Identification of a de novo missense variant in the BRI3BP gene in a Holstein calf with congenital cardiac malformation and carpus valgus

Abstract

Congenital malformations in cattle pose a diagnostic challenge with limited treatment options and are often associated with a guarded prognosis. The aim of this study was to characterize the clinicopathological phenotype of a viable calf with complex congenital heart defects and carpus valgus, and to identify a possible genetic cause using a whole genome sequencing trio approach. A 3‐month‐old female Holstein calf was referred for respiratory distress and congenital carpal deviation. Clinicopathologic findings included ventricular septal defect, ventricular dilatation, atrioventricular valve dysplasia, an overriding aorta, and unilateral carpus valgus. Genetic analysis revealed a private heterozygous missense variant in BRI3BP affecting an evolutionarily conserved residue (c.478G>A; p.Val160Ile). The variant was predicted to be deleterious and was present only in the affected calf and was absent in more than 5100 sequenced bovine genomes, including both parents, indicating a de novo origin. This study implicates an important role for the uncharacterized BRI3 binding protein in cardiac and possibly also bone development. By presenting the first BRI3BP‐related disease model, this study demonstrates the potential to gain new insights into the function of individual genes by using phenotypically well‐studied spontaneous mutants in large animals, and it provides a novel candidate gene for similar conditions in humans.

In cattle, congenital heart diseases (CHDs) are relatively rare, with a prevalence ranging from 1 to 5% of all congenital anomalies (Buczinski et al., 2010; West, 1988; Wronski et al., 2021). Nonetheless, they pose a significant diagnostic and therapeutic challenge, and the prognosis is usually guarded (Buczinski et al., 2010).

Ventricular septal defects (VSDs) are considered the most common cardiovascular malformation in calves, resulting from the failed fusion of the outflow tract cushions (Buczinski et al., 2006, 2010; Fisher et al., 2000; Lin et al., 2012; Rousseaux, 1994; West, 1988; Wronski et al., 2021). VSDs may occur alone but they are also observed in combination with other cardiac malformations, including transposition of the great vessels, persistent foramen ovale, patent ductus arteriosus, and tricuspid valve anomalies (Buczinski et al., 2010; Caivano et al., 2022; Lin et al., 2012; West, 1988). The survival of calves with these anomalies depends on the severity of the malformation with clinical presentations ranging from poor growth to exercise intolerance to severe cardiorespiratory distress (Buczinski et al., 2010; Rousseaux, 1994; Wronski et al., 2021).

Records of relevant genes causing congenital heart anomalies in cattle remain sparse (Bourneuf et al., 2017). Recently, germline de novo variants have been associated with CHDs in calves: double‐outlet right ventricle NUMB‐related in Chianina cattle and persistent truncus arteriosus GATA6‐related with in Holstein cattle (Besnard et al., 2023; Jacinto et al., 2022).

Herein, we aimed to characterize the clinicopathological phenotypes of a Holstein calf affected by a complex cardiac malformation, and to search for a possible causative genetic variant using a whole‐genome sequencing (WGS) trio‐approach.

A 3‐months‐old female Holstein calf was referred to the Clinic for Ruminants of the Vetsuisse Faculty, University of Bern because of respiratory distress and a congenital carpal deviation. The parents were not related within at least four generations and were reported to be healthy. An antigen enzyme‐linked immunosorbent assay for bovine viral diarrhea virus test was negative.

On admission, the calf appeared alert with a reduced constitution and nutritional status. Heart and respiratory rate were 120 beats/min and 40 breaths/min, respectively; rectal temperature was 38.2°C. The calf showed no signs of dehydration. The mucous membranes were pale. Thoracic auscultation revealed a pansystolic heart murmur accentuated on the right side. Clinical examination of the musculoskeletal system showed a moderate outward deviation of the right carpus without signs of pain upon palpation. Ultrasonographic examination of the heart revealed multiple cardiac anomalies, including biventricular dilation, a ventricular septal defect, displacement of the presumptive tricuspid valve, an overriding aorta, and a potentially patent ductus arteriosus. Radiologic examination of the right fore limb revealed a 13° deviation of the limb axis at the level of the distal radial epiphysis/radiocarpal joint. In addition, the proximodistal length of the lateral aspect of the epiphysis was markedly shorter than the medial side. A moderately increased and slightly inhomogeneous opacity of the distal radial metaphysis proximal to the epiphyseal line was noticed. These findings were compatible with an angular limb deformity termed Carpus valgus of the right front limb and with moderate bone remodeling at the level of the distal radial metaphyseal area.

Due to the guarded prognosis, euthanasia was selected, and a full postmortem examination was performed. Gross pathology identified moderate alveolar and interstitial pulmonary edema. The clinically reported cardiac anomalies were fully visualized, revealing marked dilation of the left ventricle and atrium, along with a VSD approximately 5 cm in diameter beneath the overriding aorta. Three AV leaflets were present in the left heart, with the chordae tendineae of the septal leaflet inserting into the right ventricle through the VSD (Figure 1a). The right heart was moderately dilated, and the expected AV valves were absent (Figure 1b). The ductus arteriosus was closed and therefore the clinically suspected patent ductus arteriosus was not confirmed. The clinically and radiologically diagnosed carpus valgus was confirmed at necropsy.

