ≥3 LOS experiments). Each dot in the violin plot represents a vulnerable locus. P values were from t-test. The baseline for blood samples is US_Control blood and the baseline for muscle samples is US_Control muscle. (b-p) Box plots with dots show DNA methylation level (y-axis) at highly vulnerable loci without obvious differences (>10%) in parental blood samples (b-f), with obvious differences in sire only (g), both sire and dam (h-i), dam only (j-o), and sibling only (p) when compared to the mean of control group. Each dot in the box plot represents a sample.

Figure 7. Distribution of ART associated differentially methylated regions (DMRs) across various genomic contents. (a-c) Muscle ES_ART vs. ES_Control DMRs. (d-f) Muscle ES_RF vs. ES_Control DMRs. (a-b and d-e) Each figure shows the total number of DMRs in the comparison and the number and percent of the hypermethylated (hyper; a and d) and hypomethylated (hypo; b and e) DMRs over each genomic context. In addition, the figures include the number and percent of DMRs that overlap with two previous studies (Li [25] and Chen [19]) for comparison purposes. (c and f) Percent of the genomic context that overlaps with DMRs. Obs = observed frequencies. Exp = expected frequencies (mean ± standard deviation; obtained from randomly shuffling DMRs across genome 10,000 times). The p values were calculated as p = n(|Exp – mean(Exp)| ≥ |Obs – mean(Exp)|)/10,000.

Figure 8. Conservation of DNA methylation at LOS-vulnerable loci between muscle and blood in ES_RF group. Y-axis = DNA methylation level. Note: ES_ART_2 is the same animal as Figure 1(d).

Li Y, Boadu F, Highsmith M, et al. Allele-specific aberration of imprinted domain chromosome architecture associates with large offspring syndrome. iScience. 2022; In press.

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Chen Z, Hagen DE, Ji T, et al. Global misregulation of genes largely uncoupled to DNA methylome epimutations characterizes a congenital overgrowth syndrome. Sci Rep. 2017;7(1):12667.

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S equencing d ata a vailability statement

The raw sequencing reads of WGBS used in this study are available in the GEO database with accession numbers (GSE199084).