Candidate genes for congenital malformations in pigs

the local breeders’ intention to enhance the selection candidates (Krupová et al., 2017). The overall ambition is to identify gene markers and pathways associated with phenotypically measurable traits to enhance the selection response. Therefore, this study aimed to briefly overview the clinical symptoms, etiopathogenesis and candidate genes of the most critical congenital malformations in Congenital malformations occur in numerous pig breeding programs. Clinical symptoms, etiopathogenesis and candidate genes of the most critical congenital malformations in pigs were briefly overviewed in the study. Based on the recent literature, identifying and evaluating the genomic regions associated with defects, such as splay legs syndrome, hernias, cryptorchidism, atresia ani, kyphosis, intersexuality, and malignant melanoma, can enhance the selection response. As promising genes were published e.g. NREP, FBXO32, and HOMER1 for splay leg syndrome, SRC, OSM , COL family, and CGRP for hernias, GNRHR , GATA2 , and RLF for cryptorchidism, and GLI2 for atresia ani. Potential candidate genes associated with defects were mainly detected in literature by the genome-wide association approach. Reviewing the studies and following the suggestions in some of papers it is indicated the necessity for molecular and more comprehensive evaluation in terms of the sample standardisation and accurate phenotyping of a broad spectrum of populations and breeds. Moreover, knowledge transmission among all livestock species and humans is recommended in literature to better understand malformation biology.


Introduction
Breeding in pigs to improve production traits can lead to physiological changes that can deteriorate animal health and negatively affect the pig farm economics (Prunier et al., 2010;Sevillano et al., 2015). Congenital malformations occur in numerous pig breeding programs worldwide (Mattsson, 2011). Splay legs syndrome, hernias, cryptorchidism and other congenital defects (e.g. atresia ani, kyphosis, intersexuality, and malignant melanoma) are defined as the most critical congenital defects. Generally, heritability rate of malformations is of 0.01-0.80 (Mattsson, 2011;Sevillano et al., 2015;Rousseau et al., 2013) and about 1-8% of animals of the given pig category is affected by these defects. A common practice to eliminate malformations is the reproductive exclusion of the affected animals (Mattsson, 2011).
The identification of structural and functional proteins, genes, and quantitative trait loci allowed the study of congenital malformations (i.e. splay legs syndrome, hernias, and cryptorchidism) and other health traits at the genome level (Schumacher et al., 2021). Therefore, the identification and evaluation of genomic regions that control congenital defects was a breakthrough for the breeding programs (Sevillano et al., 2015). Besides, the detailed knowledge of the genome using single nucleotide polymorphism (SNP) markers can be usefully applied to manage the diversity of conventional populations (Moravčíková and Kasarda, 2020).
In the Czech Republic, the negative selection of animals (or entire litters) for malformations has been applied for many years in pig breeding programs. However, the routine phenotype monitoring of malformations is provided in the population (Žáková et al., 2020) and the collection of animal tissues for the genomic evaluation of developmental defects started in accordance with the local breeders' intention to enhance the selection candidates (Krupová et al., 2017). The overall ambition is to identify gene markers and pathways associated with phenotypically measurable traits to enhance the selection response. Therefore, this study aimed to briefly overview the clinical symptoms, etiopathogenesis and candidate genes of the most critical congenital malformations in The splay legs syndrome (also known as spraddle leg syndrome) is the most frequent lame disorder in newborn piglets and the most observed inherited malformations in pigs. The syndrome has high economic and welfare consequences on the swine industry (Schumacher et al., 2021). The affected piglets are not able to stand or walk due to temporary impairment of the pelvic muscle function that occurs early after birth (Papatsiros, 2012;Hao et al., 2017). Histologically, the syndrome is expressed as myofibrillar hypoplasia and characterised by a higher proportion of less developed and smaller myofibrils (Schumacher et al., 2021). Clinically, the syndrome appears in various forms, from ataxia (sitting like a dog with a possibility to stand up) through the uncoordinated movement of rear legs (piglet is not able to get up without help) or the splaying of rear legs (movement is done just with the front legs) to a severe form, in which both the front and rear legs are splayed (piglet is not able to move). Standard treatments, such as leg binding, vitamin, and selenium intake, and appropriate care, are applied to reduce losses of affected piglets due to malnutrition, hypothermia, or crushing by the sows. Usually, piglets that survive the first week of their life recover completely (Papatsiros, 2012).

