For eight of the nine haplotypes with a significant effect on calving rate (see Mapping section), Fritz et al. (2013) searched for causal mutations via whole-genome sequence data from 25 Holstein, 11 Montbéliarde and nine Normande bulls which had made major contributions to their breed. Specifically, they filtered "for mutations that were (a) located at+or –6 Mb from the detected haplotype (b) carried in the heterozygous state by the carrier bulls and (c) absent from the non carrier bulls from the three breeds" and then examined identified polymorphisms for their likely effect on protein structure and function. For MH1, Fritz et al. (2013) provided strong evidence for a candidate causal mutation, namely a nonsense mutation (g.27956790C>T; UMD 3.1 genome assembly) in the SHBG gene (which encodes sex-hormone binding globulin), leading to p.Q52X. Reinartz and Distl (2016) reported this mutant occurring in Vorderwald cattle with Montbéliarde ancestry, including in one live animal that is homozygous for this mutant. The one live animal homozygous for this supposedly lethal variant reported by Reinartz and Distl (2016) was reinforced by an extensive study by Michot et al. (2017) who, in the results of genotyping 128,743 Montbéliarde cattle, "identified 242 homozygous carriers for the SHBG nonsense mutation (for 650 expected homozygotes), suggesting that it was not the causative mutation for MH1-linked embryonic lethality." Subsequent fine mapping of the MH1 region excluded SHBG as a candidate and eventually led Michot et al. (2017) to "g.28511199C>T (rs455876205) . . . PFAS p.R1205C" as the likely causal variant. Supporting evidence included "The absence of homozygous mutants among tens of thousands of genotyped animals, the perfect conservation of the affected residue among eukaryotes, and the critical function of PFAS and of the de novo purine synthesis pathway".
