A germline de novo variant in NUMB associated with a double-outlet right ventricle in Chianina cattle.

BACKGROUND

Congenital heart disease (CHD) comprises a wide spectrum of abnormalities in heart structure that occur in the fetus during pregnancy (Sun et al., 2015). Double‐outlet right ventricle (DORV) is a rare form of ventriculoarterial connection reported in a few cattle breeds (Caivano et al., 2021; McManus et al., 2020; Newhard et al., 2017; Nourani et al., 2009; Prošek et al., 2005; Wilson et al., 1985). However, the etiology of DORV in cattle has not yet been clarified.

ANALYSIS

DNA was extracted from ear tissue of the 10‐day‐old male Chianina DORV‐affected calf that was previously reported (Figure S1; Caivano et al., 2021), from EDTA blood from its dam and from semen from its sire. We hypothesized a genetic etiology for the present DORV. A trio‐based whole‐genome sequencing approach was performed as described before (Jacinto et al., 2022). Reads were mapped to the ARS‐UCD1.2 assembly (Rosen et al., 2020), resulting in average read depths of 16.9× in the calf, 17.9× in the dam and 21.3× in the sire, and then processed as reported earlier (Jacinto et al., 2021). This identified two heterozygous private protein‐changing variants present exclusively in the genome of the affected calf and absent in both parental genomes as well as in 5365 controls (Table S1). Only one of these variants was in a putative candidate gene for the observed phenotype. This heterozygous variant at chr10:84751870G>A (NM_001101951.1: c.416C>T) represents a missense variant in a splicing region located in exon 7 of the NUMB endocytic adaptor protein (NUMB) gene (Figure S1). The guanine to arginine substitution affects an evolutionary highly conserved amino acid (NP_001095421.1: p.Thr139Met) in the phosphotyrosine interaction domain (PTB/PID) and was predicted to be deleterious using four different tools (provean, −2.599; polyphen, 2: 56%; sift, 79%; and mapp, 57%). Sanger sequencing confirmed the presence of the heterozygous NUMB variant in the affected calf and its sire, which clearly carried the mutant allele at a low level in comparison with the wild type allele, representing a germinal mosaic (Figure S1). Moreover, we identified 30 homozygous protein‐changing variants present exclusively in the genome of the affected calf, of which only one affects a putative candidate gene (XRCC1) and was predicted to be deleterious (Chr18:51749382G>A; c.1058C>T; p.Pro353Leu). As well as three heterozygous carriers in Chianina and Romagnola cattle (Table S1), subsequent genotyping of 217 normal Chianina bulls showed an allele frequency of 6.91% and three homozygous XRCC1 mutants, which rules out a possible causal cause.

COMMENTS

We propose the heterozygous c.416C>T variant as a candidate causative variant for the observed congenital disorder and thereby NUMB as a novel candidate gene for DORV. The protein‐altering nature of this de novo mutation inherited from a mosaic sire strongly suggests the causality of the variant. The affected gene encodes a membrane‐bound protein that plays an important role in the determination of cell fates during development (O'Leary et al., 2016). In mice, homozygous NUMB mutant animals (MGI3783761) show a variety of abnormalities, including abnormal cardiac morphology, abnormal vascular regression, pericardial edema, abnormal central nervous system morphology, abnormal neuron differentiation, embryonic growth retardation and embryonic lethality during embryogenesis (Zilian et al., 2001). The calf reported herein was uniquely affected by a congenital malformation of the heart (Figure S1). We hypothesized that the observed phenotypical differences between the NUMB mutated mice and the calf in this study might be explained by the presence of two mutated alleles in mice and only one in the calf. Therefore, considering the rarity, in silico effect prediction and known function of NUMB, the identified missense variant might be considered as a possible cause for the observed phenotype, although this gene has not been previously associated with DORV in mammals, including humans.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figure S1

Table S1

Click here for additional data file.