Candidate genes for congenital malformations in pigs
The genetic aspects of the syndrome have been thoroughly evaluated in the context of etiopathogenesis (Maak et al., 2009;Hao et al., 2017). An overview of the candidate genes associated with the syndrome is presented in Table 1. The primary pathological mechanism of the syndrome includes atrophy of muscle fibres and delayed skeletal muscle maturation at birth (Schumacher et al., 2021). Therefore, three promising candidate genes, NREP, FBXO32, and HOMER1, associated with myogenic differentiation, atrophy, and muscle development should be considered for the study of myofibrillar hypoplasia in pigs. Moreover, muscle sampling should be standardised for improving phenotypic precision.

Hernias
A hernia is generally characterised as penetration of the intestine, an internal organ, or tissue through the weakened supportive muscles or body wall. The umbilical, inguinal, and scrotal hernias often occur in pigs. Clinical verification is usually needed to diagnose the inguinal and scrotal hernias; therefore, they are frequently evaluated as a single trait (Ding et al., 2009;Grindflek et al., 2006). Inguinal hernias are typically detected in the first week of life at the time of castration, and affected piglets are usually euthanized. Pigs with umbilical hernias do not appear until the nursery or the early fattening stage (Atkinson et al., 2017). Similar to the splay leg syndrome, hernias have economic consequences on the swine industry (Nowacka-Woszuk, 2021) when the feed efficiency and growth ability are reduced in affected animals (Ding et al., 2009).
From the papers intensively focused on the genetic architecture of hernias outcomes their complex background is apparent from the interaction of polygenic heredity and environmental conditions (Nowacka-Woszuk, 2021). The candidate genes associated with hernias are presented in Table 1. The most promising genes are linked to the proper growth and differentiation of cells (SRC and OSM), the function of connective tissues (COL family), the gubernacular development and function (INSL3 and MIS), and processus vaginalis (CGRP). The latter gene probably leads to a relationship between the scrotal hernia and cryptorchidism with a correlation of 0.2-0.7 (Mattsson, 2011). Both congenital defects occur often and have a sex-linked expression.

Cryptorchidism
Cryptorchidism occurs when one or both testicles of male piglets cannot descend from the intra-abdominal placement in the scrotum. The unilateral (left side) cryptorchidism mainly occurs in pigs, and the affected males should be excluded from reproduction (Mattsson, 2011;Mahmud et al., 2015). The malformation is usually diagnosed at birth or early in the postpartum period. Similar to other congenital defects, variability of the malformation prevalence is identified among the evaluated populations, breeds, parities, and litters (Sevillano et al., 2015;Mattsson, 2011).
Cryptorchidism is linked to the animal genome, anatomy, and endocrine system (Mahmud et al., 2015). An overview of the putative candidate genes associated with cryptorchidism are presented in Table 1. The most promising candidate genes are associated with the testes descent (GNRHR), the urogenital development and function (GATA2 and PDGFRA), and the modulation of foetal responses to oestrogens (AFP; Sevillano et al., 2015). Besides, RHOA that has an indirect impact on scrotal hernia also plays an essential role in cryptorchidism due to the regulation of smooth muscle tissues, which are responsible for the obliteration of processus vaginalis that precedes appropriate testicular descent. Furthermore, a mutation in INSL3 (also known as RLF) probably has an impact on the cryptorchidism incidence in male mice (Tomboc et al., 2000). Therefore, multiple genes are implicated in proper testicular descent and fertility, some of which have a surplus role.

