Long QT syndrome-associated mutations in intrauterine fetal death

Abstract

Importance: Intrauterine fetal death or stillbirth occurs in approximately 1 out of every 160 pregnancies and accounts for 50% of all perinatal deaths. Postmortem evaluation fails to elucidate an underlying cause in many cases. Long QT syndrome (LQTS) may contribute to this problem.

Objective: To determine the spectrum and prevalence of mutations in the 3 most common LQTS susceptible genes (KCNQ1, KCNH2, and SCN5A) for a cohort of unexplained cases.

Design, setting, and patients: In this case series, retrospective postmortem genetic testing was conducted on a convenience sample of 91 unexplained intrauterine fetal deaths (mean [SD] estimated gestational age at fetal death, 26.3 [8.7] weeks) that were collected from 2006-2012 by the Mayo Clinic, Rochester, Minnesota, or the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. More than 1300 ostensibly healthy individuals served as controls. In addition, publicly available exome databases were assessed for the general population frequency of identified genetic variants.

Main outcomes and measures: Comprehensive mutational analyses of KCNQ1 (KV7.1, LQTS type 1), KCNH2 (HERG/KV11.1, LQTS type 2), and SCN5A (NaV1.5, LQTS type 3) were performed using denaturing high-performance liquid chromatography and direct DNA sequencing on genomic DNA extracted from decedent tissue. Functional analyses of novel mutations were performed using heterologous expression and patch-clamp recording.

Results: The 3 putative LQTS susceptibility missense mutations (KCNQ1, p.A283T; KCNQ1, p.R397W; and KCNH2 [1b], p.R25W), with a heterozygous frequency of less than 0.05% in more than 10 000 publicly available exomes and absent in more than 1000 ethnically similar control patients, were discovered in 3 intrauterine fetal deaths (3.3% [95% CI, 0.68%-9.3%]). Both KV7.1-A283T (16-week male) and KV7.1-R397W (16-week female) mutations were associated with marked KV7.1 loss-of-function consistent with in utero LQTS type 1, whereas the HERG1b-R25W mutation (33.2-week male) exhibited a loss of function consistent with in utero LQTS type 2. In addition, 5 intrauterine fetal deaths hosted SCN5A rare nonsynonymous genetic variants (p.T220I, p.R1193Q, involving 2 cases, and p.P2006A, involving 2 cases) that conferred in vitro electrophysiological characteristics consistent with potentially proarrhythmic phenotypes.

Conclusions and relevance: In this molecular genetic evaluation of 91 cases of intrauterine fetal death, missense mutations associated with LQTS susceptibility were discovered in 3 cases (3.3%) and overall, genetic variants leading to dysfunctional LQTS-associated ion channels in vitro were discovered in 8 cases (8.8%). These preliminary findings may provide insights into mechanisms of some cases of stillbirth.

Figure 1. KCNH2 Isoforms and Molecular Position of the HERG1b R25W Mutation

HERG1a and HERG1b are 2 isoforms encoded by KCNH2 alternatively spliced transcripts (KCNH2[1a] and KCNH2[1b]). The shaded regions of the exons represent the amino acid coding region of the gene that is initiated by the ATG start codon. Full-length KCNH2 contains 15 exons. The alternatively spliced gene transcript KCNH2 has an alternate exon 1 (labeled 1b). KCNH2(1b) does not include the first 5 exons of the full-length transcript but includes identical exons 6 through 15 that are present in the full-length transcript (KCNH2). The 2 isoforms differ only by their N-termini; HERG1b has a 56 amino acid residue N-terminus in which the first 36 residues have a unique sequence (single letter amino acid abbreviations), whereas HERG1a has a longer (396 residue) N-terminus, with the rest of the protein identical in both splice isoforms. The DNA sequence chromatogram illustrates the heterozygous c.73 C>T nucleotide substitution that results in the substitution of an arginine (R) for a tryptophan (W) at amino acid residue 25 encoded by alternate exon 1b. CNBD indicates cyclic nucleotide-binding domain; PAC, PAS-associated C-terminal; and PAS, Per-ARNT-Sim.

Figure 2. KCNQ1 Genetic Variants and Molecular Position of the K_V7.1 Mutations A283T and R397W

Depicted are the novel p.A283T mutation, located between the S5 transmembrane spanning domain and the pore region (between S5 and S6 of the channel), and the mutation p.R397W, located in the C-terminal region following S6 of the protein. The DNA sequence chromatograms indicate the nucleotide changes corresponding to each mutation (c.847 G>A, p.A283T; c.1189 C>T, p.R397W). In the case of c.847 G>A, both black (G) and green (A) peaks are present at the same position indicating heterozygosity at nucleotide position 847, which predicts substitution of alanine (A) for threonine (T) at amino acid position 283 in the K_V7.1 protein. The c.1189 C>T mutation (superimposed blue and red peaks) predicts substitution of arginine (R) for tryptophan (W) at amino acid position 397 in K_V7.1

Figure 3. K_V7.1-A283T and K_V7.1-R397W Mutations Decrease Current at Positive Membrane Potentials

A, Whole-cell recordings from HEK293 cells transfected with K_V7.1-wild-type, K_V7.1-A283T, or K_V7.1-R397W are illustrated. Macroscopic currents (I_Ks) were recorded at room temperature using the following voltage-clamp protocol: 5-second depolarizations from −80-mV to 70-mV in 10-mV increments (to elicit step currents) followed by a 5-second repolarization to −50 mV (to elicit tail currents). Peak step and tail I_Ks were measured at the points indicated by the arrows. Horizontal time scale and vertical current scale in picoamperes (pA) bars applicable to all 3 families of current traces are illustrated. The vertical line indicates the zero current baseline. The mean peak step or the tail peak current I_Ks density in picoampres per picofarad (pA/pF) is plotted as a function of the step potential for cells expressing K_V7.1-wild type (n = 7), K_V7.1-A283T (n = 10), and K_V7.1-R397W (n = 11) as shown in panels B and C. The individual peak tail I-V relations were described with a Boltzmann equation (dark gray line overlaying the data points, Figure 2C) to calculate the maximally activating current (I_MAX), the midpoint potential of half maximal activation (V½), and slope factor (k) error bars indicate 95% confidence intervals. ^aP < .05 vs cells expressing K_V7.1-A283T. ^bP <.05 vs cells expressing K_V7.1-R397W.

Figure 4. Electrophysiological Properties of Wild-Type and Mutant HERG Channels

A, Whole cell recordings from Chinese hamster ovary cells cotransfected with wild-type HERG1a-DsRed and either wild-type HERG1b-GFP (HERG1a + HERG1b) or mutant HERG1b-R25W-GFP (HERG1a + HERG1b-R25W) are illustrated. Currents were recorded at room temperature using the following voltage-clamp protocol: 2-second depolarizations from −80-mV to 70-mV in 10-mV increments (to elicit step currents) followed by a 2-second repolarization to −50 mV (to elicit tail currents). Peak step and tail currents were measured at the points indicated by the arrows (pA, picoampere; pF, picofarad). Horizontal time scale and vertical current density scale bars applicable to both current traces are illustrated for the upper trace. B, Mean peak step current density in pA/pF plotted as a function of voltage for HERG1a + HERG1b (n = 16) and HERG1a + HERG1b-R25W (n = 16; P <.05 vs cells expressing HERG1a + HERG1b. C, Mean peak tail current density in pA/pF plotted as a function of voltage for HERG1a + HERG1b (n = 16) and HERG1a + HERG1b-R25W (n = 16; P <.05 vs cells expressing HERG1a + HERG1b). Error bars indicate 95% confidence intervals.