Sporadic occurrence of cleft palate has been reported in many mammalian species (Łobodzińska, 2014), and animal models have been major contributors to understanding normal and abnormal palatogenesis. Mouse models have been particularly useful for dissecting the molecular genetics and mechanisms of craniofacial development, but they are rarely useful as clinical models for post-natal or in utero cleft palate repair because mice are small, and the gestational window for post-operative healing without scarring is short (Lorenz and Longaker 2003). Dogs have been used as a large animal model for cleft palate (Martinez-Alvarez et al. 2013; Peralta et al. 2017). However, while there are several reports of dog families with CL/P or CPO as part of a syndrome of midline and/or limb anomalies (Natsume et al. 1994; Villagómez and Alonso, 1998; Kemp et al. 2009; Moura et al. 2012; Wolf et al. 2014; Wolf et al. 2015), there are few reports of familial CPO occurring as an isolated defect (Richtsmeier et al. 1994; Peralta et al. 2017). The use of dogs as a model is also supported by the observation that the incidence of CL/P in dogs can be reduced by dietary folate supplementation (Domosławska et al. 2013), as seen in some human populations (Wyszynski and Beaty 1996; Hackshaw et al. 2011). Overall, sporadic CPO is more common than CL/P or CLO in dogs, but the incidence of OFC subcategories varies significantly across breeds (Peralta et al. 2017; Roman et al. 2019). To date, however, genetic risk variants have been determined for only two OFC in dogs (Wolf et al. 2014; Wolf et al. 2015).
The molecular genetic etiology of CPO in the dog family reported here is not yet known. However, the morphological and histological analyses may provide clues. The immuno-staining for K17 and p63 appeared normal, showing that periderm and basal epithelial formation and function were not grossly affected. At gestational d39, the palate shelves are fusing in normal dog fetuses (Freiberger et al. 2021), but while the palate shelves of affected pups had elevated over the tongue, they had failed to fuse. This could be failure of the palate shelves to grow and elongate horizontally so that the epithelia did not meet at midline. Alternatively, it could be failure of the opposing shelves to fuse at the midline when the leading-edge epithelia met, with subsequent growth of the head causing a relative retraction of the shelves that left the gap. Analyses of mutant mouse models indicate that developmental regulation of mesenchymal cell proliferation, palate shelf elongation, and fusion at the medial epithelial seam involves multiple signaling pathways, including sonic hedgehog (SHH), wingless-related integration site (WNT), fibroblast growth factor (FGF), bone morphogenic protein (BMP), and transforming growth factor β (TGFβ), and that there is extensive cross-regulation between pathways (Gritli-Linde 2007; Lan and Jiang 2009; Iwata et al. 2011; Lane and Kaartinen 2014; Okello et al. 2017; Jia et al. 2017; Reynolds et al. 2019; Sweat et al. 2020). We note that some of the same signaling pathways that affect palate development are involved in limb development (e.g. SHH and FGF), and perturbations can lead to limb deformities like those observed in this CPO family (Mariani et al. 2008; Guerrini et al. 2011; Tickle and Towers 2017; Reynolds et al. 2020). Even with a shared-etiology hypothesis, the large number of genes involved in palate and limb development and the pleiotropic effects of variants in those genes, argue against a candidate gene approach to determining the etiology of CPO in this family. Rather, we propose that a whole genome approach is warranted.
Results of our breeding experiments are not consistent with a simple inheritance model that accounts for both phenotypes. The pedigree suggests that CPO is inherited as an autosomal recessive trait, but possibly with the added complexity of a suppressor allele. If there was an allele suppressing CPO, it appeared to be restricted to dog 173 because the frequency of CPO was as expected of simple recessive inheritance, except when 173 was the dam. One possible model is that a suppressor allele was inherited from 173 as a dominant allele with incomplete penetrance, and the same allele suppressing CPO also suppressed limb defects. However, this model fails to explain the complete absence of limb defects in CPO offspring from crosses with 173. The attractive hypothesis that limb defects were caused by the same recessive allele as caused CPO also requires modification because 11 of 25 offspring with CPO when 173 was not the dam did not exhibit limb defects. An alternative hypothesis is that the family sire (170) harbored a dominant TLD-causing allele that was only fully expressed in CPO offspring. And yet another possibility compatible with the data is that TLD is caused by a recessive allele that segregates independent of the CPO allele but, again, is fully expressed only in CPO offspring.
An explanation of our observations could also be that while CPO is indeed an autosomal recessive trait in this family, the limb defects were not an inherited trait, but instead resulted from insults to developing fetuses created in utero by the maternal genome. This model is fully consistent with the pedigree and is also suggested by the type of limb defects observed, notably variably expressed TLD. TLD are unique among LRD in that many are attributed to vascular disruption from placental or fetal blood clots during limb development, such as may occur in cases of maternal thrombophilia (Sadler and Rasmussen 2010; Ordal et al. 2016; Hunter 2000). This model lends itself to the observed maternal effect.
In summary, we describe a dog model for non-syndromic CPO. Backcross matings led to limb defects as a second phenotype in this family. While the two phenotypes may be related genetically, the current pedigree suggests that breeding strategies can be designed to minimize the occurrence of limb defects in these dogs. Thus, this line of dogs represents a well-characterized and highly reproducible large animal model for non-syndromic CPO. Finally, the pedigree suggests models of inheritance for the CPO and TLD phenotypes that provide a genetic framework to analyze future whole genome data.