## 4 Discussion
This was reflected in the low imputation R\({}^{2}\) values (<0.5) for the two unconfirmed variants, which indicates that very low confidence was achieved in their imputation. The fact that subsequent Sanger sequencing identified additional carriers, initially imputed as non-carriers, and that two out of four SNPs were not confirmed, demonstrates that this approach may recover missed carrier samples of poorly imputed variants and is fundamental to confirm the presence of SNPs in the studied cohort when the imputation quality is not very reliable. The two Sanger-confirmed SNPs have never been reported to be associated with AF, not even in recent studies using very large AF sample sizes with high statistical power [33; 34; 35]. Their identification in the CHRIS cohort may have been facilitated, as these two SNPs are both enriched in a family where they segregate with the AF phenotype. Specifically, in this family, three out of five individuals carrying both SNPs are AF cases. Two of them are men with a reported medical AF diagnosis, and one is an elderly woman showing an absence of P wave and taking specific cardiac drugs. On the other hand, the two carriers without AF signs are two young women. However, we cannot exclude that these two young individuals may eventually develop AF later. Interestingly, the two significant SNPs are respectively close or within the probable candidate genes, _PBX1_ and _PCCA_, both of which are expressed in atria. We disregarded the _LMXIA_ gene since its function is mostly related to the development of dopamine-producing neurons during embryogenesis. _PBX1_ acts as a transcription factor and has an important role during cardiac development, specifically regulating distinct transcriptional pathways to control great-artery patterning and cardiac outflow tract septation [36]. Mutations in this gene have recently been associated with CAKUTHED (congenital anomalies of kidney and urinary tract with or without hearing loss, abnormal ears, or developmental delay) syndrome, characterized by multiple congenital defects including congenital heart disease [37]. The involvement of other genes with a specific function in the cardiac development and associated with AF was previously reported. For example, in a recent study, which has identified 151 genes associated with AF [35], pathway and functional enrichment analyses have highlighted processes related to cardiac development as being functionally relevant in AF pathogenesis. Of note, in a genome-wide whole blood transcriptomic analysis from the Framingham Heart Study, the expression of _PBX1_ gene was found to be up regulated in AF patients [38]. The _PCCA_ gene encodes the mitochondrial propionyl-CoA carboxylase, and mutations in this gene lead to an enzyme deficiency resulting in propionic acidemia, often associated with long QT syndrome [39]. It is interesting to highlight that long-QT syndrome has also been associated with AF [40,41], apparently by inducing early afterdepolarization-mediated ectopic activity [42]. Moreover, cardiac dysfunction in Pcca-/-(A138T) mice was associated with lower systolic Ca\({}^{2+}\) release, impairment in the sarcoplasmic reticulum Ca\({}^{2+}\) load, and decreased Ca\({}^{2+}\) re-uptake by SR-Ca\({}^{2+}\) ATPase (SERCA2a) [43]. Notably, it has been reported that Ca\({}^{2+}\)-handling dysregulation can promote beat-to-beat alternation in action potential duration, favoring reentry and causing AF [44]. Indeed, Ca\({}^{2+}\) handling abnormalities are a common finding in atrial cardiomyocytes of AF patients [45, 46, 47]. These findings indicate that _PBX1_ and _PCCA_ could be considered very good candidate genes for AF. In the last decade, numerous studies have been performed to better understand the genetic determinants of AF, but there remains scant literature on the role of altered metabolic pathways in AF.