# An autosomal recessive nonsense variant in the EGFR gene induces perinatal lethality in “Blonde d’Aquitaine” calves

Sandrine Floriot, Sébastien Fritz, Vincent Herry, Christian Beauvallet, Mekki Boussaha, Giorgia Egidy, Amandine Duchesne, Marthe Vilotte,

Marie-Christine Cadiergues, Didier Boichard, et al.

► To cite this version:

Sandrine Floriot, Sébastien Fritz, Vincent Herry, Christian Beauvallet, Mekki Boussaha, et al. An autosomal recessive nonsense variant in the EGFR gene induces perinatal lethality in “Blonde d’Aquitaine” calves. BMC Veterinary Research, 2026, 22, 10.1186/s12917-025-05225-6. hal-05512782

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# An autosomal recessive nonsense variant in the *EGFR* gene induces perinatal lethality in "Blonde d'Aquitaine" calves

Sandrine Floriot¹*, Sébastien Fritz¹,², Vincent Herry³,⁴, Christian Beauvallet¹, Mekki Boussaha¹, Giorgia Egidy¹, Amandine Duchesne¹, Marthe Vilotte¹, Marie-Christine Cadiergues⁴, Didier Boichard¹, François Schelcher⁵ and Jean-Luc Vilotte¹

**Abstract**

**Background** Six cases of perinatal lethality associated with phenotypes consistent across cases, short and brush-haired appearance, alopecia and erythematous areas on the limbs and facial protuberances, excess skin all-over the body, have recently occurred in the "Blonde d'Aquitaine" cattle breed in South-Western France, suggesting the emergence of a recessive genetic anomaly.

**Results** Genetic analysis identified a nonsense variant in the *epidermal growth factor receptor (EGFR)* gene, *XP_002696936:p.Trp898Ter*, as likely responsible for this anomaly and a bull born in 1967 as one ancestor that contributed to the spread of this allele in the breed. Functional studies on affected calves and cross-species comparisons of the phenotypes associated with such a variant were consistent with the results of the genetic study.

**Conclusion** A recessive nonsense *EGFR* variant was identified as being responsible for perinatal lethality in the "Blonde d'Aquitaine" cattle breed. It enabled the introduction of a screening test for this allele, which should lead to the progressive eradication of this genetic flaw.

**Keywords** EGFR, Nonsense variant, Bovine, Perinatal lethality, Cutaneous disease

**Background**

Recent advances in herd health monitoring, with the development of dedicated national structures such as the French National Observatory for Bovine congenital Anomalies ONAB (https://doi.org/10.15454/BRKO V3), and in knowledge of genome structures have made it possible to detect the emergence of genetic anomalies and identify the underlying causal variants especially in cattle. Such identifications enable the eradication of deleterious alleles in livestock, but can also be used in translational medicine studies [24]. In cattle, apart from bullous diseases, genetic diseases with cutaneous expression include various forms of hypothrichosis (some of which are lethal), chondrodysplasia, retinal, ectodermal,

<footnote>*Correspondence:
Sandrine Floriot
Sandrine.floriot@inrae.fr
¹Université Paris-Saclay, INRAE, AgroParisTech, GABI, Jouy-en-Josas
78350, France
²Eliance, 149 Rue de Bercy, Paris Cedex 12 75595, France
³SELAS EVA, Réseau Cristal, 16 Avenue du Général De Gaulle,
Argentonnay 79150, France
⁴ENVT, Université de Toulouse, Toulouse 31076, France
⁵IHAP, Université de Toulouse, INRAE, Ecole Vétérinaire de Toulouse,
Toulouse 31076, France</footnote>
rhizomelic craniofacial and/or skeletal-cardio-enteric dysplasia (sometimes associated with dental disorders), cutaneous asthenia and ichthyoses. They are mostly reported in dairy breeds (https://www.omia.org/home/, [4, 11] for recent examples).

At the end of the 2000s, the very first cases of Blonde d'Aquitaine calves dying in the week following the birth and displaying original skin and coat lesions were observed in practice. All these affected calves originating from different farms in the South-West of France had an unusual but similar phenotype, which, despite the limited number of cases observed, suggested a disease of genetic origin and led to further investigations. Veterinarians facing this unusual clinical pattern have referred cases to the Department of bovine pathology at the National Veterinary School of Toulouse (ENVT—Toulouse, France).

