MYH3‐associated non‐syndromic palatoschisis (cleft palate, CP) in Limousine cattle

Palatoschisis (also called cleft palate, CP) is an opening or cleft in the upper lip, the roof of the palate, or both, and is a common defect in both humans and domestic animals (Mossey & Modell, 2012; Mulvihill et al., 1980). It can also occur in cattle as an isolated (non-syndromic) birth defect (OMIA 000197-9913), owing to de novo mutations (Jacinto et al., 2021; OMIA 002483-9913) or associated with familial recessive syndromes (Agerholm et al., 2016; OMIA 000070-9913). A 2-years-old Italian-bred Limousine heifer was referred for a non-syndromic form of palatoschisis involving both soft and hard palate, rhinitis and retarded growth (Figure S1). We hypothesized a genetic etiology for this congenital anomaly and performed whole-genome sequencing. Further details of the methodology are described in Appendix S1. Two homozygous private protein-changing variants were located in a putative candidate gene for the observed phenotype, myosin heavy chain 3 (MYH3) (Table S1). Inherited forms of MYH3-related human syndromes, including cleft palate, are known (OMIM 160720). The homozygous variants at chr19:29609623A>G and at chr19:29609604TGTCAGCTCAAG>T represent a missense variant (NM_001101835.1:c.2864T>C; NP_001095305.1:p.Ile955Thr) and frameshift deletion (NM_001101835.1:c.2872_2882delCTTGAGCTGAC; NP_001095305.1:p.Leu958_Leu962delinsThrGlyGlnGlyTer), respectively, in exon 24 of bovine MYH3 (Figure S2). The isoleucine-to-threonine substitution affects an evolutionarily highly conserved amino acid in the myosin tail domain, and the frameshift deletion was predicted to cause a premature stop codon. Sanger sequencing confirmed the presence of both homozygous variants in MYH3 in the case (Figure S2). Among the additional 28 private homozygous coding variants, 23 were probably common variants, while the remaining five were classified as variants of uncertain significance (Appendix S1). A recent publication from France (Vaiman et al., 2022) finally proved the assumption of causality of MYH3 variants. To estimate the prevalence of MYH3 carriers within the Swiss Limousine population, we genotyped a population sample of purebred animals, including the bulls currently used for insemination (Table S2). The frequency of the variant allele in 1167 genotyped cattle was 1.5%. We have successfully identified the most likely cause for a rare autosomal recessive disorder with only one case. Our clinicopathologic and genomic findings for the MYH3-related recessively inherited form of non-syndromic cleft palate are consistent with those recently reported by Vaiman et al. (2022) (OMIA002590-9913). Interestingly, the 2-year-old Limousine female in our study appears to be the oldest known animal with MYH3-associated non-syndromic palatoschisis. According to Vaiman et al. (2022), life expectancy is short, with most animals euthanized in the first month of life. In addition, the variant allele frequency was found to be lower in the current Swiss population compared with the French Limousine (2.4%). Whether the MYH3 missense or the loss-of-function variant, or both, is actually causal remains open. The affected gene, MYH3, encodes the myosin heavy chain 3 protein, which is known to be responsible for muscle contraction. In addition, MYH3 is expressed in a variety of human and mouse fetal tissues (Chong et al., 2015; Zieba et al., 2017). In particular, in the mouse, Myh3 has been shown to be moderately expressed in head tissues during embryonic development (MGI: 1339709). There are several factors that can lead to the development of palatoschisis, such as impaired function and position of the tongue and surrounding muscles, which can disrupt the embryonic development of the palate (Rot & Kablar, 2013). Therefore, considering the rarity, the in silico effect prediction, the role of skeletal muscle in embryonic palate formation, the known function of MYH3 and the report of Vaiman et al. (2022), the identified variants can be considered as the cause. Future studies evaluating the functional consequences will be valuable in understanding the biological impact of the MYH3 variants during craniofacial development. We thank the Interfaculty Bioinformatics Unit of the University of Bern for providing high-performance computational infrastructure. We acknowledge Isabella Aebi-Huber for expert technical assistance. We are grateful to IFN Schönow GmbH, Germany for the genotyping of the Swiss Limousine population control cohort. Open access funding provided by Universitat Bern. This study was financially supported by the Arbeitsgemeinschaft Schweizerischer Rinderzüchter, Zollikofen, Switzerland and the Federal Office for Agriculture, Bern, Switzerland. The authors declare no conflict of interest. The whole genome sequencing data are available under the study accession no. PRJEB18113 at the European Nucleotide Archive (www.ebi.ac.uk/ena; SAMEA111531537). Appendix S1. Figure S1. Figure S2. Table S1. Table S2. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.