New perspectives of genetic disorders in cattle

In the last decades a negative trend in inbreeding has accompanied the evident improvement in productivity and performance of bovine domestic population, predisposing to the occurrence of recessively inherited disorders. The objectives of this thesis were: a) the study of genetic diseases applying a “forward genetic approach” (FGA); b) the estimation of the prevalence of deleterious alleles responsible for eight recessive disorders in different breeds; c) the collection of well-characterized materials in a Biobank for Bovine Genetic Disorders. The FGA allowed the identification of seven new recessive deleterious variants (Paunch calf syndrome - KDM2B; Congenital cholesterol deficiency - APOB; Ichthyosis congenita - FA2H; Hypotrichosis - KRT71; Hypotrichosis - HEPHL1; Achromatopsia - CNGB3; Hemifacial microsomia – LAMB1) and of seven new de novo dominant deleterious variants (Achondrogenesis type II - two variants in COL2A1; Osteogenesis imperfecta - COL1A1; Skeletal-cardio-enteric dysplasia - MAP2K2; Congenital neuromuscular channelopathy - KGNG1; Epidermolysis bullosa simplex - KRT5; Classical Ehlers-Danlos syndrome - COL5A2) in different breeds, associated with a large spectrum of phenotypes affecting different systems. The FGA was based on the sequence of a clinical, genealogical, gross- and/or histopathological and genomic study. In particular, a WGS trio-approach (patient, dam and sire) was applied. The prevalence of deleterious alleles was calculated for the Pseudomyotonia congenita, Paunch calf syndrome, Hemifacial microsomia, Congenital bilateral cataract, Ichthyosis congenita, Ichthyosis fetalis, Achromatopsia and Hypotrichosis. A particular concern resulted the allelic frequency of 12% for the Paunch calf syndrome in Romagnola cattle. In respect to the Biobank for Bovine Genetic Diseases, biological materials of clinical cases and their available relatives as well as controls used for the allelic frequency estimations were stored at -20 °C. Altogether, around 16.000 samples were added to the biobank

Short communication: Prevalence of deleterious variants causing recessive disorders in Italian Chianina, Marchigiana and Romagnola cattle.

In the last two decades, the molecular cause of six monogenic autosomal recessive disorders has been identified in native Italian beef cattle: two different ATP2A1 variants for the pseudomyotonia congenita, the first in Chianina and Romagnola (PMT1) and the second in Romagnola (PMT2); a KDM2B variant for the paunch calf syndrome (PCS) in Marchigiana and Romagnola; a NID1 variant for the congenital cataract (CC) in Romagnola; a LAMB1 variant for the hemifacial microsomia (HFM) in Romagnola; an ABCA12 variant for the ichthyosis fetalis (IF) in Chianina and a FA2H variant for the ichthyosis congenita (IC) in Chianina. The aim of this study was to evaluate the potential impact of these disorders in the affected Italian populations. For this purpose, 3331 Chianina, 2812 Marchigiana and 1680 Romagnola bulls born in the last 40 years were considered. The allelic frequency (AF) of the variant for PMT1 was 1.0% in Romagnola, 4.6% in Marchigiana and 5.9% in Chianina. The AF of the variant for PMT2 was 3.3% in Romagnola and 0% in the other two breeds. The AF of the variant for PCS was 11.7% in Romagnola, 2.0% in Marchigiana and 0% in Chianina. The AF of the variants for CC, HFM, IF and IC resulted below 3%, being the variants detected only in the breed populations in which they were previously reported. Considering a selected male population in the single breed, Chianina showed carrier prevalence of 11.9% for PMT1, 7.7% for IC and 6.4% for IF. Romagnola showed carrier prevalence of 23.4% for PCS, 6.7% for PMT2, 4.1% for HFM, 3.2% for CC and 2.0% for PMT1. Marchigiana showed carrier prevalence of 9.1% for PMT1 and 4.0% for PCS. With respect to the Romagnola cattle, the concerning presence of a total of five defect alleles in the population hampers a general approach based on the prevention of carriers from artificial insemination. However, identification of carriers may allow conscious mating to prevent the risk of homozygous descendants as well as the spread of heterozygous offspring. Therefore, systematic genotyping for all seven known harmful alleles is recommended to prevent risk mating between carriers, in particular to avoid the occurrence of affected offspring

Congenital syndromic Chiari-like malformation (CSCM) in Holstein cattle: towards unravelling of possible genetic causes.

