A DIO2 missense mutation and its impact on fetal response to PRRSV infection

Abstract

Background: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted.

Results: A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection.

Conclusions: The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.

Keywords: Deiodinase; Fetus; Nonsynonymous mutation; PRRSV; Swine; Thyroid.

Fig. 1

Identification of a missense variant (Asn91Ser) in porcine DIO2. (A) Sequenced exons are indicated in the context of DIO2 gene structure with neighboring genes on Sus scrofa chromosome (SSC7) 101–105 Mb region (Ensembl Release 105 (Dec 2021) used). Coding sequence (red colored box) encompasses the two sequenced exons (blue box). A single nucleotide variant site (light blue arrow) in the coding region on SSC7:103,102,041 is a missense variant changing asparagine (Asn) to serine (Ser) at position 91 of porcine DIO2 protein. This variant is present as a dinucleotide polymorphism with a synonymous variant (bolded left to the missense variant) on SSC7:103,102,040. AA or BB is genotype of DRGA0008048 (DRGA) SNP which is an intergenic variant ~ 14 Kb distal to DIO2. The alternative allele (GC) is labelled as D while the reference (REF) allele (AT) is C to distinguish the variant from DRGA. (B) Map of AA and BB genotypes for each SNP within the exonic region of DIO2 in gilts and sires used in trial-2. SNP labels correspond to labels in additional file 1; SNP24-SNP3, SNPs in the 3 prime untranslated region; SNP1 and SNP2, Asn91Ser variant and the neighboring synonymous variant. Each cell denotes genotype at each SNP site. Light yellow cells, homozygous genotype for reference (ref) allele. Sky blue cells, heterozygous genotype. Dark blue cells, homozygous genotype for alternative (alt) allele. Number of gilts and sires was shown for each row indicating a combination of all genotypes across SNP loci. (C) Haplotypes in the sequenced DIO2 region in AA versus BB genotype by DRGA SNP. The most probable haplotypes were determined by manual assignment. * denotes frequency of each copy of haplotypes within DRGA genotype (AA versus BB) and within parents (gilts versus sires). Gray cells, alternative allele. White cells, reference allele. Edited with BioRender

Fig. 2

Estimated marginal means (EMM) of natural log transformed fold change (FC) of fetal DIO2 expression. (A) fetal heart; (B) fetal kidney. Data is grouped by Asn91Ser genotype for the subset of the trial-2 [14]. Fold change was calculated relative to control fetuses from non-inoculated litters and transformed by natural logarithm. Colored dots and associated vertical lines are EMM and 95% confidence interval, respectively. Overlapping gray dots are observed values. 5 CD fetuses deviating over log FC > 1 are highlighted with the dark gray dots in (A). P values for coefficient of genotype are labeled in the plots. Fetal counts in each genotype: (A) CC = 87, CD = 23; (B) CC = 81, CD = 23. Ratio of brain to liver weight (P = 0.001) or brain weight (g) (P = 0.015) included as a covariate in the regression model for heart log FC or kidney log FC, respectively

Fig. 3

Estimated odds ratios (OR) and 95% confidence interval (CI) of fetuses being viable or live. CC is homozygous genotype for the reference allele for Asn91Ser mutation of DIO2, CD for heterozygous genotype. Red dotted vertical lines indicate OR = 1. The Benjamini-Hochberg (BH) adjusted P values are applied to all possible pairwise comparisons (N = 6) of levels of Asn91Ser and trial (A), and Asn91Ser and fetal sex (B). CC genotype or trial-1 was used as the reference group (ref). Different x-axis scales were used across figures. (A) Contrasts for the interaction between Asn91Ser (CD vs. CC) and trial (trial-2 vs. trial-1) for fetal viability. Trial-1, Yang et al’s GWAS [6]; trial-2, follow-up study to Yang et al.’s GWAS [14]. Fetal counts in each genotype within each trial: CC = 41, CD = 37, in trial-1; CC = 71, CD = 74, in trial-2. (B) Contrasts for the interaction between Asn91Ser (CD vs. CC) and sex (male vs. female) for fetal viability. Fetal counts in each genotype within each sex: CC = 57, CD = 47, in female; CC = 55, CD = 64, in male. (C) Contrast for Asn91Ser (CD vs. CC) for fetal survival

Fig. 4

Estimated marginal means (EMM) of fetal viral loads and circulating thyroid hormones by Asn91Ser genotype (CC, CD) for each trial (trial-1 [6], trial-2 [14]). (A) viral load (VL) in serum (log10 copies/µl); (B) viral load (VL) in thymus (log10 copies/mg); (C) T4 level (nmol/L); (D) T3 level (nmol/L). Large central dots and associated vertical lines represent the EMM and 95% confidence interval, respectively. Overlapping small dots are raw data. Different y-axis scales used between (C) and (D). P values for coefficients of genotype or trial are shown in the plots if significant at P < 0.05. Fetal counts in each genotype within each trial: CC = 24, CD = 21, in trial-1; CC = 47, CD = 59, in trial-2 (1 CD fetus from trial-2 excluded for T3 level after residual analyses)

Fig. 5

Estimated marginal means (EMM) of fetal growth by Asn91Ser genotype (CC, CD) for each trial (trial-1 [6], trial-2 [14]). Large central dots and associated vertical lines represent the EMM and 95% confidence interval, respectively. Overlapping small dots are raw data. (A) fetal body weight (g); (B) fetal crown rump length (CRL) (cm); (C) fetal brain weight (g); (D) fetal ratio of brain to liver weight. (A, B) The Benjamini-Hochberg (BH) adjusted P values are applied to all possible pairwise comparisons (N = 6) of levels of Asn91Ser and trial and labeled for the genotypic or trial effects at P < 0.10) (C, D) P values for coefficients of genotype or trial are shown in the plots if significant at P < 0.05. Fetal counts in each genotype within each trial: (A, B) CC = 41, CD = 37, in trial-1; CC = 71, CD = 74, in trial-2; (C, D) CC = 26, CD = 24, in trial-1; CC = 47, CD = 59, in trial-2 (1 CD fetus from trial-2 excluded for the ratio of brain to liver weight after residual analyses)

Fig. 6

Fetal cortisol, T4, and T3 levels in sera by fetal classification and trial. (A) Data from both trials (trial-1 [6] and trial-2 [14]) were merged into each fetal classification. Asn91Ser genotypes were also combined at each level of fetal classification due to lack of significant differences on cortisol levels stratified by trial or fetal classification. Large central dots and associated vertical lines represent median values and equi-tailed two-sided nonparametric 95% confidence intervals, respectively, for each fetal classification. Overlapping small dots are raw data. Different superscripts denote a significant difference in median values between groups, based on Benjamini-Hochberg (BH) adjusted P value < 0.05 from pairwise two-sample permutation tests across groups of fetal classification. (B, C) Fetal observations for cortisol and T4 or T3 level colored by each group of fetal classification with different shapes for trial, respectively. Fetal counts in each level of fetal classification across trials: UNIF = 28, LV-VIA = 25, HV-VIA = 23, HV-MEC = 14