loss of popdc2 in mice results in sinus pauses and bradycardia, and morpholino - mediated knockdown of popdc2 in zebrafish results in atrioventricular ( av ) block. we identified bi - allelic variants in popdc2 in four families with a phenotypic spectrum consisting of sinus node dysfunction, av conduction defects, and hypertrophic cardiomyopathy. using homology modeling, we show that the identified variants are predicted to diminish the ability of popdc2 to bind camp. in in vitro electrophysiological studies, we demonstrated that, in contrast with wild - type popdc2, variants found in affected individuals failed to increase trek - 1 current density. while muscle biopsy of an affected individual did not show clear myopathic disease, it showed significantly reduced abundance of both popdc1 and popdc2, suggesting that stability and / or membrane trafficking of the popdc1 - popdc2 complex is impaired by pathogenic variants in either protein. single - cell rna sequencing from human hearts demonstrated that co - expression of popdc1 and popdc2 was most prevalent in av node, av node pacemaker, and av bundle cells. using population - level genetic data of more than 1 million individuals, we show that none of the familial variants were associated with clinical outcomes in heterozygous state, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow - up. our findings provide evidence for bi - allelic variants in popdc2 causing a mendelian autosomal recessive cardiac syndrome. the rhythmic contraction of the heart is orchestrated by the cardiac pacemaker and conduction system. electrical activity in the heart arises in the sinus node, located in the right atrium near the entrance of the superior vena cava. the electrical impulse then spreads through the atria to the atrioventricular ( av ) node and is subsequently propagated through the bundle of his and bundle branches to the purkinje fibers from where it spreads throughout the ventricles. cardiac conduction defects ( ccds; mim: 115080 ) are primarily the consequence of age - related degeneration, structural heart disease, or post - operative complications. presentation of ccds in the young should raise suspicion of a genetic disor der. rare variants in genes encoding cardiac ion channels ( e. g., scn5a, mim: 600163; trpm4, mim: 606936; and hcn4, mim: 605206 ), transcription factors ( e. g., tbx5, mim: 601620; nkx2 - 5, mim: 600584 ), constituents of the inner nuclear membrane ( e. g., lmna, mim: 150330; emd, mim: 300384 ), gap junction proteins ( e. g., gjc1, mim: 608655 ), and others ( e. g., gnb5, mim: 604447; tnni3k, mim: 613932; prkag2, mim: 602743 ) have been implicated in inherited ccd presenting in isolation or in presence of other cardiac or extracardiac features. however, many affected individuals with early - onset ccd remain genetically unexplained. bi - allelic variants in popdc1 ( also known as bves, mim: 604577 ), encoding the popeye domain - containing protein 1, are associated with muscular dystrophy and av block ( mim: 604577 ). in mice, knockout of popdc1 or popdc2 resulted in stress - induced sinus pauses and sinus bradycardia. in zebrafish, morpholino knockdown of popdc1 or popdc2 resulted in second - degree av block and bradycardia. the twik - related potassium channel 1 ( trek - 1, encoded by kcnk2; mim: 603219 ) is an established interacting protein of popdc2 ( mim: 605823 ) and co - expression of popdc2 and trek - 1 has been shown to result in a 2 - fold higher trek - 1 current in comparison to expression of trek - 1 alone. here, we provide evidence for bi - allelic loss - of - function ( lof ) variants in popdc2 as the cause of an autosomal recessive syndrome in four families, consisting of a phenotypic spectrum including sinus node disease and av conduction defects with hypertrophic cardiomyopathy ( hcm; mim: 192600 ). family a was referred to the department of human genetics of the amsterdam umc ( amsterdam, the netherlands ) for genetic testing and counseling for ccd and hcm. to follow up on the findings from exome sequencing in this family, we studied 78 individuals that were diag nosed with a similar clinical presentation to family a ( i. e., ccds and hcm, cohorts 1 – 3 ). in addition, we studied 96 hcm individuals without ccds ( cohort 4 ). in all 174 individuals, genetic testing had ruled out causative variants in established arrhythmia and / or cardiomyopathy genes. families c and d were identified via a genetic and phenotypic match through decipher and genematcher, respectively. the study protocol was approved by the amsterdam university medical center research ethics committee and the local institutional review boards of contributing centers. signed informed consent was obtained from the affected individuals or their parents. details on case recruitment and dna - sequencing methods for each family can be found in the supplemental information and table s1. to ensure the privacy of the affected individuals and their families, ( 1 ) ages are presented as non - overlapping age ranges ( i. e., 0 – 5, 6 – 10, and 11 – 15 years ), ( 2 ) pedigrees were modified, ( 3 ) information related to ancestry / country or origin / nationality are not reported, and ( 4 ) clinical descriptions were minimized. the censoring undertaken for privacy reasons does not affect the ability to evaluate the presented data. sex was not considered as a biological variable. two homology models were generated using either swiss - model or alphafold2 multimer. the swiss - model web server used a cyclic amp ( camp ) - regulatory protein from yersinia pestis ( 6dt4 ) as a template to generate the homology model for the popeye domain of popdc2. a dimer of full - length popdc2 was generated using alphafold multimer, and the intrinsically disor dered c - terminal residues 275 – 364 were deleted to simplify figure presentation. a final homology model of popdc2 was created by replacing residues 128 – 213 of the alphafold multimer model with residues 128 – 213 of the swiss - camp model after superimposing the individual popeye domains. alphamissense was used to generate the predicted pathogenicity of single - amino - acid substitutions and deleted regions. alphamissense uses language modeling to understand amino - acid distributions based on sequence context, then it incorporates structural information using an alphafold - derived system to consider a protein ’ s three - dimensional form when assessing a variants ’ impact. it also utilizes weak labels from population frequency data to refine predictions without human biases. using these models, we evaluated the consequence of popdc2 variants found in families a – d and five variants that occurred homozygously in individuals from the genome aggregation database v2. 1. 1 ( gnomad ), which are not expected to cause disease ( table s2 ) source paper: pmc12256823 single-nucleus rna - sequencing ( snrna - seq ) data and visium spatial gene expression data were obtained from a previously published study. processed data of single - cell rna - sequencing ( scrna - seq ) / snrna - seq and visium data are available for browsing gene expression and download from the heart cell atlas ( https: / / www. heartcellatlas. org ). annotate d, log - normalized count matrices for both modalities were downloaded and specifically analyzed for expression of popdc1, - 2, and - 3 using scanpy package for python run in jupyter notebook. original histological annotation of tissue sections was used. the cell - state annotation was adapted from the original study. all atrial cardio myoc ytes were pooled in one category, and all ventricular cardio myoc ytes were pooled together. from the conduction system cells, sinoatrial node pacemaker ( san p ) cells and purkinje cells are shown separately; due to low cell numbers, atrioventricular node pacemaker ( avn p ) and bundle cells are shown together.