# Methods

## Phenotype description

Methodical clinical and paraclinical investigations were carried out at ENVT to assess the various possible causes of an unusual lesion pattern that prompted the referrals by the veterinarians. A thorough physical examination and a dermatological examination with photographs were carried out for each case. When the calf examined was alive, blood samples were taken to test for Bovine Viral Diarrhoea (BVD) virus and *Neospora* infections during pregnancy, trace element status, plasma biochemistry (including retinol and thyroid hormones) and haemogram. Plasma, serum and EDTA were cold-stored negative and ultra-negative. After natural death or euthanasia by sedation with xylazine hydrochloride (0.2 mg/kg intravenously, Rompun 2%, Elanco, Sèvres, France) followed with embutramide, mezebonium and tetracaine (T61, Intervet, Beaucouzé, France—0.1 mL/kg intravenously into the jugular vein), the calves were necropsied and samples of the following organs were preserved in 10% neutral buffered formalin for 24-48 h and embedded in paraffin according to standard protocols for histopathological examination: skin from various anatomical zones, thymus, thyroid, adrenal gland, liver, kidney, myocardium (interventricular septum), brain, lung, cornea, abomasum, jejunum, ileum colon (all three only if euthanasia) and any macroscopic lesions detected. Skin samples taken from two calves of the same age and breed that died from unrelated causes served as controls.

## Histological analyses of the skin

Skin samples from control and affected animals were fixed (10% neutral buffered formalin for 24-48h) and embedded in paraffin according to standard histological protocols. Five-micron thick sections were mounted on Superfrost® plus slides. Sections were deparaffinized and stained with HES (haematoxylin, eosin, safran) to

observe the skin morphology and with a Roan solution (nuclear red, orange yellow and aniline blue) to examine hair follicles. Digitally scanned (Pannoramic SCAN, 3DHISTECH) slides with a 20× objective were visualized using CaseViewer (3DHISTECH). Immunofluorescence was performed with rabbit Ki67 (1:1000, Abcam), KRT14 (1:200, Invitrogen), mouse IgG2A α-SMA (1:200; Dako) and mouse IgG1β-catenin (1:200, Transduction Laboratories) antibodies after heat-antigen retrieval pH6. Nuclear counterstaining was performed with 4',6'-diamidino-2-phenylindole (DAPI) (1:1000, Invitrogen) and sections examined using a Zeiss Axio Observer ZIM ApoTome microscope (Carl Zeiss). The lack of the first antibodies resulted in the absence of labelling. Images were processed using the AxioVision computer program version 4.6 (Carl Zeiss). All images shown are representative of the skin samples studied and correspond to individual sections from the z-series stack.

## Ultrastructural examination by transmission electron microscopy

Immediately after sampling, punch biopsies from two calves (one affected and one control) were fixed and examined as previously described [7].

## Sample selection and DNA extraction

Affected animals were photographed and reported to the ONAB by the breeder and veterinarian. Samples were taken during the necropsy procedure and stored at -80°C for further analysis. Blood or liver samples were used for DNA extraction. DNA was extracted from blood samples using the Genisol Maxi-Prep kit (AbGene, UK) or from liver using phenol/chloroform extraction (Miller SA). DNA quality was checked on 2% agarose gel electrophoresis. DNA concentration was measured using NanoDrop ND-1000 spectrophotometer.

## Homozygosity mapping

At the time of analysis, DNA samples were available for three cases only. Genome wide homozygosity scan was carried out using genotyping data from these three affected calves and from control Blonde d'Aquitaine cattle. Cases were genotyped with the BovineSNP50 Beadchip v2 [19]. The control population included the whole Blonde d'Aquitaine population genotyped at the time of the analysis (24,149), excluding the cases. Animals were genotyped with various Illumina SNP chips including the BovineSNP50 Beadchip v1 and v2 and different versions of the Eurogenomics EuroG10K chip [3] (Illumina Inc., San Diego, CA) including between 10,234 and 23,330 SNP. An imputation step was carried out with FImpute software [26] to recover complete information from 53,469 SNP. Resulting real or imputed genotypes were subsequently analysed using an in-house implementation
of the HOMAP (homozygosity mapping) tool [8], as in Vaiman et al. [29]. First, this method counts the genotypes at each marker separately in cases and controls, computes allelic frequencies in the control population, and determines the most frequent allele (let's say A) in cases. Then the test is computed as the sum over all cases of -2logalc(freq(A)) if the case genotype is AA and log10(5) otherwise The test has no theoretical distribution but behaves as a likelihood ratio test [8].

