cardio myoc yte - rich structures showed higher popdc2 expression compared to cardio myoc yte - poor regions ( e. g., cardiac skeleton or fat ) ( figure 5 b ). we then interrogate d the co - expression pattern of popdc1 / bves and popdc2, as ( 1 ) swan et al. described the necessity of popdc1 - 2 co - expression for their trafficking to the cell membrane, which is required for their proper function; and ( 2 ) variants in popdc1 / bves - popdc2 have also been found to affect the stability and / or membrane trafficking of the complex as corroborated here in the muscle biopsy of the affected individual ii - 3 from family a ( figure 4 ). the most prominent area where popdc1 / bves and - 2 expression was seen co - localizing in the same visium spots was the av node ( figure 5 c ). the sinus node expressed popdc2, but expression of popdc1 was sparse ( figure 5 b ). to validate these findings and to explore popdc1 and - 2 co - expression within the same cell, we analyzed snrna - seq data from eight regions of adult human hearts, including the conduction system. popdc2 expression was almost entirely restricted to atrial and ventricular cardio myoc ytes compared to other cell types ( figure 5 d ). other genes from the popdc family ( i. e., popdc1 and popdc3 ) also showed a preferential expression in cardio myoc ytes, but their expression was less prevalent than popdc2. the snrna - seq object was then sub - set to cardio myoc ytes only and expression of popdc1 - 3 interrogate d. popdc2 was expressed in all cardio myoc yte cell states, while popdc1 showed the highest expression in the pacemaker cells ( p cells ) of the av node and the av bundle cells ( figure 5 e ). the co - expression of popdc1 and - 2 within the same single - cell was most prevalent in av node p cells and av bundle cells, which concurs with the spatial transcriptomic data ( figure 5 f ). we then also assessed the cardiac expression of popdc2 in mice utilizing previously published scrna - seq data of the sinus node and av node / his region, dissected from embryonic - day - 16. 5 mouse hearts. consistent with the phenotype observed in the affected individual, popdc2 was expressed in cardio myoc ytes as well as sinus node and av node / his - region cells ( figure s6 ), corroborating observations from previous studies in mice and our observations reported here in human hearts ( figures 5 a – 5f ). we investigated whether heterozygous carriers of the popdc2 variants found in families a – d ( i. e., p. gln172 _ tyr176delinshis, p. arg263his, p. arg263cys, p. trp188ter, and p. leu37serfs 20 ) showed ( sub - ) clinical manifestations of the recessive popdc2 syndrome in four large population biobanks ( total n = 1, 089, 031 ) with genetic data, namely decode genetics in iceland ( n = 173, 025 ), uk biobank ( n = 428, 503 ), copenhagen hospital biobank and the danish blood donor study in denmark ( n = 487, 356 ), and intermountain in utah, usa ( n = 138, 006 ). we searched their association with ( 1 ) av block, ( 2 ) bradycardia, ( 3 ) cardiac arrest, ( 4 ) hcm, ( 5 ) myoc arditis, ( 6 ) pacemaker implantation, ( 7 ) sinus node dysfunction, and ( 8 ) heart rate. we detected 426 heterozygous carriers of the five familial popdc2 variants ( p. gln172 _ tyr176delinshis, n = 62; p. arg263his, n = 34; p. arg263cys, n = 156; p. trp188ter, n = 162; and p. leu37serfs 20, n = 12 ) among 1, 089, 031 participants in the included biobanks but no homozygotes or compound heterozygotes. none of the variants showed statistical association with ( sub - ) clinical outcomes after bonferroni correction, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow - up ( table s6 ). we then conducted genetic burden analyses, wherein rare variants in popdc2 are aggregated, with the phenotypes above and muscular dystrophy as this is a clinical hallmark of recessive syndromes associated with the two other members of the popdc family ( i. e., popdc1 and popdc3 ). we found a significant association with bradycardia after bonferroni adjustment ( p < 1. 4 × 10 ) for the following variant sets: lofcadd ( p = 3. 3 × 10, odds ratio [ or ] 1. 6, lof and missense when predicted deleterious with cadd phred score ≥25 ) and lof1misid ( p = 2 × 10, or 1. 59, lof and missense when predicted deleterious by at least one of the following prediction methods: metasvm, metalr, or cadd phred score ≥25; table s7 ). although no association with a clinically relevant phenotype was detected, an association with slow heart rate was seen in heterozygous carriers in the general population. whether this has clinical relevance remains to be explored. we identified bi - allelic variants in popdc2 in four families that presented with a phenotypic spectrum consisting of sinus node dysfunction, av conduction defects, and hcm. using homology modeling, we show that the identified popdc2 variants are predicted to diminish the ability of popdc2 to bind camp. in in vitro electrophysiological studies, we demonstrated that, while co - expression of wt popdc2 with trek - 1 increased trek - 1 current density, popdc2 harboring variants found in the affected individuals failed to increase trek - 1 current density. scrna - seq from human hearts demonstrated that co - expression of popdc1 and - 2 was most prevalent in av node, av node pacemaker, and av bundle cells. sinus node cells expressed popdc2 abundantly, but expression of popdc1 was sparse. together, these results concur with predisposition to av node disease in humans with lof variants in popdc1 and popdc2 and presence of sinus node disease in popdc2 but not in popdc1 - related disease in human. our findings provide evidence for bi - allelic variants in popdc2 as the cause of a mendelian autosomal recessive cardiac syndrome. several observations support the causality of the identified popdc2 variants ( encoding p. gln172 _ tyr176delinshis, p. arg263his, p. arg263cys, p. trp188ter, and p. leu37serfs 20 ). ( 1 ) the cardiac arrhythmia phenotype of the affected individuals harboring these variants in the homozygous or compound heterozygous state is similar to observations of sinus pauses and bradycardia that were made in mice lacking popdc2 and to av block observed in zebrafish after morpholino knockdown of popdc2. ( 2 ) the variants affect highly conserved residues and are predicted damaging by multiple in silico prediction tools. ( 3 ) the variants are extremely rare in gnomad and were not found in homozygous state. ( 4 ) a change affecting the paralogous residue of arg263 ( families b and c ) in popdc3, p. arg261gln, has been associated with muscular dystrophy ( mim: 605824 ). the dimeric homology model of popdc2 predicted all disease variants we identified in the homozygous or compound heterozygous state to critically affect camp binding. one variant leads to the substitution of five residues with a single histidine residue ( p. gln172 _ tyr176delinshis; figure 2 b ), and another two ( p. trp188ter / p. leu37serfs 20; figure 2 c ) are expected to produce a truncated protein, all of which are expected to critically affect the ability of popdc2 to bind camp. in addition, both transcripts could be subject to nonsense - mediated decay, which would result in no protein product being made. while the p. arg263his and p. arg263cys variants are not predicted to disrup t protein folding, they are expected to eliminate a key interaction predicted to stabilize camp binding. based on these predictions, we hypothesize that camp binding is critical for popdc2 function and, when affected, causes disease. although heterozygous popdc2 variants have been proposed to increase susceptibility for cardiac conduction disease in humans by rinne et al., no mendelian recessive disor der has been linked to this gene so far.