Abstract
Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis1,2,3,4. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K+ channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H+ ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.
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References
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Acknowledgements
We are grateful to the patients and their families who contributed to this study. We thank I. Jantke, S. Cecchetti and S. Venanzi for skillful technical assistance, T. Kock for site-directed mutagenesis, A. Hasse for CHO cell transfection and injection, R. Bähring, J.M. Schröder and E. Neumann for help with the oocyte experiments, P. Meinecke for discussing clinical phenotypes and A. Podolska for help with ATP6V1B2 sequencing. G.B., L.S. and M.T. acknowledge CINECA for computational resources (whole-exome sequencing data and structural analyses). The KCNH1/heag1 clone was kindly provided by S.H. Heinemann (Friedrich Schiller University Jena). This work was supported by grants from the Deutsche Forschungsgemeinschaft (KO 4576/1-1 to F.K. and KU 1240/5-1 to K.K.), Istituto Superiore di Sanità (Ricerca Corrente 2013 to M.T.), Ministero della Salute (Ricerca Finalizzata RF-2010-2312766 to B.D.) and Ospedale Pediatrico Bambino Gesù (Gene-Rare to B.D.).
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Contributions
F.K. performed whole-exome sequencing data analysis and validation, molecular screening and genotyping and wrote the manuscript. V.C. performed whole-exome sequencing data analysis and validation and wrote the manuscript. C.K.B. contributed the electrophysiological studies and wrote the manuscript. L.S. and G.B. performed the homology modeling and structural analysis. A.C. and M.A. contributed to the whole-exome sequencing data processing and analysis. E.F., S.P., M.L.D. and T.T.M.N. carried out the molecular screening and/or genotyping. P.G., G.C.K., V.L., D.M., L.D.V.N., P.T., S.M.W., B.D. and A.P. recruited and clinically characterized the study subjects and collected the biological samples. P.M.C. performed whole-exome sequencing data analysis, validation and genotyping. M.T. and K.K. conceived the project, analyzed and interpreted the data, and wrote the manuscript. All authors contributed to the final manuscript.
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Integrated supplementary information
Supplementary Figure 1 Sequence electropherograms of individuals with KCNH1 or ATP6V1B2 mutation.
Sequence electropherograms showing the de novo origin of the identified KCNH1 and ATP6V1B2 missense mutations in subjects 1–8 (upper and lower panels, indicated by red arrows). The heterozygous state of three mutations was documented in peripheral leukocytes, hair bulb and/or buccal cells of subjects 4, 5 and 7, indicating germline origin. An additional previously annotated (ExAC database) heterozygous KCNH1 variant, c.125T>C (p.Ile42Thr), was present in subject 5 and his healthy mother (indicated by blue arrows). By cloning the KCNH1 exon 7–containing amplicon of subject 2 followed by sequencing, we determined the haplotypes and found that the two identified de novo changes c.974C>A and c.1066G>C are in cis (wild-type allele and mutated KCNH1 allele in the middle panel; mutated nucleotides are framed).
Supplementary Figure 2 Multiple protein sequence alignments around the KCNH1 and ATP6V1B2 amino acid substitutions from different species.
Alignment of the regions flanking the detected missense variants in orthologous proteins, showing the evolutionary conservation of amino acids S325, G348, L352, V356, I467 and G469 in human KCNH1 (NP_002229.1) and of R485 in human ATP6V1B2 (NP_001684.2). Multiple alignments were gathered from http://www.ncbi.nlm.nih.gov/homologene/. Conserved residues have a red background, and non-conserved residues have a gray background. Amino acid sequence alignments demonstrate high (S325 and V356 in human KCNH1) or complete (G348, L352, I467 and G469 in human KCNH1 and R485 in human ATP6V1B2) evolutionary conservation of the altered residues.
Supplementary Figure 3 Voltage dependence of wild-type and mutant KCNH1 channel activation.