## Analysis of whole-genome sequences

The genome of the bull 44,784, identified as carrying at a heterozygous status the haplotype associated with the anomaly was previously sequenced in 2014 (16× sequencing coverage resulting in 13×alignment coverage, ENA EBI project PRJEB47654, accession number ERS27090267, SAMEA number SAMEA120389581). DNA was extracted from a sperm straw provided by AURIVA breeding company. This whole genome sequencing had been performed at the INRAE GetPlaGe platform (http://genomique.genotoul.fr/) on a HiSeq 2500 Illumina sequencer producing 150-bp-long paired end reads, according to the manufacturer's protocol. More recently, reads were realigned against the ARS-UCD1.2 bovine reference genome [25] and processed in accordance with WGS alignment guidelines of the 1000 Bull Genomes Project [15]. Small genomic variants located within the haplotype region were identified as previously described [5] using the GATK-Haplotype-Caller software [21]. Variants were then filtered against the sequences of the “1000 bull genomes” project (run 9) and against the 1600 sequences available at INRAE. Variants present in homozygous state in Blonde d'Aquitaine or with a non-zero frequency in any other breed were filtered out.

In order to investigate their biological effect, genomic variants were annotated using the Ensembl variant effect predictor (VEP) tool [22] using the dbSNP build 143. The effect of the amino acid changes was predicted using the SIFT score [16] implemented in the VEP software.

## PCR analysis and DNA sequencing

Primers were designed in exons along the entire length of *EGFR* gene based on the *Bos_taurus_ARS-UCD1.2/bosTau9* assembly using the Primer3 software (https://p.rimer3.org/). Details on the PCR primers are in Supplementary Table 1. PCR reactions were performed using the GO-Taq Flexi polymerase kit (Promega) according to the manufacturer's instructions on a Mastercycle pro thermocycler (Eppendorf). Aliquots of PCR products were analysed by electrophoresis on agarose gels. The remaining amplicons were purified and sequenced by Eurofins (Eurofins MWG, Ebersberg Germany).

## Genotyping

DNA from affected calves was amplified by PCR using primers flanking exon 22 of the *EGFR* gene (EGFR-I21F and EGFR-I23R, Supplementary Table 1). Genotyping was determined by sanger sequencing of the PCR-amplified DNA to confirm the presence or absence of the variant.

## RT-PCR and semi-quantitative RT-PCR analyses

Total RNA was extracted from the liver of wild-type and affected animals using the RNeasy Lipid tissue Mini Kit (Qiagen) and a DNase I treatment. One µg RNA was reverse-transcribed using a Superscript™ VILO™ cDNA Synthesis kit (Thermofisher), with random primers according to the manufacturer's instructions. cDNA was amplified by PCR for 35 amplification cycles with PCR primers for the *EGFR* gene (EGFR_E20F and EGFR_E23R, Supplementary Table 1). For semi-quantitative PCR analysis, 30 amplification cycles were performed with PCR primers for *EGFR* (EGFR_E28F and EGFR_3UTR) and *GAPDH* (GAPDH_E2E3F and GAPDH_E2E3R) genes (Supplementary Table 1). The *GAPDH* gene was used for normalization based on its observed expression stability in liver.

## Western blot analysis

Frozen ear pieces from healthy and affected calves were crushed in liquid nitrogen and cytoplasmic proteins were extracted with 0.5 mL of a classic RIPA buffer containing 5 mM DTT, 5 mM EDTA and protease Inhibitor cocktail (Roche). Samples were vortexed 5 to 6 times for 30 s and kept on ice for 45 min. Then, they were centrifuged at 16,000×g for 30 min at 4°C, and the supernatants were collected in Eppendorf tubes. Protein concentrations were quantified with the 2-D Quant kit (Cytiva). Aliquots of 15 µg of protein were mixed with 2×Laemml and boiled for 5 min before freezing on ice. Two volumes of 10 µL of MW Protein Precision Plus Standard (Bio-Rad) were aliquoted. Samples and standards were loaded onto a 4–15% SDS-PAGE gradient (Bio-Rad) and electrophoresed at 80 Volts for 20 min and 175 Volts for 3 h. Afterwards, proteins were electro-transferred onto a nitrocellulose membrane with a Trans-Blot Turbo (Bio-Rad) at 2.5 A, 25 Volts maximum for 10 min. The membrane was rinsed in MilliQ water for 2×5 min and the membrane-bound proteins were stained in a concentrated Red Ponceau solution. MilliQ water rinsed residual Ponceau and the membrane was imaged on a ChemiDoc Touch Imager system (Bio-Rad). After 15 min of destaining in TBS-Tween 20 0.1% (TBST), hydrophobic sites of the membrane were blocked with 5% nonfat dry milk in TBST for 1 h at room temperature. Primary hybridization was done overnight at 4°C with either a polyclonal anti-EGFR antibody (Cell Signal. Tech., #2232) diluted at
1/1,000 in TBST containing 5% BSA, as recommended, or with a mouse monoclonal anti-EGFR antibody (Santa-Cruz sc-120) diluted at 1/500 in 1% BSA. After three washes in TBST at room temperature, the membranes were incubated for 1 h with either an anti-rabbit horse-radish peroxidase (HRP)-conjugated secondary antibody or with an anti-mouse HRP-conjugated secondary antibody, each diluted 1/10,000. After three washes in TBST at room temperature, signals were detected using the ECL prime enhanced chemiluminescence system (Cytiva) on the ChemiDoc Touch Imager system (Bio-Rad).

