Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance

Abstract

Sinoatrial node dysfunction and deafness (SANDD) syndrome is rare and characterized by a low heart beat and severe-to-profound deafness. Additional features include fatigue, dizziness, and episodic syncope. The sinoatrial node (SAN) drives heart automaticity and continuously regulates heart rate. The CACNA1D gene encoding the Ca_v1.3 protein expressed in inner hair cells, atria and SAN, induces loss-of-function in channel activity and underlies SANDD. To date, only one variant c.1208_1209insGGG:p.(G403_V404insG) has been reported for SANDD syndrome. We studied five Pakistani families with SANDD and characterized a new missense variant p.(A376V) in CACNA1D in one family, and further characterized the founder variant p.(G403_V404insG) in four additional pedigrees. We show that affected individuals in the four families which segregate p.(G403_V404insG) share a 1.03 MB haplotype on 3p21.1 suggesting they share a common distant ancestor. In conclusion, we identified new and known variants in CACNA1D in five Pakistani families with SANDD. This study is of clinical importance as the CACNA1D founder variant is only observed in families from the Khyber Pakhtunkhwa (KPK) province, in Pakistan. Therefore, screening patients with congenital deafness for SAN dysfunction in this province could ensure adequate follow-up and prevent cardiac failure associated with SAN.

Fig. 1

Pedigree drawings for the families included in this study. a DEM4730, b DEM4713, c DEM4690, d DEM4591, and e DEM4318. Squares represent males and circles represents females; filled symbols represent individuals with SANDD and clear symbols represent the unaffected family members. Double lines show consanguineous marriages. Below each family member of DEM4730 for which a DNA sample is available, their CACNA1D c.1127 C > T genotype is shown. For DEM4713, DEM4690, DEM4591, and DEM4318, the CACNA1D c.1208_1209insGGG genotype is shown for family members from whom a DNA sample is available

Fig. 2

Clinical presentation of affected individual IV:2 from family DEM4730 with SANDD syndrome. a ECG recordings from a control (60 bpm) and b affected family member (IV:2 from DEM4730) with SANDD syndrome (48bpm) c audiogram for the affected family member IV:2 from DEM4730. Pure-tone audiometry was performed between 250 and 8000 Hz and x represents the results for the left ear and o for the right ear

Fig. 3

Overview of CACNA1D protein domains and sequence/protein data on variant c.1127 C > T; p.(A376V) a Schematic presentation of the predicted transmembrane helices in CACNA1D (NP_000711.1) (adapted from the results of TMHMM2.0) and depiction of amino acid positions of the new missense variant p.(A376V) in S5–S6 linker of D1 and previously reported variant p.(G403_V404insG), located in the S6 helix of Cav1.3. D1–D4 indicates the four homologous domains of α1 subunit. The box indicates the variant found in this study for SANDD syndrome. The dotted line indicates the location of Trp372, which lies in the S5 helix, and forms a hydrophobic interaction with Val376 in the mutated protein (as depicted by 3D modeling). b Sanger sequencing electrograms and segregation of CACNA1D variant c.1127 C > T; p.(A376V) in family DEM4730. c Predicted three-dimensional structure of the CACNA1D protein. The extracellular domain is colored in cyan and the Ala376 residue is shown by a stick model. d Superimposed structure of template (red) and homology model of CACNA1D (cyan). e Close-up view of the structure showing the wild type f and mutant protein. g Comparison of LTCC paralog α1 subunit isoforms by Clustal omega (Ensemble ids ENSP00000288139, ENSP00000355192, ENSP00000365441, ENSP00000382563 for Cav1.3, Cav1.1, Cav1.4, and Cav1.2 respectively) (upper panel). Sequence alignment of amino acids across CACNA1D orthologs in various species by Homologene (lower panel), showing that the alanine 376 residue (in green) is conserved in paralogs and across species

Fig. 4

Haplotype analysis of affected individuals from families with SANDD syndrome that segregate the CACNA1D c.1208_1209insGGG variant. Affected individuals in four families share the same haplotype associated with the c.1208_1209insGGG founder variant (a 1.03 Mb region between 53.32 and 54.35 Mb)