Assessment of paralogue annotation for improving diagnostic accuracy in CALM1, CALM2, and CALM3 genes

The calcium (Ca²⁺) sensor calmodulin (CaM) genes CALM1, CALM2, and CALM3 were recently included in the American College Medical Genetics and Genomics (ACMG) secondary findings (SF) list, given their significance in causing long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). These three genes share identical protein sequences, posing potential challenges in variant interpretation. Using paralogue annotation (PA) to classify pathogenic variants and variants of uncertain significance (VUS) across these three paralogue genes, we performed a systematic, semi-automated curation of the CALM1, CALM2, and CALM3 variants. The analysis identified 173 unique CALM variants from ClinVar and Mastermind databases (75 CALM1, 59 CALM2, and 39 CALM3 variants). After paralogue annotation, we identified 126 unique variants in each of the three genes-378 cDNA variants in total. Out of 126 unique variants for each CALM gene, 63 were VUS, 62 were likely pathogenic/pathogenic (LP/P), and one was conflicting (192 VUS, 186 P/LP, and 3 C in total). Twelve unique variants in the CALM1, CALM2, or CALM3 genes had conflicting classifications between VUS and LP/P calls, which were resolved as LP/P. The application of paralogue annotation and variant curation resulted in an increased number of likely pathogenic/pathogenic variants (111% increase). Additionally, our analysis confirms that the majority of known pathogenic variants are predominantly located within the C-lobe of the CaM protein. This study highlights the benefits of paralogue annotation for accurate variant interpretation in CALM1, CALM2, and CALM3 genes, suggesting that a reduction in missed diagnoses is associated with calmodulinopathies.