## Results

### Phenotypic description

Between 2011 and 2015, five Blonde d'Aquitaine calves, one male and four females, were fully investigated. All

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Phenotypic aspects of affected calf. **A** Excess skin. **B** Large, stiff and curved hair shafts. **C** Alopecic lesions and erythema. **D** Hypotrichotic aspect of the muzzle. **E** Keratitis occasionally observed</figcaption></figure>

five cases were from different farms in south-west France. All calves had a reduced birth weight (35 to 39 kg for a standard weight between 45 and 48 kg), they all had low vivacity and difficulty in suckling. They died within a few days of birth or were humanely euthanized. Apart from skin abnormalities, only a tachycardia and a swinging heart were detected on physical examination. All qPCR were negative for BVD virus and *Neospora*. Haematology and biochemistry profiles were unremarkable. Levels of vitamin A, iodine/thyroid hormones and copper, zinc and selenium were variable and most likely related to the status of each farm of origin. Similarly, no gross findings were detected during the *post-mortem* examination, nor microscopic abnormalities of the various organs sampled, with the exception of the skin. All animals had light tan or milk chocolate coats, darker than the standard wheat- or cream-colour of the breed. The coat was short, and of brush-haired appearance, which gave the French term “veaux paillassons”, in English “doormat calf syndrome”. In addition, skin appeared folded all over the body (Fig. 1 A), as if there was excessive skin, but without apparent hyperextensibility. The hair shafts were generally large in diameter, stiff and curved (Fig. 1 B). Their density appeared normal, or possibly increased. On close examination, some hairs appeared larger, longer and more curved than others. All were easy to epilate, which could explain the alopecic lesions and erythema on areas of friction such as joint protuberances or the face (Fig. 1 C). The muzzle, the periocular region, and the ear pinnae were hypotrichotic (Fig. 1 D). The eyelashes, and to a lesser extent the vibrissae, were stiff and large in diameter. In addition, distichiasis was regularly observed. Keratitis, likely secondary to the distichistic eyelashes, was occasionally observed (Fig. 1 E). Otherwise, the surface of skin itself looked normal, without any scaling.

