KDM2B‐associated paunch calf syndrome in Marchigiana cattle

Abstract Background Chianina, Romagnola, and Marchigiana are the 3 most important Italian breeds of cattle raised in the Apennine Mountains. Inherited disorders have been reported in the Chianina and Romagnola breeds but not in the Marchigiana breed. Recently, a case resembling recessively inherited KDM2B‐associated paunch calf syndrome (PCS) in Romagnola cattle was identified in Marchigiana cattle. Hypothesis/Objectives To characterize the features of the observed congenital anomaly, evaluate its possible genetic etiology, and determine the prevalence of the deleterious allele in the Marchigiana population. Animals A single stillborn Marchigiana calf was referred for clinicopathological examination because of the presence of PCS‐like morphological lesions. Methods The animal was necropsied and the calf and its parents were genotyped. A PCR‐based direct gene test was applied to determine the KDM2B genotype and 114 Marchigiana bulls were genotyped. Results The pathological phenotype included facial deformities, enlarged fluid‐filled abdomen, and hepatic fibrosis. The affected animal was the offspring of consanguineous mating and homozygous presence of the KDM2B missense variant was confirmed. Both parents were heterozygous for KDM2B and the prevalence of carriers in a selected population of Marchigiana bulls was <2%. Conclusions and Clinical Importance The characteristic malformations and genetic findings were consistent with the diagnosis of PCS and provide evidence that the deleterious KDM2B variant initially detected in Romagnola cattle also occurs in the Marchigiana breed.

Animals (OMIA) database, 4 and identification of recessive pathogenic variants allows targeted genotyping and the avoidance of high-risk matings of carrier animals. 5 Although many are unknown and routine screenings are not performed, recessive variants can quickly spread through a population by widespread use of popular carrier bulls in artificial insemination (AI). This practice led to the appearance of worrisome pathogenic variants in the Chianina and Romagnola breeds. [6][7][8][9][10] In Chianina cattle, the c.491G>A missense variant in the ATP2A1 gene, encoding the SERCA1 pump, was reported to cause autosomal recessive pseudomyotonia (PMT), a congenital muscle function disorder (OMIA 001464-9913). 9 Among the ranked Chianina sires in the years 2007 to 2011, the prevalence of heterozygous PMT carriers was 13.6%, illustrating the scale of the problem. 11 Subsequently, ATP2A1-associated PMT also was reported in the Romagnola breed associated with allelic heterogeneity of the ATP2A1 gene. 10 In addition to the previously reported c.491G>A missense variant originating from accidental introgression of a Chianina PMT carrier, 2 additional rare ATP2A1 missense variants (c.632G>T and c.857G>T) also were found to be causative for the disorder. 10 Ichthyosis fetalis caused by a missense variant in the ABCA12 gene (harlequin ichthyosis; OMIA 002238-9913) is another lethal recessively inherited disorder that occurs in the Chianina breed. 12 A less severe form of congenital ichthyosis also was reported in Chianina cattle, but no causative genetic variant thus far has been found. 13 More recently, rare recessively inherited congenital bilateral immature nuclear cataracts have been found in Romagnola cattle (OMIA 001936-9913) and are associated with a large deletion affecting the coding region of the NID1 gene. 14 Previously, an outbreak of a lethal multiorgan developmental dysplasia was described in 65 Romagnola cattle (OMIA 001722-9913) and determined to be caused by a c.2503G>A missense variant in the KDM2B gene. 6,7 On the basis of the phenotype, this disorder was named "paunch calf syndrome" (PCS). Affected calves usually are stillborn or die within hours of delivery. 6,7 The KDM2B gene encodes a histone demethylase that acts as an important transcription regulator affecting organ development and cell differentiation. The disease-associated variant leads to an amino acid exchange in a highly conserved domain, thus explaining the phenotypic effect of the genetic variant. 7 The PCS phenotype mainly is characterized by facial dysplasia, an enlarged and pendulous abdomen ("paunch") with considerable abdominal effusion and hepatic fibrosis. 6,7 Additional lesions, such as cleft palate, lack of the medial dew claws of ≥1 limbs, SC edema, perihepatic cysts, and cardiac malformations are found in some cases. 6  were applied to formalin-fixed liver samples.

| MATERIALS AND METHODS
Additionally, a pedigree analysis of the affected calf was performed. We harvested cutaneous tissue from the calf, as well as EDTA blood from its parents. Desoxyribonucleic acid was extracted and the 3 subjects were genotyped for the c.2503G>A variant in the KDM2B gene, as previously described 7

| Clinical phenotype
The affected calf had an enlarged head caused by a SC swelling that was especially evident in the periocular and the submandibular regions. The splanchnocranium was shortened and the frontal region asymmetric. The tongue was swollen and protruded from the mouth.
The eyes were enlarged with evident scleral injection and conjunctival F I G U R E 1 Stillborn Marchigiana calf showing multiple congenital anomalies. Note the shortened face and distended ventral abdomen resembling paunch calf syndrome reported in Romagnola cattle. Note the swollen hind legs and the right fetlock externally rotated edema. The ventro-abdominal region was enlarged and pendulous and generalized swelling was noticed. The hind legs also were swollen and the right fetlock was externally rotated (Figure 1).

| Pathological phenotype
The swellings were caused by generalized SC infiltration of serosanguineous fluid, as evidenced at necropsy. Similar fluid collection also was present in the peritoneal, pleural, and pericardial cavities.
The liver was larger than normal, with an irregular surface and firm consistency. Upon sectioning, the cut surface of the liver had numerous bands of whitish, collagenous connective tissue, and a lobular structure partitioned by fibrous septa, which also were visible inside the lobules. Two round 5-mm diameter cysts, containing red-

| Genetic analysis
Pedigree analysis of the stillborn Marchigiana calf disclosed the presence of a consanguineous mating, because the sire of the affected calf was also the sire of its dam (Figure 3). In light of this inbreeding, autosomal recessive inheritance seemed a likely explanation for the occurrence of the congenital anomaly. The homozygous presence of the

| DISCUSSION
The clinical and pathological findings described in the calf of our study completely overlapped with the phenotype reported for PCS of the Romagnola breed. Therefore, our report constitutes the first description of a KDM2B homozygous mutant case of PCS in Marchigiana This speculation was confirmed by the genotyping of 114 bulls used for AI. Nevertheless, additional data might be useful to make a more precise estimate. For this purpose, DNA isolated from skin biopsy samples or semen should be used to determine the individual's genotype without concern about cattle-specific leukochimerism possibly causing a false genotype. 16  Our report serves to alert breeders of Marchigiana cattle about the possible emergence of PCS in the future and will permit the avoidance of carrier matings by systematic genetic testing of potential sires.

ACKNOWLEDGMENTS
The data reported in this work are classified and stored in the tissue bank of the Institute of Genetics, Vetsuisse Faculty, University of Bern. Data concerning the genotyping of the specific bulls are property of the breeding association.

CONFLICTS OF INTERESTS DECLARATION
Authors declare no conflict of interests.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.