BACKGROUND Chiari malformation type II (CMII) was originally reported in humans as a rare disorder characterized by the downward herniation of the hindbrain and towering cerebellum. The congenital brain malformation is usually accompanied by spina bifida, a congenital spinal anomaly resulting from incomplete closure of the dorsal aspect of the spinal neural tube, and occasionally by other lesions. A similar disorder has been reported in several animal species, including cattle, particularly as a congenital syndrome. A cause of congenital syndromic Chiari-like malformation (CSCM) in cattle has not been reported to date. We collected a series of 14 CSCM-affected Holstein calves (13 purebred, one Red Danish Dairy F1 cross) and performed whole-genome sequencing (WGS). WGS was performed on 33 cattle, including eight cases with parents (trio-based; group 1), three cases with one parent (group 2), and three single cases (solo-based; group 3). RESULTS Sequencing-based genome-wide association study of the 13 Holstein calves with CSCM and 166 controls revealed no significantly associated genome region. Assuming a single Holstein breed-specific recessive allele, no region of shared homozygosity was detected suggesting heterogeneity. Subsequent filtering for protein-changing variants that were only homozygous in the genomes of the individual cases allowed the identification of two missense variants affecting different genes, SHC4 in case 4 in group 1 and WDR45B in case 13 in group 3. Furthermore, these two variants were only observed in Holstein cattle when querying WGS data of > 5,100 animals. Alternatively, potential de novo mutational events were assessed in each case. Filtering for heterozygous private protein-changing variants identified one DYNC1H1 frameshift variant as a candidate causal dominant acting allele in case 12 in group 3. Finally, the presence of larger structural DNA variants and chromosomal abnormalities was investigated in all cases. Depth of coverage analysis revealed two different partial monosomies of chromosome 2 segments in cases 1 and 7 in group 1 and a trisomy of chromosome 12 in the WDR45B homozygous case 13 in group 3. CONCLUSIONS This study presents for the first time a detailed genomic evaluation of CSCM in Holstein cattle and suggests an unexpected genetic and allelic heterogeneity considering the mode of inheritance, as well as the type of variant. For the first time, we propose candidate causal variants that may explain bovine CSCM in a certain proportion of affected calves. We present cattle as a large animal model for human CMII and propose new genes and genomic variants as possible causes for related diseases in both animals and humans

A large deletion in the COL2A1 gene expands the spectrum of pathogenic variants causing bulldog calf syndrome in cattle.

BACKGROUND Congenital bovine chondrodysplasia, also known as bulldog calf syndrome, is characterized by disproportionate growth of bones resulting in a shortened and compressed body, mainly due to reduced length of the spine and the long bones of the limbs. In addition, severe facial dysmorphisms including palatoschisis and shortening of the viscerocranium are present. Abnormalities in the gene collagen type II alpha 1 chain (COL2A1) have been associated with some cases of the bulldog calf syndrome. Until now, six pathogenic single-nucleotide variants have been found in COL2A1. Here we present a novel variant in COL2A1 of a Holstein calf and provide an overview of the phenotypic and allelic heterogeneity of the COL2A1-related bulldog calf syndrome in cattle. CASE PRESENTATION The calf was aborted at gestation day 264 and showed generalized disproportionate dwarfism, with a shortened compressed body and limbs, and dysplasia of the viscerocranium; a phenotype resembling bulldog calf syndrome due to an abnormality in COL2A1. Whole-genome sequence (WGS) data was obtained and revealed a heterozygous 3513 base pair deletion encompassing 10 of the 54 coding exons of COL2A1. Polymerase chain reaction analysis and Sanger sequencing confirmed the breakpoints of the deletion and its absence in the genomes of both parents. CONCLUSIONS The pathological and genetic findings were consistent with a case of "bulldog calf syndrome". The identified variant causing the syndrome was the result of a de novo mutation event that either occurred post-zygotically in the developing embryo or was inherited because of low-level mosaicism in one of the parents. The identified loss-of-function variant is pathogenic due to COL2A1 haploinsufficiency and represents the first structural variant causing bulldog calf syndrome in cattle. Furthermore, this case report highlights the utility of WGS-based precise diagnostics for understanding congenital disorders in cattle and the need for continued surveillance for genetic disorders in cattle

MYH3-associated non-syndromic palatoschisis (cleft palate, CP) in Limousine cattle.

We have successfully identified the most likely cause for a rare autosomal recessive disorder with only one case. Our clinicopathologic and genomic findings for the MYH3-related recessively inherited form of non-syndromic cleft palate are consistent with those recently reported by Vaiman et al. (2022) (OMIA002590-9913). Interestingly, the 2-year-old Limousine female in our study appears to be the oldest known animal with MYH3-associated non-syndromic palatoschisis. According to Vaiman et al. (2022), life expectancy is short, with most animals euthanized in the first month of life. In addition, the variant allele frequency was found to be lower in the current Swiss population compared with the French Limousine (2.4%)

A Missense Variant in PLP2 in Holstein Cattle with X-Linked Congenital Mast Cell Tumor.