Microscopical examination of the skin biopsies is illustrated in Figs. 2 and 3 (optical microscopy) and in Fig. 4 (transmission electron microscopy). Optical microscopic examination showed diffuse, mild to moderate orthokeratotic hyperkeratosis, with a compact stratum corneum and no significant acanthosis. The superficial dermis was composed of fine collagen fibres, with no significant inflammatory component. The hairs were abnormal, with numerous very large hair follicles; most of them in the telogen phase, with very fine follicular sheaths and irregular contours, containing a voluminous hair with equally irregular contours, and whose cuticle and cortex were often mineralised. There were small hair follicles in the anagen phase, with few lesions compared with the large ones, with moderate mineralisation of the follicular sheaths. Sebaceous glands were small and sweat glands were ectatic (Figs. 2 and 3 b). The use of ROAN staining provided better contrast of collagen fibres and hair follicle structure (Fig. 3). Hair follicles sometimes
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Histological analysis of skin sections. Skin sections were HES-stained. **a** control calf, **b** affected calf. EP: Epidermis, D: Dermis, HF: Hair follicle, HB: Hair bulb, SG: Sebaceous gland, SC: Stratum corneum (Keratin). Bars: 100 µm</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3** Histology (ROAN staining) of skin sections. Skin sections were stained with ROAN solution. **a,b,c** control calf skin sections, (**d,e,f**) affected calf skin sections. EP: Epidermis, D: Dermis, HF: Hair follicle, HB: Hair bulb, SG: Sebaceous gland, SC: Stratum corneum (Keratin), M: Medulla, C: Cortex, Cu: Cuticle, IRS: Internal root sheath, ORS Outer root sheath. Bars: 100 µm</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4** Transmission electron microscopy analysis of dermis and superficial epidermis. **a,b,c** control calf, (**d,e,f**) affected calf. ILL: Intercellular Lipid Lamellae, CD: Corneodesmosome, C: Cornéocyte, SG: Stratum Granulosum, SC: Stratum Corneum</figcaption></figure>
appeared enlarged and non-cylindrical in cross-section (Fig. 3f). Collagen fibres appeared disorganised, not following the longitudinal axis of the skin. At higher magnification papillary layer of the dermis was loose and below, sweat glands looked collapsed among numerous enlarged hair follicles (Figs. 2 a,b; 3 a,d) with finer and irregularly contoured follicular sheaths (Fig. 3 c,f). Electron microscopy showed abnormal corneocytes that appear thicker, the intra-corneocyte matrix was less compact and appeared to contain residues and clusters of intermediate filaments. Corneodesmosomes persisted over the entire height (~three cell layers) of the stratum corneum (Fig. 4 a,d). They looked fewer and shorter. The intercorneocyte spaces were reduced. In the dermis, collagen fibers were disorganised and shorter. Immunofluorescent labelling revealed the presence of cytoplasmic KRT14 not only in the basal layer of epidermis and hair follicle sheath but also in suprabasal layers. Variants in the genes for these keratins are associated with epidermolysis bullosa simplex. Nuclear Ki67, a marker of proliferation, was detected in cuboidal cells located at the basal membrane and suprabasal flat cells, as well as in different layers of the hair follicle sheath. Membrane β-catenin

was detected in the majority of cell types (supplementary Fig. 1). These observations suggested that the process of proliferation is being spatially sustained.

The similarity of phenotypes among affected calves, the presence of both sexes among them and their belonging to different farms with no common first-degree relatives led to the hypothesis of an autosomal recessive anomaly. The genetic cause of this anomaly was then sought through homozygosity mapping and sequencing.

**Genomic analysis and identification of the likely causative variant**

To fine map the location of this defect, a genome wide homozygosity scan was carried out (see Methods). The most significant haplotype shared by the genotyped cases was found on the beginning of BTA22 (Fig. 5). The confidence interval was defined by the homozygous haplotype shared by all individuals, from chr22: g.178967_1945967967 on the ARS-UCD1.2 reference genome [25]. Within this confidence interval, the maximum LRT value (depending on genotypes distribution in this group and on allelic frequencies in the control population) was observed at position chr22: g.810412.

<figure><img src="image_7.png" /><figcaption>**Fig. 5** Results of homozygosity mapping on the ARS-UCD1.2 reference genome. Top figure: Evidence for linkage (y axis) is measured as log(1/P), leading to the identification of a approximately 1.9-Mb shared region of BTA 22; chr22: g.178967_1945967967 on the ARS-UCD1.2 reference genome [25]. Bottom figure: Zoom in on the first 12 Mb of bta22, showing the -log(P) and the number of discordant markers, allowing to define the limits of the confidence interval</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 6** Pedigree for the recessive trait for one of the affected calves. The pedigree chart showing the close relationship between the affected animal (black symbols). Circles and rectangles represent female and male sex, respectively. The available genotypes are indicated. Over nine generations, the paternal and the maternal paths trace to a male ancestor born in 1967 indicating possible monogenic recessive inheritance</figcaption></figure>

<table><caption>**Table 1** Genomic protein-changing variants identified within the BTA22 haplotype</caption><thead><tr><th>Chromosome ARS-UCD1.2 reference genome [25]</th><th>Polymorphism</th><th>Effect</th><th>SIFT Prediction</th><th>Gene:</th></tr></thead><tbody><tr><td>22</td><td>CM008189<br>: g.238350: G > A</td><td>missense variant</td><td>MODERATE</td><td>URGCP: ENS-BTAG00000009453</td></tr><tr><td>22</td><td>CM008189<br>: g.239627: T > C</td><td>missense variant</td><td>MODERATE</td><td>URGCP: ENS-BTAG00000009453</td></tr><tr><td>22</td><td>CM008189<br>: g.387409: C > A</td><td>Missense variant & splice-region_</td><td>MODERATE</td><td>DBNL: ENS-BTAG00000017547</td></tr><tr><td>22</td><td>CM008189<br>: g.916239: C > T</td><td>Nonsense variant:<br>XP_002696936:p.Val876Ter</td><td>HIGH</td><td>EGFR: ENS-BTAG00000011628</td></tr></tbody></table>

Identification of the haplotype carrying the variant enabled genealogical analyses to be carried out based on the genotyped ancestors available. While for most of the calves these analyses could not go back more than a few generations, for one of them it allowed identification a bull born in 1967 that had thus contributed to the spread of this anomaly in the breed (Fig. 6).

