A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model

Abstract

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.

Fig 1. Genome scan for the milk somatic cell count trait LSCS in a grand-daughter design of 1009 dairy sheep identifies a highly significant QTL on chromosome OAR3.

(A) Manhattan plot for likelihood ratio test profile for LSCS trait based on haplotype-based association analyses on the 26 ovine autosomes. (B) Global likelihood ratio test (LRT) profile for LSCS trait on chromosome OAR3 based on both linkage and haplotype-based association analyses. The 5% genome-wide thresholds are indicated for association (solid line) and linkage (dotted line) analyses. (C) Localisation of the 207 SNP in the OAR3 QTL confidence interval. SNP were identified using whole genome sequencing in a trio of rams.

Fig 2. Bioinformatics characterization of Socs2 and SOCS2 p.R96C mutation.

(A) Structure of the Socs2 gene. (B) SOCS2 is highly conserved across species. The figure was obtained with SOCS2 protein sequences from human, mouse, cattle, pig and sheep species with weblogo software (http://weblogo.threeplusone.com/). Amino-acids are coloured according to their chemical properties. A red arrow shows R96 position. (C) The site of the mutation in the SOCS2 protein structure based on a model predicted by Homotopy Optimization Method, HOPE. (D) Close-up of the mutation. The protein is colored grey, the side chains of both the wild-type and the mutant residue are shown in green and red respectively. The mutation is located within the SH2 domain and encodes an arginine (a polar positively charged) to cysteine (mostly hydrophobic) substitution, which can disturb this domain and abolish its function.

Fig 3. Real-time binding of SOCS2-WT and SOCS2-p.R96C proteins on immobilized pY-GHR.

(A) Binding analysis was performed on immobilized GHR and scramble pY-peptides (370 RU) at a final concentration of 800 nM. (B) Single Cycle Kinetics analysis was performed on immobilized GHR and scramble peptides (75 RU) with five injections of analyte at 100nM, 300nM, 900nM, 2.7μM, and 8.1μM. Analyte injections lasted for 120 s each and were separated by 184-s dissociation phases. The last injection was followed by an extended dissociation period of 10 min. The two sensorgrams recorded for a given analyte were fitted globally to a 1:1 interaction (black curves). Each sensorgram represents a differential response where the reference channel has been subtracted and is expressed in RU as a function of time in seconds.

Fig 4. Effect of Socs2 genotype on milk somatic cell counts (LSCS), Milk Yield and Fat Content in 468 rams.

(A, B and C) The lsmeans (error bars indicate standard errors) for LSCS (A), Milk Yield (B) and Fat Content (C) from a mixed model including the genotype and sire effect. Traits are expressed as the standard deviation of daughter yield deviations (DYD). The lower case letters (a, b, c) show significant differences in the trait between genotypes. For LSCS, the heterozygotes(C/T) and homozygotes (T/T) were significantly different from wild-type homozygous (C/C) as determined by t-test at p < 0.05. For Milk yield and Fat content, heterozygous (C/T) were significantly different from C/C homozygotes as determined by t-test at p < 0.05.

Fig 5. Effect of Socs2 genotype on body weight in eighteen sheep.

The lsmeans (error bars indicate standard errors) for body weight from the mixed model with repeated measures over a 3-year phase including 3 lambing periods. The asterisk shows significant differences in the trait between three genotypes. * For time point 8 to 11, homozygotes (T/T) were significantly different from wild-type homozygous (C/C) as determined by t-test at p < 0.05. ** For time point 11, homozygotes (T/T) were significantly different from wild-type homozygous (C/C) as determined by t-test at p < 0.01. *** For time point 12, homozygotes (T/T) were significantly different from wild-type homozygous (C/C) as determined by t-test at p < 0.001 and heterozygous (C/T) were significantly different from wild-type homozygous (C/C) by t-test at p < 0.05.

Fig 6. Effect of Socs2 genotype on body size in eighteen sheep.

Lsmeans are from a GLM models including Socs2 genotype (C/C, C/T and T/T) age at scoring and sire effects. The asterisk shows significant overall effect of Socs2 genotype at * p <0.05 and ** p <0.01. Different letters superscript (a,b) show a significant difference between genotypes at p <0.05.