Congenital tumors occur infrequently in cattle. The aim of this study was to detail the clinicopathological phenotype of a Holstein calf with a congenital mast cell tumor and to identify the genetic cause by a whole-genome sequencing (WGS) trio-approach. An 18-day-old male Holstein calf was clinically examed and revealed multifocal, alopecic, thick and wrinkled skin lesions over the entire body. At 6 months of age, the general condition of the calf was characterized by retarded growth, poor nutritional status, and ulceration of the skin lesions. Histopathological examination revealed a primary cutaneous, poorly differentiated embryonal mast cell tumor with metastases in the lymph nodes and liver. Genetic analysis revealed a private X-linked variant in the PLP2 gene (chrX:87216480C>T; c.50C>T), which was present only in the genomes of the case (hemizygous) and his mother (heterozygous). It was absent in the sire as well as in 5365 control genomes. The identified missense variant exchanges the encoded amino acid of PLP2 at position 17 (p.Thr17Ile), which is classified as deleterious and affects a protein that plays a role in tumor growth and metastasis. Therefore, we suggested that the detected PLPL2 variant could be a plausible cause for this congenital condition in the affected calf

Malattie congenite del sistema nervoso del bovino

Bovine congenital diseases of thenervous system In calves, congenital (condition existing at or before birth) dis-orders of the nervous system are not rare: in the past they wereestimated to account for about 20% of all congenital defects.Unfortunately, the scarcity of surveillance programs and thelow economical value of young animals hinder submission ofclinical cases to diagnostic centers; hence their real prevalencegoes underestimated. Moreover, many defects can be identi-fied only by necropsy or histopathology.Congenital neurological conditions may be due to external ter-atogens or to genetic defects.Teratogens are biologic, toxic, chemical, physical or metabol-ic and can act directly on the developmental processes or canalter patterns of gene expression, inhibit cell interactions, orblock morphogenetic cell movements. Genetic defects are most-ly represented by autosomal inherited recessives and are usu-ally based on inborn errors of metabolism and on lack or dys-function of hormones, receptors, enzymes, structural proteins,neurotransmitters and ion channels (metabolic defects, stor-age defects, disturbed neurotransmission, channelopathies). Theincreased use of intensive breeding programs, based on the wide-spread use of selected elite sires, and the consequent increaseof the \u201cinbreeding grade\u201d, has allowed the homozygous pres-ence of recessive variants - responsible for malformation - inthe inbred progeny of many breeds.Genetic causes includes also \uabde novo\ubb pathogenic variants thatmay occur in one of the germ cells (sperm or egg) of one ofthe parents, or arise in the fertilized egg itself during early em-bryogenesis.The defects characterized by structural deformities of the nerv-ous structures are termed \u201ccongenital malformation\u201d and arethe result of disorders of tissue development such as: agene-sis (complete absence of an organ), aplasia (absence of an or-gan with retention of the organ rudiment), hypoplasia (in-complete development of an organ), dysraphic anomaly (fail-ure of opposed structures to undergo adhesion and fusion), in-volution failure (persistence of an embryonic structure that nor-mally disappears during development), division failure (in-complete cleavage of embryonic tissues owing to lack of pro-grammed cell death), atresia (failure of an organ rudiment toform a lumen), dysplasia (abnormal organization of cells in atissue), ectopia (error of morphogenesis in which an organ islocated outside its correct anatomic site), dystopia (error of mor-phogenesis in which an organ is retained at a site where it residedduring a stage of development). The majority of these disor-ders involve also the nervous system. The defect characterized by inborn alteration of physiologicalfunctions or metabolic processes may not be expressed or rec-ognized until later in life, so being erroneously interpreted asacquired diseases.The article presents the most common congenital defects of thenervous system in calves. Due to the fact that for the majori-ty of the congenital diseases the exact cause remains unknown,it is intention of this article to prompt farmers and veterinar-ians to consider - whenever possible - the submission of suchcases to diagnostic centers for further studies and investigation