Small genomic variants were identified (see Methods) and the effects of amino acid changes were predicted using the SIFT score [16]. Four variants located within the candidate regions were found to have a predicted significant biological effect (Table 1). They were then compared to those present in both our local small genomic variations database [5] and those present in the 1000 bull genomes project database [9]. Only one variant was found to be absent from both databases. This variant is a nonsense variant within exon 22 of the EGFR-encoding gene, XP_002696936:p.Trp898Ter, and was therefore considered the likely causal variant (Table 1, Fig. 7 and supplementary Fig. 2). This variant induces a STOP codon at position 898 of the EGFR protein, which

normally has 1208 amino acids. It is located within the catalytic domain of this protein tyrosine kinase (aa 704–1016).

The five affected calves were genotyped for the *EGFR*-exon 22 by sequencing analysis of the amplified exon 22 and were found homozygous for this variant in line with the recessive nature of the phenotype. During the course of this study, in 2022, a sixth affected calf, a male, was suspected and reported to the ONAB. Similar DNA analysis confirmed that this animal was also homozygous for the *EGFR* nonsense variant, making the identification of this variant as causal highly probable. Unfortunately, this new case did not allow us to refine our genealogical studies.

This genotype was not found in the other (n = 24,155) animals of this breed. The variant has been designed on the EuroG10k V7 Eurogenomics Illumina chip in 2017. Of the 700 cattle genotyped in this breed in 2018, no other homozygous and 8 heterozygous carriers of the anomaly have been identified (1.14%, allelic frequency 0.57%), showing a limited dissemination of the variant
<figure><img src="image_9.png" /><figcaption>**Fig. 8** Nonsense mediated EGFR mRNA decay. RT-PCR of *EGFR* mRNA expression of in liver samples from control (C) and affected (Aff) calves. Top row: Numbers refer to calf identification. Housekeeping *GAPDH* gene was used as an internal control. Molecular weight markers (base pairs, bp) are indicated (right margin) next the PCR product lengths</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 9** EGFR Western blot analysis. Western blot analysis of ears protein extracts from two affected (Aff1, Aff2) and one control (C) calves. Top figure: schematic representation of the EGFR protein and of the location of the epitopes of the two antibodies used (N-Term and C-Term, see Mat & Meth section).
*: location of the stop-codon caused by the variant in affected calves. The antibody used is indicated on the left of the Western result. MW: Molecular weight. The sizes of the MW bands are indicated on the right. The detected EGFR protein band is surrounded by a rectangle. The Ponceau red staining of the membrane shown at the bottom of the figure indicates equal amount of protein per well</figcaption></figure>

shortly afterwards (Fig. 7c). No other alteration was observed in the *EGFR* transcripts of the affected animals (data not shown).

Finally, we looked for the presence of EGFR protein by western blotting in skin and liver samples from healthy and diseased calves using two antibodies, one directed against the N-terminal region of the protein, the other against its C-terminal region. While both antibodies detected the protein in extracts from healthy calves, no protein was detected in affected calves (Fig. 9 and supplementary Fig. 4). Although we cannot formally exclude the expression of very low levels of a C-terminally truncated EGFR protein in affected calves, under the detection limit of the Western, these results are consistent with transcript analysis and suggest that the variant confers on

the animals a phenotype identical to that of a knockout at the *EGFR* locus.

## Discussion

The appearance in the South-West of France of several cases of perinatal calf lethality in the “Blonde d’Aquitaine” cattle breed, all with similar phenotypes, led to the hypothesis of a recessive genetic anomaly in this specific breed. The genetic analyses that we undertook identified a nonsense variant in exon 22 of the *EGFR* gene that is likely responsible for this disease, XP_002696936:p.Trp898Ter. In fact, all affected calves are homozygous for this variant, and this genotype is not found in other animals of this breed. Only 8 heterozygous carriers of the anomaly were identified out of the 700
“Blonde d’Aquitaine” cattle genotyped in 2018, highlighting its low dissemination. This variant was also absent in other breeds of cattle (Table 2). Analysis of the “Blonde d’Aquitaine” breed’s genotyped animals identified a bull born in 1967 as a carrier of this variant and as an ancestor of one of the affected calves. Unfortunately, it has not been possible to trace the genealogy of all affected cases and we could not therefore identify a common ancestor.

