Arthrogryposis and macroglossia have long been known as congenital abnormalities observed in several cattle breeds [1
]. Arthrogryposis is characterized by joints contractures with different degrees of severity, which can affect one to four legs, with various associated clinical signs, the most frequent being cleft palate [3
]. More than one etiologic event, such as plant toxicosis [4
], prenatal viral infections, and a possible hereditary component, have been reported as responsible for the disease occurrence [5
]. Less information is available for macroglossia, which consists in the swelling of the tongue that may interfere with the calf’s ability to nurse. The defect is thought to have a genetic basis, but no scientific evidence is available so far.
For both defects, double muscling is considered as a predisposing factor. Already back in the 1963, Lauvergne et al. [6
] listed rickets-like troubles and macroglossia among the clinical signs displayed by the hypertrophied animals. The observation that the manipulation of the myostatin gene and, more specifically, the downregulation of its expression resulted in a series of adverse effects, including leg problems and macroglossia, which seems to confirm the negative influence of double muscling [7
]. Moreover, macroglossia is one of the primary features of the human Wiedemann-Beckwith syndrome (OMIM 130650), which is clinically similar to muscular hypertrophy in cattle [8
Both arthrogryposis and macroglossia have been reported for decades in the hypertrophied Piemontese cattle breed. Since the end of 1980s, the National Association of the Piemontese cattle Breeders (ANABORAPI) started to select against these two pathologies by culling Artificial Insemination (AI) bulls with a high percentage of affected progeny. A decrease from 2.74% to 0.34% and from 2.36% to 0.28% in the occurrence of arthrogryposis and macroglossia, respectively, were obtained in the period 1990–2017 (Supplemental Figure S1
) as a consequence of this selection strategy (ANABORAPI).
These data seem to support the hypothesis of a genetic background for the defects, but the few investigations in the Piemontese breed did not give conclusive results. Huston et al. [3
] suggested that, in the Piemontese, arthrogryposis could be determined by an incompletely penetrant recessive allele, with higher penetrance in males, which seems to be consistent with the ANABORAPI data (Supplemental Figure S1
). However, a genome-wide association study carried out on Piemontese calves affected by arthrogryposis and macroglossia genotyped with a medium density (50 K) single nucleotide polymorphism (SNP) BeadChip did not detect clear signals of association for both pathologies [9
]. On the contrary, recent studies detected variations in single genes as responsible for arthrogryposis in Angus [10
], Swiss Holstein [11
], Belgian Blue [12
], and Red Danish [13
] cattle breeds.
Therefore, the aims of this study were: (i) to test the hypothesis of a similar monogenic determinism for arthrogryposis in the Piemontese cattle breed by genotyping affected and healthy animals with a high-density chip never used in previous studies and applying genome-wide association study (GWAS), FST and canonical discriminant analysis (CDA) procedures, and (ii) to investigate with the same approach the genetic background of macroglossia, for which no information is available so far.
The present study provides new data on the genetics of arthrogryposis and the first insight into the analysis of macroglossia in the Piemontese breed. An important systematic bias in GWAS often reported in the literature is caused by population stratification due to ethnic/breed admixture and/or close relationships among individuals of case-control studies [21
]. In our case, a limited inflation of the statistical tests was observed; thus, a genomic-control approach was adopted, since the individuals included in the design belonged to the same breed and were weakly or not related [21
Interestingly, the combined use of case-control GWAS, FST
, and CDA highlighted several markers potentially associated with the investigated syndromes. The use of multiple approaches is generally advised in genome-wide analysis [17
]. The use of CDA was recently proposed as an effective tool for improving the discovery rate either alone or in a combination with GWAS, especially when the sample size is reduced [25
As for arthrogryposis, the results depict a situation different from what was observed in the other investigated breeds [10
], where variations in single genes were identified as responsible for the disease. In fact, our data did not evidence a single strong association with the pathology, while they highlighted a number of significant markers located on different chromosomes, suggesting a polygenic mechanism underlying the disease. The joint role of these markers is supported by their ability to separate the three groups of animals according to their health status.
None of the markers for arthrogryposis identified in the Piemontese breed are located within or near the genes reported as causing the disease in the other breeds. In this respect, it is important to underline that also the causal variations found in those breeds were of different types and in different genes: a large deletion encompassing three genes (BTA 16) in Angus [10
], a missense mutation in the MYBPC1
gene (BTA 5) in Swiss Holstein [11
], a splicing variant in the PIGH
gene (BTA 10) in Belgian Blue [12
], and a small deletion in the CHRNB1
gene (BTA 19) in Red Danish [13
]. This implies that the genetic determinism of arthrogryposis is not the same in the affected breeds. On the other hand, it must be considered that a large variability in the phenotypic expression of what is called “arthrogryposis” was observed in the breeds studied so far, from lethal consequences, as in Belgian Blue or Angus breeds, to less severe problems, as in the Piemontese. Additionally, at least six types of arthrogryposis with different clinical signs and grades of severity were reviewed by Huston et al. [3
] in cattle. Such heterogeneity makes it difficult to clearly define the trait that could explain the differences observed at the genetic level. In all cases, however, the findings of the different studies are compatible with the autosomal recessive mode of inheritance suggested since the earliest studies. The incidences of the two pathologies in the Piemontese breed in the last decades also showed a trend compatible with the case of selection against the recessive phenotype, and this led us to hypothesize the existence of a monogenic determinism similar to what was observed in the other cattle breeds. However, the present data do not support this hypothesis, suggesting that a more complex mechanism is responsible for the disease in the Piemontese breed.
For macroglossia, no previous genetic data exist. The results of the current study highlight a situation comparable to that obtained for arthrogryposis, so that a multifactorial mechanism can be hypothesized also for macroglossia.
The identified SNPs were located within or close to 33 genes, of which 9 and 13 were exclusive for arthrogryposis and macroglossia, respectively, and 11 common to the two pathologies. This is worthy of note, considering that, in the Piemontese breed, both pathologies are sometimes observed in the same animal. Thus, the findings of this study might suggest that the putative candidate genes common to both diseases could be involved in basic physiological processes common to both defects.
In the case of arthrogryposis, seven of the relevant SNPs mapped in coding genes, whereas, for macroglossia, 11 SNPs were located within coding genes. In some cases, it is unclear from the gene annotations their possible involvement in the pathologies. Instead, for other genes, a possible role can be hypothesized, as their products are part of processes related to muscular or nervous tissue developments whose defects are included among the common causes of the pathologies here considered [26
Among these genes, Netrin3 (NTN3
) encodes a member (NTN3) of a family of extracellular proteins that act as chemotropic guidance cues for migrating cells and axons during neural development [27
]. In mice, it was demonstrated that NTN3 is expressed in muscle cells, and therefore, it may play a role in guiding peripheral axons to their corrected muscle targets [28
(potassium voltage-gated channel subfamily H member 1) and KCNH2
(potassium voltage-gated channel subfamily H member 2) genes code for proteins that belong to a complex protein superfamily widely distributed during embryonic development and involved in a wide variety of cell functions. In mice, the two genes are co-expressed in the skeletal muscle during embryogenesis, including the cranial, thoracic, and limb regions [29
]. In man, KCNH1 was shown to be involved in myoblast fusion, a complex process that includes withdrawal from the cell cycle, cell-cell interactions, adhesion, alignment, and a final membrane fusion to form the multinucleated skeletal muscle fiber [30
A possible role can be also suggested for the KANK3
(KN motif and ankyrin repeat domains 3) gene strongly expressed in different body compartments, including the skeletal muscle, and involved in the control of cytoskeleton formation by negatively regulating actin polymerization [31