Autosomal Cholesterol Deficiency in a Holstein Calf

Cholesterol deficiency (CD) is an autosomal recessive defect in Holstein cattle caused by a mutation in the apolipoprotein B gene (APOB). This paper reports the clinical and pathological phenotype of a case of CD in a 5-months-old Holstein calf. Retarded growth, chronic, intermittent diarrhea, stomatitis, hypocholesterolemia and low blood triglycerides concentrations were the most important clinical and ancillary findings. Histopathologically, inflammation of the digestive organs was the most evident sign. Blood from the patient, the dam, sister1, sister2 and semen of the sire were tested for APOB mutation: the calf resulted homozygous, whereas the dam and the sire resulted heterozygous carriers. Both sisters were APOB mutation free. Cholesterol deficiency should be considered in the differential diagnosis of chronic diarrhea and failure to thrive in Holstein calves

A Heterozygous Missense Variant in the COL5A2 in Holstein Cattle Resembling the Classical Ehlers–Danlos Syndrome

Classical Ehlers–Danlos syndrome (cEDS) is a heritable connective tissue disorder characterized by variable degrees of skin hyperextensibility and fragility, atrophic scarring, and generalized joint hypermobility. The purpose of this study was to characterize the clinicopathological phenotype of a cEDS-affected Holstein calf and to identify the causative genetic variant associated with the disorder by whole-genome sequencing (WGS). A 3-day-old female Holstein calf was referred because of easily induced skin detachment and hyperextensibility in the neck. A complete clinical investigation was performed in the calf, dam, and maternal-grandmother. The calf and dam showed hyperextensibility of the neck skin and atrophic scarring; additionally, the calf presented skin fragility. Moreover, the histopathology of biopsies from the calf and its dam showed that the collagen bundles in affected skin areas were wavy, short, thin, and surrounded by edema and moderate to severe acute hemorrhages. Genetic analysis revealed a private heterozygous missense variant in COL5A2 (c.2366G>T; p.Gly789Val) that was present only in the calf and dam. This confirmed the diagnosis of cEDS and represents the first report of a causal variant for cEDS in cattle and the first COL5A2-related large animal model. Genodermatoses represent inherited disorders of the skin that mostly follow a monogenic mode of inheritance. Heritable connective tissue disorders such as classical Ehlers–Danlos syndrome (cEDS) belong to this group of human rare diseases that sporadically occur in other species. Herein, affected cattle are reported showing skin lesions including cutis laxa clinically and pathologically resembling cEDS in humans. Microscopic findings in the deeper dermis were consistent with collagen dysplasia. Whole-genome sequencing (WGS) identified a most likely disease-causing mutation in the COL5A2 gene. The COL5A2 gene is known to be associated with dominant inherited cEDS forms in mice and humans, but so far, it was not shown to cause a similar phenotype in domestic animals. The disease phenotype examined herein showed co-segregation with the identified missense variant within the maternal line across two generations and is most likely due to a spontaneous mutation event. Rare non-lethal disorders such as cEDS in livestock are mostly not diagnosed, but might affect animal welfare and thus lower the value of affected animals. WGS-based precision diagnostics allows understanding rare disorders and supports the value of surveillance of cattle breeding populations for harmful genetic disorders

A Nonsense Variant in Hephaestin Like 1 (HEPHL1) Is Responsible for Congenital Hypotrichosis in Belted Galloway Cattle.

Genodermatosis such as hair disorders mostly follow a monogenic mode of inheritance. Congenital hypotrichosis (HY) belong to this group of disorders and is characterized by abnormally reduced hair since birth. The purpose of this study was to characterize the clinical phenotype of a breed-specific non-syndromic form of HY in Belted Galloway cattle and to identify the causative genetic variant for this recessive disorder. An affected calf born in Switzerland presented with multiple small to large areas of alopecia on the limbs and on the dorsal part of the head, neck, and back. A genome-wide association study using Swiss and US Belted Galloway cattle encompassing 12 cases and 61 controls revealed an association signal on chromosome 29. Homozygosity mapping in a subset of cases refined the HY locus to a 1.5 Mb critical interval and subsequent Sanger sequencing of protein-coding exons of positional candidate genes revealed a stop gain variant in the HEPHL1 gene that encodes a multi-copper ferroxidase protein so-called hephaestin like 1 (c.1684A>T; p.Lys562*). A perfect concordance between the homozygous presence of this most likely pathogenic loss-of-function variant and the HY phenotype was found. Genotyping of more than 700 purebred Swiss and US Belted Galloway cattle showed the global spread of the mutation. This study provides a molecular test that will permit the avoidance of risk matings by systematic genotyping of relevant breeding animals. This rare recessive HEPHL1-related form of hypotrichosis provides a novel large animal model for similar human conditions. The results have been incorporated in the Online Mendelian Inheritance in Animals (OMIA) database (OMIA 002230-9913)