FIGURE 1.

Complex cardiac malformation in a calf. (a) Left heart. The ventricle and atrium are markedly dilated. A ventricular septal defect (arrowheads) measuring 5 cm in diameter is visible apical of the left septal atrioventricular (AV) valve. The chordae tendinae (asterisks) of the left AV valve extend from the left heart to the right heart through the ventricular septal defect. (b) Right heart. The right ventricle is moderately dilated. The right AV valves are absent. Chordae tendinae of the left septal AV valve insert in the right ventricular endocardium (asterisk). The pulmonary trunk (dot) was morphologically unremarkable.

Altogether, the clinical and pathological findings were consistent with a complex congenital cardiac malformation associated with unilateral carpus valgus.

We hypothesized a possible genetic etiology for this disorder and performed a genetic analysis. Genomic DNA was isolated from liver tissue of the affected calf, and from its parents (EDTA‐blood from the dam and semen from the sire) using standard methods and WGS was performed as previously described (Jacinto et al., 2021). Reads were mapped to the ARS‐UCD1.2 assembly (Rosen et al., 2020). Single‐nucleotide variants and small indel variants were called and an evaluation of possible larger structural variants was investigated as previously described (Jacinto et al., 2024).

The genome sequence data have confirmed the hypothesized relationship of the studied trio. Assuming a rare monogenic protein‐changing variant as the cause of this disorder, we filtered the single‐nucleotide variant data and identified no homozygous but three heterozygous private variants with a predicted high or moderate effect on three different genes present in the affected calf and absent in both parents and all controls (Table S1). The three identified variants were confirmed to be true in the genome of the affected calf using Integrative Genomics Viewer software. Among these three, only one variant was predicted to be deleterious affecting a potential candidate gene (Appendix S1). It was a heterozygous missense variant in BRI3BP exon 3 (chr17:g. 50813902C>T) (Figure 2a–c). The detected BRI3BP variant (c.478G>A) alters the encoded amino acid of BRI3BP residue 160 (p.Val160Ile) included in the Phobius transmembrane region of the negative regulator of p53/TP53 domain (Figure 2d). Furthermore, the valine to isoleucine substitution affects an evolutionarily conserved amino acid (Figure 2e) and was predicted to be deleterious using several tools (PredictSNP1 score: 61%; MAPP score: 77%; PolyPhen‐2 score: 65%; SIFT score: 53%). No evidence of possible larger structural variants was observed.

FIGURE 2.

A heterozygous missense variant in BRI3‐binding protein (BRI3BP) in a calf with a complex cardiac malformation and unilateral carpus valgus. (a) BRI3BP gene structure showing the location of the variant on chromosome 17, exon 3 (red arrow). (b) Integrative Genomics Viewer screenshot showing the Chr17: G. 50813902C>T variant heterozygous in the affected calf (shown below) and homozygous wildtype in both parents (top left: sire; top right: dam) revealed by whole‐genome sequencing. (c) Multiple sequence alignment of the BRI3BP protein encompassing the region of the p.Val160Ile variant reveals complete evolutionary conservation across species. The following National Center for Biotechnology Information proteins accessions for BRI3BP were used: NP_001092557.1 (Bos taurus), NP_542193.3 (Homo sapiens), XP_002798882.3 (Macaca mulatta), NP_084028.1 (Mus musculus), NP_001017487.1 (Rattus norvegicus), NP_001035209.1 (Gallus gallus), NP_001082804.1 (Danio rerio), and NP_001017089.1 (Xenopus tropicalis). (d) Schematic representation of the bovine BRI3BP protein. The position of the p.Val160Ile variant is indicated by the red arrow.

We have performed a comprehensive clinical, pathological, and genetic evaluation of a Holstein calf with a complex cardiac malformation, including a VSD, AV valve dysplasia, an overriding aorta and biventricular and atrial dilatation associated with unilateral carpus valgus. Pathomorphologic evidence suggestive of cardiogenic decompensation, such as hepatic centrilobular necroses due to hypoxia or lesions indicating cardiac congestion, was absent. The pulmonary edema, given the lack of histological evidence of chronicity, was probably a perimortal event associated with euthanasia, and a cardiogenic etiology was deemed unlikely.

