we noted an autosomal recessive mode of inheritance in families a and c wherein the identified variants in popdc2 were inherited from each of the unaffected parents. while we expect a recessive inheritance, we could not assess the inheritance pattern in individuals 2 and 6 ( from family b, ii - 4; and d, ii - 1; figure 1 a ) as dna of ( one of ) the parents was not available. in total, we report five popdc2 variants ( refseq transcript: nm _ 001308333. 2; protein id: np _ 001295262. 1 ), of which two are missense variants ( c. 788g > a [ p. arg263his ], c. 787c > t [ p. arg263cys ] ), one in - frame insertion deletion ( c. 516 _ 527del [ p. gln172 _ tyr176delinshis ] ) and two are expected to result in protein truncation ( c. 563g > a [ p. trp188ter ], c. 110 _ 113del [ p. leu37serfster20 ], table s2 ). the maf of popdc2 variants identified in affected individuals ranged from 1. 4 × 10 to 1. 3 × 10 in the gnomad v4. 0 ( accessed march 2024; table s2 ). furthermore, none of the variants were found homozygous in 730, 947 exomes and 76, 215 genomes from gnomad, suggesting that homozygosity for these variants is not well tolerated. in line with this, all variants are predicted damaging by multiple in silico prediction tools ( table s2 ). intriguingly, in families b and c, the same residue is affected: p. of note, a homozygous variant ( c. 787g > a [ p. arg261gln ] ) in popdc3, which affects the paralogous residue of p. arg263 in popdc2 ( families b and c ), has been associated with muscular dystrophy without cardiac arrhythmia ( mim: 604577 ). in family c, the three affected siblings homozygous for p. arg263cys inherited the variant from the heterozygous and clinically unaffected parents ( ii - 1, ii - 2, and ii - 3 in figure 1 a ). we performed a look - up of the p. arg263cys variant in recent whole - genome sequencing dataset consisting of young affected individuals ( n = 226 ) receiving a pacemaker because of av block, and none of the individuals was homozygous for p. arg263cys, nor were there any individuals with bi - allelic variants in popdc2 ( note s2 ). in family d, we identified both p. trp188ter and p. leu37serfs 20 variants that are expected to result in lof due to premature truncation of the protein and / or nonsense - mediated decay ( individual ii - 1 in figure 1 a ). the cardiac arrest accompanied by sinus bradycardia and first - degree av block he displayed fitted the conduction disease seen in the other popdc2 - affected individuals. however, although the cardiac biopsy was negative for myoc arditis, a potential causal role of myoc arditis cannot be ruled out, as the disease can be focal and patchy, potentially reducing sensitivity due to sampling error. to explore the structural and functional consequences of the homozygous and compound heterozygous popdc2 variants, we generated a predicted structural model of popdc2, as no experimentally determined structures are currently available. swiss - model and alphafold multimer were used since these protein - structure - prediction programs could generate dimeric models of popdc2, and dimerization has been shown to be critical for the activity of popdc1. as a complementary approach, alphamissense was used to predict relative pathogenicity scores of the identified single - amino - acid substitutions and deleted regions. alphafold multimer generated a high - confidence dimer ( figure s2 a ) for full - length popdc2 with a dimeric transmembrane domain compose d of six transmembrane α helices ( three α helices from each subunit ) and two camp - binding popeye domains that also contained an extensive dimer interface ( figure 2 a ). in the alphafold model, arg263 ( altered in families b and c ) was pointing into the camp - binding pocket of the popeye domain from the adjacent subunit. swiss - model was the only program able to model a camp - bound state of the popeye domain ( figure s2 b ). to generate a model of the camp - bound state of full - length popdc2, we merged the alphafold multimer and swiss - model predictions by swapping the popeye domains ( see section material and methods ). this created a merged model of popdc2 that included the transmembrane domain and the popeye domains bound to camp ( figure 2 a ). based on the structural model, all variants ( p. gln172 _ tyr176delinshis, p. arg263his, p. arg263cys, and p. trp188ter / p. leu37serfs 20 ) are predicted to diminish the ability of popdc2 to bind camp. the first variant ( p. gln172 _ tyr176delinshis, family a ) replaces five residues with a single histidine residue. the deleted residues are predicted to form one beta strand that is part of a three - stranded beta sheet at the base of the camp - binding pocket ( figure 2 b ). replacement of the five residues with a single histidine residue would significantly alter the structural integrity of the camp - binding popeye domain and directly affect the ability of popdc2 to bind camp. the second and third variants, p. arg263his ( found in family b ) and p. arg263cys ( found in family c ), are also both predicted to interfere with proper camp binding, as the positively charged arg residue is predicted to be near the negatively charged cyclic phosphate group of camp ( figure 2 b ). change of the arg to either his or cys would eliminate a key interaction predicted to stabilize camp binding and thus reduce or eliminate the ability of popdc2 to bind camp. in support of arg263 being involved in a specific interaction to stabilize camp binding, alteration of arg263 to any other residue is predicted to be pathogenic by alphamissense, with pathogenicity scores of 0. 82 and 0. 79 for the p. arg263his and p. arg263cys variants, respectively. the compound heterozygous variants are also predicted to eliminate camp binding by popdc2 ( figure 2 c ). p. leu37serfster20 is a truncated protein that completely lacks the camp - binding popeye domain and would also generate an incomplete transmembrane domain. p. trp188ter would retain the dimeric transmembrane domain, but truncate the popeye domain, thus leaving an incomplete domain incapable of binding to camp ( figure 2 c ). in addition, both variants could be subject to nonsense - mediated decay, which would result in no protein product being made. notably, residues with high pathogenicity scores ( > 0. 5 ) all cluster in the regions of the popdc2 structure that are involved in either dimerization or camp binding ( figures 2 d and 2e ), which emphasizes the importance of camp binding and dimerization for popdc2 function. consistently, other variants within popdc2, found homozygously in gnomad, and without a clear disease association ( p. arg17val, p. val29ile, p. arg98cys, p. val270ile, and p. ala321thr ), are all positioned away from the camp - binding pocket and are predicted to be benign by alphamissense with low pathogenicity scores < 0. 1 ( figures 2 e and 2f ). taken together, the disease-associated variants reported here are primarily located in regions of the popdc2 structure that are critical for protein function. we then aimed to functionally characterize the popdc2 variants. trek - 1 is a recognized interacting protein of popdc2, and co - expression of popdc2 and trek - 1 has been shown to increase trek - 1 current in comparison to expression of trek - 1 alone. furthermore, cardiac - specific trek - 1 - deficient mice display a sinus node phenotype characterized by bradycardia with frequent episodes of sinus pauses, partially resembling the phenotype in the affected individuals with bi - allelic popdc2 variants presented here. we therefore hypothesized that the effect of the p. gln172 _ tyr176delinshis ( family a ) and p. arg263his ( family b ) variants is mediated through modulation of the trek - 1 current. we did not test the variants found in family c ( p. arg263cys ) and family d ( p. trp188ter / p.