Although unlikely due to the fact that 1) no healthy
cow carrying the *EGFR* variant at homozygosity was
observed, 2) the absence of viral infection declared in cat-
tle on the farms at the time the affected calves were born,
we cannot formally exclude as a possible cause of this dis-
ease other viral infections than BVD, such as bluetongue
virus (BTV) or Schmallenberg virus (SBV) infections
which were not tested. This may constitute a limitation of
our study. However, cutaneous lesions in neonates have
never been reported in relation to transplacental BTV or
SBV infections.

Analyses of tissue samples from affected calves sug-
gest the likely absence of EGFR protein expression,
even of a C-terminally truncated one, in tissues of
these animals. Indeed, only a weak expression of tran-
scripts from the mutated allele was observed, probably
due to their degradation by RNA decay [17, 31] for
reviews), and accordingly Western analyses failed to
reveal the presence of a protein. These animals could
thus be considered as EGFR-knockout cows. Although
superficial overall, the retrospective analysis of the
phenotypes observed in these calves seems consistent
with the diseases associated with EGFR knockout vari-
ants in humans or mice. In both species, EGFR gene
knockout or EGFR loss of function leads to perina-
tal lethality [6, 13, 14, 20, 23, 27, 28], as observed in
bovine. Low birth weight, resulting from intrauterine
growth failure, and respiratory failure were reported in
Egfr-inactivated mice [23, 27, 28] and in affected chil-
dren [13, 14, 20]. Moreover, in view of its expression
profile, a role for EGFR during gestation is also sus-
pected in cattle [1]. Although these phenotypes were
not quantified, the affected calves showed an emaci-
ated appearance and suffered from tachycardia, two
features reminiscent of the above-mentioned diseases.
The absence of EGFR protein also induces significant
skin defects in humans and mice, reflecting immature
epithelial development. Mice present “waived”-hair
that look similar to the “brushed”-hair observed in
affected calves (Fig. 1). Histological analysis of the skin
from EGFR-deficient animals revealed disorganized
follicles, few of which reach their entire development,
in both mice and cows [23] and Fig. 2). Interestingly,
a wavy coat phenotype was also observed in mice
carrying a dominant, non-lethal at heterozygosity,
allele (D833G) of the Egfr-gene, the Velvet allele, that

mostly abolished its kinase activity [10]. Thus, even if
a C-terminally truncated EGFR protein was expressed
in affected calves, this protein will also lack kinase
activity and will induce a phenotype that is consistent
with that reported in *Egfr*<sup>Velvet</sup> mice. In EGFR defi-
cient patients, recurrent skin infections were reported
alongside skin erosions and ecchymoses [20]. Clinical
examination of some affected calves also revealed mul-
tifocal necrotic and ulcerative dermal changes.

In mice, the genetic background seems to influence the
fate of EGFR-deficient animals, with lethality occurring
during embryonic development or shortly after birth [27,
28]. In humans, lethality appears to be mainly perinatal,
although children may survive for several years thanks to
the medical care provided [20]. To our knowledge, there
is no documented case of miscarriage associated with an
EGFR deficiency of the foetus in humans, although, as in
the mouse, its important role in the development of the
fetoplacental unit is recognized [12, 18]. In the bovine
Blonde d'Aquitaine breed, the most recent carrier bulls
are used in crossbreeding with dairy cows and prevent to
address the possibility of intrauterine lethality associated
with a homozygous EGFR-mutated genotype.

In mice and human, the EGFR deficiency induces
multi-organ abnormalities. The fatal outcome a few
days after the calves were born also supports this
hypothesis, although no abnormalities were detected
during the *post-mortem* examination. More in-depth
studies would be needed if other calves carrying this
homozygous variant were born in the future. On
the other hand, the identification of this variant has
enabled the introduction of a screening test for this
allele, which should lead to the progressive eradication
of this genetic flaw in the “Blonde d’Aquitaine” breed.
At present, the allelic frequency of the variant is only
0.004% in the Blonde d’Aquitaine population, the only
breed where this allele is detected.