In this study, we suggest a possible genetic cause for the observed cardiac malformations associated with unilateral carpus valgus in this Holstein calf, with a dominant acting de novo mode of inheritance. Simple recessive inheritance could be ruled out as we found no evidence of evident inbreeding and for a rare homozygous variant affecting the coding region of an annotated gene, given the unique phenotype and assuming no similarly affected case among more than 5100 control genomes. The trio‐based WGS approach identified three protein‐changing variants that were exclusively heterozygous in the genome of the affected calf. We speculate that these variants arose de novo post‐zygotically in the developing embryo or were inherited from low germinal mosaic parent. After gene function analysis, considering the variant alleles' absence in a global control cohort and in silico effect predictions, we identified the heterozygous de novo missense variant affecting BRI3BP as a likely pathogenic variant for the observed phenotype.

BRI3BP encodes the BRI3‐binding protein (BRI3BP), which is highly expressed in the heart, in particular in the aorta, ventricles, and interventricular septum (Doll et al., 2017). According to Disease Novelty (TIN‐X) from Pharos interface, BRI3BP is predicted to be associated with CHDs, including VSDs, atrial heart defects as well as musculoskeletal disorders, including connective tissue defects (Kelleher et al., 2023). Moreover, BRI3BP presents a functional interaction with 14 different genes with an interaction score higher than 0.7 (Kelleher et al., 2023). Among these interactors, two (EFTUD2, TOR1A) have been associated with heart defects and five (EFTUD2, TOR1A, SIGMAR1, SMPD3, PTRH2), have been reported to be associated with limb defects in humans and/or mice. Specifically, pathogenic heterozygous variants in EFTUD2 cause mandibulofacial dysostosis Guion‐Almeida type in humans (OMIM 610536), where affected patients present, among other malformations, atrial and/or ventricular septal defects, and hand malformations including contractures and angular deformities (Lines et al., 2012). In human patients, heterozygous and homozygous variants in TOR1A can cause torsion dystonia‐1 (OMIM128100) and arthrogryposis multiplex congenita (OMIM 618947) respectively (Fan et al., 2023;Kariminejad et al., 2017 ; Saffari et al., 2023). Patients with arthrogryposis multiplex congenita are born with congenital joint contractures and may suffer from respiratory insufficiency and cardiac arrest (Kariminejad et al., 2017; Saffari et al., 2023). Pathogenic variants in SIGMAR1 are known to cause recessive distal hereditary motor neuronopathy in humans (OMIM605726), in which patients show pes cavus and pes varus in addition to distal muscle weakness and atrophy (Ververis et al., 2020). Finally, SMPD3 and PRTH2 have been associated with abnormal limb morphology and limb joint contracture in mutant mice, respectively (Guenet et al., 1981; Kairouz‐Wahbe et al., 2008). Based on these findings, we speculate that the observed phenotype in the calf described in our study may be due to an impaired interaction between BRI3BP and the aforementioned interactors. However, it cannot be excluded that the phenotype exhibited by this calf is due only to the dysfunction of the mutant BRI3BP protein. Further experimental studies would be required to functionally validate the postulated causative role of the candidate variant for the observed cardiac malformations and carpus valgus in the calf. Therefore, future studies should aim at exploring the role of pathogenic variants in BRI3BP and their role in the development of cardiac and skeletal anomalies.

Herein, we have uncovered a novel phenotype of a complex congenital cardiac malformation and carpus valgus in cattle, associated with a likely pathogenic heterozygous de novo missense variant in the bovine BRI3BP gene. We propose that this is the first BRI3BP variant associated with a congenital condition in a mammalian species, adding this relatively uncharacterized gene to the list of candidates for similar disorders. The findings from our study may serve as a starting point for further research into the function and mechanisms of BRI3BP in the context of congenital heart and bone defects. Our study highlights that the genetics of inherited disorders in well‐phenotyped large animals, such as cattle, is a valuable model system for studying fundamental aspects of gene function.

AUTHOR CONTRIBUTIONS

Chang He: Formal analysis; investigation; methodology; validation; visualization; writing – original draft; writing – review and editing. Llorenç Grau‐Roma: Conceptualization; investigation; methodology; supervision; writing – review and editing. Robin Schmid: Investigation; methodology; validation; visualization; writing – review and editing. Irene M. Häfliger: Data curation; methodology; software; validation. Mireille Meylan: Conceptualization; data curation; resources; supervision; visualization; writing – review and editing. Cord Drögemüller: Conceptualization; funding acquisition; project administration; resources; supervision; validation; visualization; writing – review and editing. Joana G. P. Jacinto: Conceptualization; formal analysis; investigation; methodology; project administration; supervision; validation; visualization; writing – original draft; writing – review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

FUNDING INFORMATION

This study was financially supported by the Arbeitsgemeinschaft Schweizerischer Rinderzüchter (ASR), Zollikofen, Switzerland, the Federal Office for Agriculture (FOAG), Bern, Switzerland.

Supporting information

Appendix S1.

Table S1.

DATA AVAILABILITY STATEMENT

The WGS data are available under study accession no. PRJEB18113 at the European Nucleotide Archive (https://www.ebi.ac.uk/ena; sample accessions SAMEA111531532 [affected calf], SAMEA111531533 [dam], and SAMEA111531535 [sire]).