A novel mutation in the HCN4 gene causes symptomatic sinus bradycardia in Moroccan Jews

Abstract

Objectives: to conduct a clinical, genetic, and functional analysis of 3 unrelated families with familial sinus bradycardia (FSB).

Background: mutations in the hyperpolarization-activated nucleotide-gated channel (HCN4) are known to be associated with FSB.

Methods and results: three males of Moroccan Jewish descent were hospitalized: 1 survived an out-of-hospital cardiac arrest and 2 presented with weakness and presyncopal events. All 3 had significant sinus bradycardia, also found in other first-degree relatives, with a segregation suggesting autosomal-dominant inheritance. All had normal response to exercise and normal heart structure. Sequencing of the HCN4 gene in all patients revealed a C to T transition at nucleotide position 1,454, which resulted in an alanine to valine change (A485V) in the ion channel pore found in most of their bradycardiac relatives, but not in 150 controls. Functional expression of the mutated ion channel in Xenopus oocytes and in human embryonic kidney 293 cells revealed profoundly reduced function and synthesis of the mutant channel compared to wild-type.

Conclusions: we describe a new mutation in the HCN4 gene causing symptomatic FSB in 3 unrelated individuals of similar ethnic background that may indicate unexplained FSB in this ethnic group. This profound functional defect is consistent with the symptomatic phenotype.

Figure 1. Family members

A–C. Family trees suggesting autosomal-dominant inheritance. Solid symbols represent affected family members. Open symbols indicate family members not carrying the mutant gene. Gray symbols represent patients whose clinical and genetic status is unknown. (Patients A-III-11, H-II-4, V-I-2, V-II-4, and V-II-5 were considered affected based only on a history of bradycardia without genetic screening due to lack of cooperation. Patients A-I-2 and A-I-6 died at 28y from unknown causes). D. This chart compares age with heart rate of carrier- and noncarrier family members. It demonstrates clear separation of two groups: affected and nonaffected family members, regarding minimal and mean heart rate on Holter recordings, independently of patient age. Low heart rate segregates near completely with positive genotype. Two exceptions are discussed in the text. Family member A-II-9 was not included in the graph due to technically unreadable Holter recordings. She was asymptomatic and was found to be a noncarrier.

Figure 2. The A485V mutation

A. Top: In this patient a C to T heterozygote change resulted in an alanine to valine amino acid change in position 485. Bottom: Control showing normal sequence. B. Multiple alignment of the A485V mutation region, showing the conserved alanine in position 485 in 3 different species (human, rat and mouse). The multiple alignment was done using the ClustalW software.

Figure 3. Results in Xenopus oocytes

Expression of HCN4 wild-type, HCN4 A485V homotetramer and HCN4 WT/A485V heterotetramer in Xenopus oocytes. A. Currents recorded from oocytes injected with RNAs of HCN4 WT, A485V, or WT and A485V together. The extracellular solution contained 24 mM K⁺. B. Enlargement of traces of WT tail currents shown in A, recorded during the voltage step to 30 mV. Right, the voltage protocol. Holding potential was −40 mV and currents were elicited by 5-s hyperpolarizing voltage steps between −30 and −130 mV in 10-mV increments, followed by a 3-s step to 30 mV. C. I–V relationship of WT (full circles, n=4), A485V (empty circles, n=4) and HCN4 WT/A485V (full reverse triangles, n=4) channels, recorded at the end of the voltage steps between −30 and −130 mV. D. Reversal potential measurement of the HCN4 currents of WT (full circles, n=4) and A485V (empty circles, n=3). The measurement shows tail current amplitude at the indicated voltages after a 5-second pre-pulse to −130 mV. (*P<0.05, same results obtained in two additional experiments).

Figure 4. Comparison between the two mutations in the pore region

Comparison between HCN4 channels with A485V and G480R point mutations expressed in Xenopus oocytes. The currents of the HCN4 WT and homotetramer mutants were recorded in two separate experiments. The graph compares the currents of WT HCN4 (black, n=4) and A485V homozygote (red, n=4) to the currents of WT HCN4 (green, n=5) and G480R homozygote (yellow, n=4) at −80 mV, as well as at −120 mV. At −120 mV, the current of the homozygote mutants relative to the WT at the same experiment is 2.5 % and 20 % for A485V and G480R, respectively. (* P<0.05, **P<0.01,***P<0.001).

Figure 5. Results in HEK273T cells

Expression of HCN4 WT and HCN4 A485V homotetramers in HEK293T cells. A, Western blot of total protein using anti-HCN4 (top) or anti-Calnexin (bottom) antibodies in WT HCN4 transfected HEK293T cells, A485V HCN4 transfected cells and pCDNA3 transfected cells. Blot exposure time was 1 min. The area of each band was estimated using the Scion software, and was estimated as following: 11356 and 5716 for total HCN4 WT (*) and total A485V HCN4 (**), respectively. B, Currents recorded from cells transfected with DNA of HCN4 WT (n=4) or A485V (n=5). The extracellular solution contained 54 mM Na⁺ and 90 mM K⁺. Right, voltage protocol. Holding potential was −10 mV and currents were elicited by 5-s hyperpolarizing voltage steps between 0 and −140 mV in 20-mV increments.