A de novo mutation in KRT5 in a crossbred calf with epidermolysis bullosa simplex

Abstract A 6‐day‐old Belgian Blue‐Holstein calf was referred because of a syndrome resembling epidermolysis bullosa simplex (EBS). The clinical phenotype included irregular and differently sized erosions and ulcerations spread over the body, in particular on the limbs and over bone prominences, as well as in the nasal planum and oral mucosa. Blisters were easily induced by rubbing the skin. The skin lesions displayed a clear dermal‐epidermal separation at the level of the basal cell layer. Post mortem examination revealed erosions in the pharynx, proximal esophagus, and rumen. Whole‐genome sequencing revealed a heterozygous disruptive in‐frame deletion variant in KRT5 (c.534_536delCAA). Genotyping of both parents confirmed the variant as de novo mutation. Clinicopathological and genetic findings were consistent with the diagnosis of KRT5‐related EBS providing the second example of a spontaneous mutation causing epidermolysis bullosa in cattle.

The majority of the previous reports focused on disorders with a recessive inheritance. However, single cases because of dominant acting de novo variants might occur sporadically without impact on breeding. At present, this obvious genetic heterogeneity could be analyzed in cattle using whole-genome sequencing (WGS)-based precision diagnostics. 28 Therefore, the purpose of this study was to characterize the clinical and pathological phenotype of an EBS-affected calf, and to evaluate its possible genetic etiology using WGS.

| CASE DESCRIPTION
A 6-day-old (46 kg) male Belgian Blue-Holstein crossbred calf was submitted for clinical investigation because of ulcerations of the skin and nasal planum since short time after birth. The animal was delivered after a gestation period of 287 days.
The cutaneous lesions were characterized by widespread irregular erosions and ulcerations of various sizes on most parts of the body ( Figure 1A), but in particular on the limbs ( Figure 1B) and over bony prominences. Upon handling, the epidermis easily separated leaving a blister with a black colored, nonhemorrhagic base indicating a separation superficial to the stratum basale. Peracute blister were easily induced by rubbing the skin by an eraser after having cut the hair locally. Older lesions consisted of ulcerations covered by crusts and occasional acute hemorrhage. On the nasal platum, lips and nares extensive ulcerations were present; the calf also showed a purulent nasal discharge ( Figure 1C). Moreover, the animal seemed to be in pain when walking on a hard surface. The aspect resembled EB and therefore was further referred to the Danish surveillance program for genetic diseases in cattle for further examination. Because of the poor prognosis and the painful situation, the calf was euthanized for welfare reasons by IV administration of an overdose of pentobarbital. In addition to the skin lesions, gross pathologic examination revealed erosions in the oral cavity, pharynx, proximal esophagus, and rumen. The epithelium on the dorsal surface of the tongue was thickened and with furrows ( Figure 1D). The incisor teeth were disorganized and not completely erupted and the surrounding parts of the mandibles appeared thickened and cystic. The hoofs seemed intact, yet when sawed longitudinally, the capsule was partly separated from the dermal lamella with hemorrhage in the interface.
Immediately before euthanasia, the skin covering the dorsal part of the pelvis was gently scrubbed with an eraser with blister formation. Skin biopsies from this area and from other representative cutaneous lesions were taken immediately after euthanasia for histological analysis, whereas the necropsy was completed at the university a few hours later. Additional specimens for histological analysis were then collected, including the oral mucosa, pharynx, rumen, reticulum, and major internal organs. All collected samples were fixed in 10% neutral buffered formalin, trimmed, processed, embedded in paraffin wax, sec-  To confirm that the c.534_536delCAA variant in KRT5 was a de novo mutation, the affected genomic region was amplified by polymerase chain reaction (PCR) and Sanger sequenced in the affected calf, its Belgian Blue sire and Holstein dam based on DNA extracted from EDTA blood of the dam, and from both EDTA blood and semen of the sire. PCR products were amplified using flanking primers for the KRT5 exon 1 deletion with 5 0 -AGGCATCCAAGAGGTCACCG-3 0 (forward primer) and 5 0 -TAGCACATATCCCACACTCATGG-3 0 (reverse primer).
Sequence data were analyzed using Sequencher 5.1 (GeneCodes). Analyzing the sequencing data, we concluded that only the EBS-affected calf was heterozygous for the KRT5 variant and the dam and sire were both homozygous for the wild type allele in all analyzed samples including both semen and blood of the sire (Figure 3).

| DISCUSSION
The clinical and pathological findings in the calf were consistent with  36,37 and muscular dystrophy. 38,39 In human, intermediate EBS with muscular dystrophy is often associated with enamel hypoplasia. 39 Furthermore, beyond the characteristic skin lesions, it might include diffuse alopecia, short stature, slow weight gain, punctate keratitis, urethral strictures, muscular dystrophy, and degenerative changes with increased connective tissue. 35 Such findings were not observed in the studied calf.
In human medicine, a de novo missense variant in KRT5 resulting in an amino acid exchange (p.Asn177Ser) in the HIM has been reported in a patient with localized EBS. 40