From the 15 ACM-related genes, none was present in the gene set of Fam1, while TTN was in the gene set of Fam2 (Table 2). Specifically, the affected male patient Fam2.II.1 and his healthy father Fam2.I.1 harbored three distinct heterozygous missense variants in TTN. The two rare variants ENSP00000434586.1:p.Gln24857His (isoform N2B) and ENSP00000434586.1:p.Arg23483His (isoform N2B) were predicted deleterious by PROVEAN and LR.PFS3. Both variants were confirmed by Sanger sequencing (see Additional file 1). The third TTN variant ENSP00000434586.1:p.Ile3716Val (isoform N2B) is common, not conserved, and was predicted neutral by PROVEAN and LR.PFS3. TTN encodes for titin, the largest human protein, which has over 300 highly repetitive independently folding domains, including 152 immunoglobulin like, 132 fibronectine 3 (Fn3), 19 Kelch, 14 tetratricopeptide repeat, and 15 solenoid domains . The variant Arg23483His is located in the 125th of the 132 Fn3 domains (PF00041), while Gln24857His is located inside titin’s only serine kinase domain (PF00069), a structurally conserved protein domain that plays an important role in the regulation of cell proliferation, apoptosis and cell differentiation. The location of this variant and other functional residues in the protein structure of the kinase domain is visualized in Fig. 3. The mutated residue Gln24857His is located opposite of the active site and results in a charge and polarity change. The variant was further predicted to destabilize the protein structure by MAESTROweb (ΔΔG = 1.433, confidence = 0.8). In addition, the wild type residue glutamine was highly conserved in a multiple sequence alignment of homologous sequences from 63 species computed by ConSurf.Table 2Fam1 and Fam2 genes and corresponding variants that overlap with the ACM genes or the PKP2-related genesFamilyGene seta Gene rankb Gene nameHGVSpVariant consequencePROVEAN predictionc Population frequencyd Genotypee CommentFam2ACM–TTNENSP00000434586.1:p.Gln24857Hismissensedeleteriousrarehet in Fam2.II.1 and Fam2.I.1Mutations in TTN can cause ACM .Fam2ACM–TTNENSP00000434586.1:p.Arg23483Hismissensedeleteriousrarehet in Fam2.II.1 and Fam2.I.1Mutations in TTN can cause ACM .Fam2ACM–TTNENSP00000434586.1:p.Ile3716Valmissenseneutralcommonhet in Fam2.II.1 and Fam2.I.1Mutations in TTN can cause ACM .Fam1PKP2 (GO: neg. Reg. cell prolif.)1DAG1ENSP00000312435.2:p.Leu86Phemissenseneutralrarehet in Fam1.III.2, Fam1.III.3, Fam1.II.1β-dystroglycan binds to Hippo pathway effector Yap to inhibit cardiomyocyte proliferation in mice .Fam1PKP2 (GO: heart developm.)2TCF25ENSP00000263347.7:p.Ser390Phemissenseneutralrarehet in Fam1.III.2, Fam1.III.3, Fam1.II.1Negatively regulates SRF, whose increased expression causes cardiomyopathy in mice .Fam1PKP2 (GO: neg. Reg. cell prolif.)3DAB2IPENSP00000259371.2:p.Asp10Glymissenseneutralrarehet in Fam1.III.2, Fam1.III.3, Fam1.II.1One variant in DAB2IP has been associated with coronary heart disease .Fam1PKP2 (GO: neg. Reg. cell prolif.)4CTBP2ENSP00000357816.5:p.Gly70Argmissenseneutralcommonhet in Fam1.III.2, Fam1.III.3, Fam1.II.1Ctbp2-null mice have defective heart morphogenesis. CTBP2 may be a regulator of Wnt-mediated gene expression .Fam2PKP2 (GO: neg. Reg. cell prolif.)1IRF1ENSP00000384406.1:p.Asn259Sermissensedeleteriousrarehet in Fam2.II.1 and Fam2.I.1IRF1 is associated with cancer and a negative regulator of coronary artery smooth muscle cells  (OMIM *147575).Fam2PKP2 (GO: reg. of bicell. Tight. junction assembly)2MYO1CENSP00000412197.2:p.Gln766Lysmissenseneutralrarehet in Fam2.II.1 and Fam2.I.1OMIM *606538Fam2PKP2 (GO: cardiac muscle cell action pot.)3DMDENSP00000367948.2:p.Arg2151Trpmissenseneutralcommonhemi in Fam2.II.1, Fam2.I.1; het in Fam2.I.2Recessive mutations in DMD can cause muscle dystrophy (OMIM *300377).Fam2PKP2 (GO: heart development)4MKKSENSP00000382008.2:p.Ile339Valmissenseneutralrarehet in Fam2.I.2 and Fam2.II.2Recessive mutations in MKKS can cause Bardet-Biedl syndrome (OMIM *604896).Fam2PKP2 (GO: neg. Reg. cell prolif.)5NOTCH2ENSP00000256646.2:p.Asp1327Glymissenseneutralcommonhet in Fam2.I.1 and Fam2.II.1This variant has been reported causal for Congenital heart disease as compound heterozygote with L2408H, which is absent in Fam2 .Fam2PKP2 (GO: heart development)6PKD1ENSP00000456672.1:p.Arg198Trpmissenseneutralrarehet in Fam2.II.1 and Fam2.I.1Dominant mutations have been associated with polycystic kidney disease (OMIM *601313).Fam2PKP2 (PPI / GO: pos. Reg. sodium ion)7SCN5AENSP00000398962.2:p.His558Argmissenseneutralcommonhet in Fam2.I.1, Fam2.I.2, Fam2.II.2, Fam1.II.3This variant has been reported causal for isolated conduction disease as compound heterozygote with T215I, which is absent in Fam2 .Fam2PKP2 (GO: neg. Reg. cell prolif.)8MYOCDENSP00000341835.4:p.Gln304delinframe deletionneutralcommonhet in Fam2.II.1 and Fam2.I.1Cardiac muscle-specific transcriptional coactivator of serum response factor. Mutations have been associated with hypertrophic cardiomypathy (OMIM *606127).Fam2PKP2 (PPI)9DSC1ENSP00000257198.5:p.Cys848Phemissenseneutralcommonhet in Fam2.II.1 and Fam2.I.1Desmosomal protein desmocolin 1 (*OMIM 125643).Fam2PKP2 (PPI)10DROSHAENSP00000339845.3:p.Ser321Leumissenseneutralcommonhom in Fam2.I.2, Fam2.II.2; het in all others except Fam1.II.2Ribonuclease III. Mutations have been associated with cancer (OMIM *608828). a ACM: known ACM genes; PKP2: PKP2 related genes b PKP2-related genes are listed according to their rank from top to bottom cdeleterious: PROVEAN score < −2.5; neutral: PROVEAN score > = 2.5 dcommon: ExAC AF > = 0.01; rare: ExAC AF < 0.01 or NA; ExAC: Exome Aggregation Consortium ehet: heterozygous; hom: homozygous; hemi: hemizygous. If an individual is not listed, his/her genotype is homozygous reference Fig. 3Structure of the titin kinase domain (PDB 1tki chain A). Functional residues are represented as ball and stick, D24874 is the catalytic aspartate. The ATP binding site includes residue K24783 as well as the nearby yellow loop. The calcium/calmodulin binding helix is colored blue, the helix in orange blocks the ATP binding site in this autoinhibited conformation. Residue Q24857 is solvent exposed on the side opposite to the functional residues