Balancing selection of a frame-shift mutation in the MRC2 gene accounts for the outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle

Abstract

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed.

Figure 1. Clinical spectrum exhibited by CTS cases.

Crooked tail, growth retardation, stocky head, extreme muscular hypertrophy, spastic paresis of the hind limbs, straight hock, scoliosis.

Figure 2. Positional identification of the c.2904_2905delAG MRC2 mutation causing CTS, and its effect of the Endo180 protein.

(A) Gene content of the 2.4 Mb interval in which the CTS mutation was located using identity-by-descent mapping. The triangles correspond to five SNPs used to refine the CTS locus position, with corresponding number of recombinant individuals out of 105 CTS cases. The resulting non-recombinant (NR) interval is marked by the red horizontal line. (B) Structure of the MRC2 gene within that interval. (C) Domain composition of the wild-type (WT) and mutant (MUT) Endo180 protein. (D) Sequences traced obtained from genomic DNA of a homozygous wild-type, carrier and homozygous CTS animal showing the deletion of the ApG dinucleotide in the mutant.

Figure 3. Nonsense-mediated RNA decay of c.2904_2905delAG mutant MRC2 transcripts.

(A) Direct sequencing of MRC2 amplicons spanning the CTS mutation obtained from genomic DNA and pulmonary cDNA of a heterozygous animal, showing the virtually exclusive detection of wild-type allele amongst transcripts (the position of the deleted nucleotides is underlined in red, the sequencing direction is represented by a triangle). (B) Comparing MRC2 mRNA levels in the lung of +/+, +/CTS and CTS/CTS animals. Data are shown for two amplicons at the 5′ and 3′ ends of the mRNA, respectively. Error bars correspond to standard errors over three replicates per sample.

Figure 4. Effect of the CTS mutation on the levels of full-length Endo180.

Western blot results from lung of animals of the three genotypes. Hu: human control sample. MW: molecular weight marker. The 55 Kd band corresponds to non-specific binding of the CAT2 antibody to tubulin, used as control for the amount of protein loaded.

Figure 5. Distribution of the number of simulations (out of 10,000) yielding 45 carriers out of 160 Précieux descendants (Y-axis), as a function of the rate of transmission of the mutation from heterozygous carriers (X-axis).

Three curves are given corresponding to frequencies of the mutation outside of the Précieux lineage of 0, 1, and 5%. The dotted red vertical line corresponds to a transmission rate of 67%, maximizing the number of simulations yielding 45 carriers for a mutation frequency (outside of the Précieux lineage) of 1%, considered to be an upper bound in BBCB.