- In order to identify private genetic and structural variants, we screened whole genome sequencing data of the affected calf and unaffected relatives including parents, a maternal and paternal halfsibling.. - A defect centrally in the front skull covered with a membrane extended into the intracranial cavity. - Filtering whole genome sequencing data revealed a private frameshift variant within the candidate gene ZIC2 in the affected calf. - The origin of this variant is most likely due to a de novo mutation in the germline of one parent or during very early embryonic development. - The ab- sence of the olfactory tract (arhinencephaly), partial fu- sion (synophthalmia), complete fusion (cyclopia) or the total absence (anopthalmia) of ocular globes are pheno- types of FND found in cattle [1]. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - Full list of author information is available at the end of the article. - The head showed severe craniofacial alterations such as ab- normal development of the maxillary processes and face skull (Fig. - Cattle Hereford Holoprosencephaly, arhinencephaly, hypotelorism Cho et al. - Japanese Brown Median cleft of the face, shortened upper jaw, ocular hypertelorism, Moritomo et al.. - Holstein Cyclopia, holoprosencephaly Kim et al. - Cyclopia with prosencephalic aplasia, brachygnathia superior and arrhinia ÖZcan et al. - Crossbreed Cyclopia, absent muzzle and all the skeletal structures of the nose, protruding tongue Malik et al. - Crossbreed Mild hypotelorism, microphthalmia, cheiloschisis, arhinencephaly Osman et al.. - Synophthalmia, arrhinia Nourani et al.. - Holstein Shortening of the nasal structures, micrognatia superior, shortened mandibles, protrusion of the tongue, bilateral eye prolapse, brain malformations. - Agerholm et al.. - Pandey et al.. - Murrah Cyclopia with cleft palate and complete absence of muzzle Singh et al. - Albarella et al.. - Sheep Texel Aprosencephaly with otocephaly Brachthäuser et al.. - Domed head with a short nasal bone and curved mandible Dantas et al.. - Cyclopia (monkey face lamb disease) Welch et al. - Goat Balady Cyclopia with small upper jaw and large protruded lower jaw Rashed et al.. - The orbits were rudi- mentary and placed on the front side near to the midline of the face instead of the lateral sides of the head. - 1 Macroscopic pictures of the skull of the affected calf (a, b) showing severe deformations and absence of facial structures. - Centrally on the skull a defect was present (asterisk), insert showing a higher magnification of the defect after removal of the skin. - 2 Computed tomography of the skull of the affected calf (a, c) and a control animal (b, d). - a In the rostrocaudal view the absence of the maxilla, nasal and incisive bones and the non-fused os frontale were striking features. - Only parts of the brain stem were devel- oped. - The malformations of the forebrain included an absence of the prosencephalon (including telencephalon and diencephalon) and a hypo- plastic mesencephalon and rhombencephalon. - In the oral cavity, incisors and canini were developed, but pre- molars were missing. - A median cleft palate of the soft and hard palate due to a complete median disclosure was evident. - The sire of the present case was a natural service. - An autosomal re- cessive mode of inheritance, where both, the dam and the sire, are heterozygous mutant, is very unlikely due to the sporadic occurrence of this case and 120 progeny within 2 years from matings of the same bull in this herd. - A pater- nal dominant germline mutation also seems unlikely be- cause of the large number of progeny and only one case among 120 births. - The sole candidate gene with a variant identified in the case was ZIC2. - g.76742066TC>T) in exon 4 of the candidate gene ZIC2 (Fig. - 4) was heterozygous in the affected calf and homo- zygous wild type in both parents and all other Limousin herdmates and private controls. - 3 Pedigree of the Limousin calf, which was affected with a frontonasal dysplasia phenotype. - The se- quence of the mutant protein has a likely amino acid (aa) exchange of prolin to arginine at position 543 (p.Pro543fs. - The frameshift mutation was predicted to cause a truncation of the Zinc Finger Protein Zic and Gli (PTHR19818) domain at codon 543 using ORFfinder.. - Congenital FND, arhinencephaly and severe defects of the central nervous system were the main and character- istic findings in the present case. - Con- trasting to the present case, bone structures of the head were completely absent in this lamb. - 4 Gene model of the wild type (a) and mutant (b) ZIC2 according to UMD3.1. - The location of the ZIC2:g.80722845 TC>T (ARS-UCD1.2:. - HPE also heart defects like inter-ventricular communica- tion orifice were found similar to the complex heart malformations seen in the