The *EGFR* nonsense variant identified in this study
induces damaging effects on gene and protein expres-
sion. The correlation between genotype and phenotype
is complete and this variant is not found in all control
cows of the other breeds analysed and not at homozy-
gosity in other Blonde d'Aquitaine cattle besides the
6 affected calves. These 6 calves come from different
parents. Furthermore, loss-of-function *EGFR* variants
have been described in other species and associated
with similar damaging phenotypes. Thus, despite the
above-mentioned potential limitation of this study, all of
these observations suggest that this variant is a patho-
genic variant according to the classification guidelines
described by Boeykens et al [2].
## Conclusions

This manuscript reports the identification of a recessive nonsense *EGFR* pathogenic variant responsible for perinatal lethality in the "Blonde d'Aquitaine" cattle breed. The phenotypes associated with this genotype were reminiscent of those described for similar human or mouse *EGFR* variants. A screening test has been designed for this allele, and the progressive eradication of this genetic disease is in progress.

### Abbreviations

Bp Base pairs

EGFR Epidermal Growth Factor Receptor

RT Reverse Transcription

PCR Polymerase Chain Reaction

UTR Untranslated Region

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12917-025-05225-6.

Supplementary Material 1: Supplementary Figure 1: Immunofluorescence analysis of proliferation marks in affected cow skin. Staining with the basal keratinocytes marker KRT14 (purple) and pericyte marker αSMA (green) (a,b) and proliferation marker Ki67 (purple) and adherens junctions β-catenin membrane marker (green) (c, d) in representative regions of epidermis (a, c) and hair bulb (b, c)). Arrowheads point to nuclei labelled with Ki67 at the basal membrane and above.

Supplementary Material 2: Supplementary Figure 2: Integrative Genomic Viewer screenshot. Integrative Genomic Viewer screenshot showing heterozygosity for a substitution at position g.916,239 of BTA22 in the whole-genome sequence of the carrier 44784. Alignment coverage was shown at the upper side of the figure and gene name at the lower side of the figure. The affected EGFR amino acid is also indicated.

Supplementary Material 3: Supplementary Figure 3: Quantification of *EGFR* transcripts by semi-quantitative PCR. Quantification of the RT-PCR bands obtained for *EGFR* gene expression (Figure 8). The relative values were adjusted according to the RT-PCR levels obtained on the *GAPDH* transcripts. Quantifications were performed using the ImageJ software.

Supplementary Material 4: Supplementary Figure 4: EGFR Western Blot Analysis. Full illustration of the Western Blot Analysis of ears protein extracts from two affected (Aff1, Aff2) and one control (C) calves. Same legend as in Figure 9.

Supplementary Material 5.

### Acknowledgements

The authors are grateful to the partners of the 1000 bull genomes consortium for their collaboration. The authors thank all veterinarians who referred the affected animals and especially Jean-Marie Nicol and the MIMA2 microscopy platform (https://doi.org/10.15454/1.5572348210007727E12).

### Authors' contributions

SaF performed all molecular biology analyses and participated in biochemical and histological studies. SaF, MB and DB carried out the genetic analyses. VH, MCC and FS performed the clinical investigations. CB, GE and MV carried out the biochemical and histological analyses. SaF, AD, DB and JLV led this study. SaF and JLV wrote the manuscript and all co-authors participated in its review.

### Funding

This work is part of the BOVANO project (ANR-14-CE19-0011) funded by the French Agence Nationale de la Recherche (Paris, France) and Apis-Gene.

### Data availability

The data are presented in the main manuscript and the materials described are available upon request.

## Declarations

### Ethics approval and consent to participate

The present work complies with INRAE Animal Care Committee's rules and the requirements of Directive 86/609 of the European Community Council. No experiment was realized on life animals. All diagnostic and therapeutic procedures were performed by licensed veterinarians in the course of routine veterinary health management. All protocols (clinical and laboratory) implemented were in accordance with international principles and protocols for animal usage.

Consent to participate was not applicable as calves were client-owned and the owners all expressed their interest in finding out the cause of the disease. Affected animals were reported to the ONAB (www.onab.fr) by their owners or their veterinarian with the full consent of the owner.
The semen sample of the ancestor was provided by AURIVA breeding company for sequencing purpose. All genotypes of the control population were produced for genomic selection purpose and their use was allowed for this study.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

Received: 2 September 2025 / Accepted: 9 December 2025

Published online: 07 January 2026

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