Other congenital malformations
Atresia ani, kyphosis, congenital tremor syndrome, as well as intersexuality and miscellaneous abnormalities (i.e. anatomic defects of head, heart, and tail) also occur as congenital malformations in pigs (Mattsson, 2011). Of these, the frequency of congenital neoplastic diseases (neoplasms), such as sarcomas, fibro-papillomatosis, hereditary malignant lymphoma, and melanoma, is relatively low (Misdorp, 2003;Morey-Matamalas, et al., 2021). The potential candidate genes for various defects and neoplasms are presented in Table 1. Some candidate genes have been specifically identified for spontaneous melanoma that is frequently observed in Duroc breed and crosses and are related to the occurrence (PLEKHA5, TRAFD1, and LIMK2), clinical ulceration of tumour (DST), and progression through metastasis (SPATA31D1 and EPB41L4A-AS2; Bourneuf et al., 2018).
Atresia ani (AA) is the developmental defect of the missing anus usually accompanied by other anomalies (Cassini et al., 2005). Morphologically, the porcine AA is characterised by abnormal development of the hindgut, which is probably controlled by an oligogenic or polygenic background (Hori et al., 2001). GLI2 has been recognised as a major positional candidate gene for AA in pigs (Cassini et al., 2005;Qiushi et al., 2013), whereas significant linkages to AA were obtained for some markers on chromosomes 1, 3, and 12 (Wiedemann et al., 2005). From the last studies dealing with this porcine malformation (Qiushi et al., 2013;Jin et al., 2013) resulted proposal to provide further investigations to explicate the genomic mutations involved in AA.
Kyphosis is a porcine back-curvature defect, which is based on deformation in the hemi-vertebrae. The affected piglets have a humpy back or a dipped shoulder and consequently show a slower growth rate, a lower probability of reaching the slaughter weight (Mattsson, 2011), and a deteriorated carcass value (Lindholm-Perry et al., 2010). Several SNPs and potential mutations in coding regions of candidate genes have been identified (Lindholm-Perry et al., 2010); however, the most promising candidate genes associated with kyphosis are PLOD1, SOX9, and probably ADAMTS18. Besides, the HOX gene family and genes located in the TGF-beta superfamily may control the incidence of kyphosis in pigs (Rohrer et al., 2015).
Congenital tremor syndrome is specified as a rhythmic tremor of newborn piglets caused by the genetic background or environmental conditions during the intrauterine foetus development, leading to myelin deficiency, especially in the spinal cord, and approximately 50% morbidity (Mittsson, 2011). The syndrome appears in two types based on the presence of histopathological lesions in the central nervous system: type A with lesions and type B without lesions (Stenberg et al., 2020). Based on causality, type A can be further divided into five subtypes (A-I to A-V). A-I is characterised by the presence of swine fever virus, whereas A-II by that of atypical porcine pestivirus (APPV) or porcine circovirus-II (PCV-II).
Previous studies have primarily focused on A-II to identify and understand the causative agents (Stenberg et al., 2020).
Intersexuality (sex reversal) is a sex congenital disorder characterised by the atypical development of the reproductive system. The "female" gonosomes (XX) in affected animals are considered true hermaphrodites or pseudo-hermaphrodites that vary in the activity and presence of both female and male gonads and genitalia (Mittsson, 2011). Therefore, the chromosomal, somatic, and gonadic sex of defected animals could be different (Rousseau et al., 2013). The economic consequences of intersexuality are related to sterility as well as the higher probability of boar taint and infections. SOX9 (mentioned above in the context of porcine kyphosis incidence) was recognised to affect sexual and skeleton development in some studies (Rousseau et al., 2013;Brening et al., 2015;Szczerbal et al., 2019).

Conclusions
The most commonly occurring congenital malformations in pigs along with all the known associated candidate genes were overviewed in the study to establish a background for genomic evaluation. In compliance with the general recommendation, the affected animals are usually excluded from reproduction. The research already focused on the genomic etiopathology of the congenital malformations in pigs has open space to explore their structural and functional genetic background and environment interaction. Promising findings of the candidate genes associated with malformations, mainly using the genome-wide association approach, come to an agreement that it is necessary to provide further and more comprehensive investigation. The trait (phenotype) accurate definition, measurement and description accompanied by the gene identification for a broader spectrum of populations and breeds should be provided to further gain in selection progress. Overall, knowledge transmission among all livestock species and humans is recommended in literature as critical to better understand malformation biology.