present case of our Limousin calf [3].. - The WGS data analysis of the present case retrieved a heterozygous frameshift variant in ZIC2, which is a strong candidate gene for HPE in human and mouse.. - We validated the wild type genotypes in the parents, two half-siblings and controls of the affected Limousin calf but were not able to get Sanger sequences from a further tissue sample of the affected calf. - The reason Sanger se- quencing failed is that DNA degraded because sampling could only be done from the insufficiently cooled body about 2 weeks after birth of the affected calf. - The subfamily PTHR19818:SF18 is part of the. - The zinc-finger domains of the Zic and Gli factors bind to the identical target sequences as the transcription factors, which me- diate hedgehog signals [32]. - addition, ZIC2 is one of the most commonly heterozy- gous mutated genes in human HPE patients [35, 36]. - In the present study, we identified a de novo frameshift variant in ZIC2 for lethal frontonasal dysplasia in a Li- mousin calf. - According to the report of the owner, the calf was the first case of any skeletal malformations seen in this cattle herd. - The sire of the affected calf was a natural service Limousin bull, registered in the Limousin herd book. - This bull sired all heifers and cows of this herd in the calving period when the case was observed. - The dam and the sire of the calf were in healthy condition and had normal appearance. - Further, all half-siblings of the affected calf were in healthy condition and had no congenital abnor- malities. - We collected tissue samples from the affected calf and EDTA-blood samples from the vena jugularis of the sire and dam of the affected calf, and further of two maternal half-siblings, 9 paternal half-siblings and three unrelated control animals. - Necropsy and histopathological examinations were performed for the affected calf with particular reference on the malforma- tions of the head and brain. - Samples of the membrane covering the defect on the front skull were taken, fixed in 10% formalin and embedded in paraffin. - Quality control of the isolated DNA was performed and ensuing libraries from these samples were prepared according to the manufacturers protocols using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA).WGS was performed on an Illumina NextSeq500 (Illumina, San Diego, CA, USA) in a 2 × 150 bp paired-end mode. - Quality control of the WGS data was done using fastqc and the reads were trimmed using PRINSEQ (V . - Variant calling was done for the five Limousin samples and further 89 WGS data from cattle of the breeds Holstein, Fleckvieh, Braunvieh, Vorderwald, German Angus, Galloway, Limousin, Cha- rolais, Hereford, Tyrolean Grey and Miniature Zebu.. - The resulting vcf-file was used for screening private vari- ants in the FND case. - bp (ARS-UCD bp) from the vcf-file using SAS, version 9.4 (Statistical Analysis System, Cary, NC, USA) to identify all coding and noncoding variants of the affected calf.. - We fil- tered for variants, which were homozygous mutant in the affected Limousin calf and heterozygous mutant in the dam and sire and heterozygous or homozygous wild type in both half-siblings using SAS, version 9.4. - In addition, we screened for variants, which were heterozy- gous mutant in the affected Limousin calf but homozy- gous wild in both parents and both half-siblings. - Structural variants were filtered out which were heterozygous mu- tant in the affected calf and homozygous wild type in all other animals or which were homozygous mutant in the affected calf and heterozygous in parents and not homo- zygous mutant in its half-sibs. - To verify the previously investigated protein effect of the ZIC2 variant, we applied the PolyPhen-2 (Poly- morphism Phenotyping v2) tool. - To visualize predicted changes in the ZIC2 protein, we used ORFfinder (NCBI, www.ncbi.nlm.nih.gov/orffinder). - The resulting amino acid (aa) sequences of the wild type protein (NSBTAP UMD3.1) and predicted mutant protein were compared and aligned using ClustalW2, version 2.1 [45]. - Only one variant (printed in bold) on BTA 12 within the critical candidate gene ZIC2, which was associated with holoprosencephaly in mammalian animals, exclusively occurred in the affected calf. - MB, JM and OD conceived and designed the experiments of the study. - WGS data of the calves, the sire, the dam and the maternal and paternal half-siblings were deposited in NCBI Sequence Read Archive under the pro- ject number PRJNA526664 (SAMN11107014, SAMN11107015, SAMN11107016, SAMN11107017, SAMN11107018). - We obtained written informed approval from the owner of the Limousin cattle to use samples and photographs from his animals for current research, publication and further investigations. - 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