(a) KCNH1 channels were expressed in CHO cells, and families of wild-type (WT) and L352V current traces recorded with the depicted pulse protocols are shown. Zero current is indicated by dashed lines and arrowheads. (b) Mean (±s.e.m.) normalized instantaneous tail current amplitudes as a function of the preceding test pulse potential. Lines represent first-order Boltzmann functions fitted to the data points. The voltage dependence of channel activation was analyzed from instantaneous tail current measurements at +40 mV (for KCNH1 WT and I467V for all experiments and for G348R and S325Y/V356L for four experiments each) or at –20 mV (for L352V, G348R and S325Y/V356L). No significant differences were found between the potentials for half-maximal G348R or S325Y/V356L channel activation determined with the two different constant pulse potentials. (c) Table with means ± s.e.m. of the potential of half-maximal channel activation and the slope factor k derived from fits of first-order Boltzmann functions to the normalized instantaneous tail current amplitudes of the individual experiments. n, number of experiments; *, significantly different from WT with P < 0.05; ***, significantly different from WT with P < 0.001. Values were tested for significant differences compared to WT data with one-way ANOVA and post-hoc Bonferroni t test.
Supplementary Figure 4 Analysis of the activation and deactivation kinetics of wild-type and mutant KCNH1 channels expressed in CHO cells.
(a) The time course of channel activation was analyzed at +40 mV from experiments as shown in Figure 4 by fitting a double-exponential function to the current traces, yielding the fast and the slow time constant of current activation at +40 mV as well as the amplitudes of the two current components (Af and As). Note that the preceding holding potential was –80 mV, except for the mutant L352V, where a holding potential of –100 mV was used. Compared to wild-type KCNH1 channels, the time course of current activation was accelerated for all mutant channels. For G348R, I467V and L352V, both activation time constants were significantly decreased, and for the double mutant S325Y/V356L, the slow time constant decreased significantly in combination with a higher relative contribution of the faster activating current component. (b) Families of wild-type and G348R current traces recorded with the depicted deactivation protocol. Zero current is indicated by a dashed line. (c) The time course of KCNH1 channel deactivation was analyzed at –120 mV from experiments as shown in b. Compared to wild-type channel, the deactivation time course of all mutant channels was significantly slowed. Current decay upon hyperpolarization to –120 mV was fitted with a double-exponential function, yielding the fast and the slow time constant of current deactivation as well as the amplitudes of the two current components (Af and As). Af and As were extrapolated to segment start, because the first 0.5 to 1 ms of the pulse segment at –120 mV was not used for fitting to minimize contributions of capacitive currents. (a,c) The number of experiments is given in the lower bar plots; **, significantly different from wild-type channel with P < 0.01; ***, significantly different from wild-type channel with P < 0.001. Values were tested for significant differences compared to wild-type data with a two-tailed heteroscedastic t test and Bonferroni correction for multiple testing.
Supplementary Figure 5 Voltage dependence of S325Y and V356L KCNH1 channel activation.
(a,b) Mean values (±s.e.m.) of normalized current amplitudes (a) and whole-cell conductance (b) for S325Y (n = 14) and V356L (n = 11). Data points in a are connected by lines; the dark gray lines in b represent fits to the data points using equation (2). Corresponding data for the double mutant S325Y/V356L and for wild-type (WT) channels are shown for comparison as light gray and black lines, respectively (data from Fig. 4). Fit parameters are given in Supplementary Table 4.
Supplementary Figure 6 Expression and coexpression of wild-type (WT) and mutant (G469R) KCNH1 channels in Xenopus laevis oocytes.
(a) Families of current traces recorded with the depicted pulse protocol. For the G469R mutant, depolarizing pulses failed to induce voltage-dependent outward currents and coexpression of the mutant with wild-type channels suppressed the amplitude of KCNH1 outward currents at more positive potentials. Zero current is indicated by dashed lines and arrowheads. (b) Means (+s.e.m.) of the current amplitude recorded at the end of the test pulse to +40 mV. Absolute current values were normalized to the average current amplitude obtained for wild-type KCNH1 channels. ***, significantly different from WT with P < 0.001 (one-way ANOVA and post-hoc Bonferroni t test); n.s., not significantly different. The amount of injected cRNA is indicated, and the number of experiments is given in parentheses. Similar results were obtained in experiments using other batches of oocytes.
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Kortüm, F., Caputo, V., Bauer, C. et al. Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome. Nat Genet 47, 661–667 (2015). https://doi.org/10.1038/ng.3282
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DOI: https://doi.org/10.1038/ng.3282
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