KCNE1 mutations D76N and Δ70 reduce I_Ks current density with minimal effects on activation kinetics.

<p>A) rates of activation, and B) current density from CHO cells transiently expressing Myc-tagged KCNQ1 (Q1) either without KCNE1 (E1), or with wild-type FLAG-tagged E1, E1-D76N, or E1-Δ70. Asterisks (*) indicate statistical significance to P<0.001. n = 6. C) Immunoblot showing KCNQ1 protein levels when expressed alone or co-expressed with KCNE1 (wild-type and mutant). Western blot analysis performed with mouse anti-Myc antibody (Santa Cruz) for KCNQ1, and mouse anti-FLAG antibody (Sigma) for KCNE1. Left lane in each gel shows signal from cells that are untransfected.</p

KCNE1-D76N and KCNE1-Δ70 shift the voltage dependence of activation of I_Ks.

<p>A) voltage-dependent activation curves, and B) normalized activation curves. Curves were fitted using a Boltzmann function. V_h for KCNQ1 = −30.3±1.0 mV, KCNQ1/KCNE1 = 9.5±4.2 mV, KCNQ1/KCNE1-D76N = 43.5±3.0 mV, KCNQ1/KCNE1-Δ70 = 71.5±7.0 mV. n = 6, p<0.001 for both mutants compared to wild-type KCNE1.</p

Biophysical characteristics of KCNE1 mutants.

<p>N = 6.</p><p>Abbreviations: V_h voltage at which half the channels are activated; ΔG(C→O) change in Gibbs free energy for the closed and open states during channel activation; ΔE_a(O→C) change in activation energy for the transition from open to closed states relative to wild-type KCNE1. (ND, not done).</p

KCNE1-D76N and KCNE1-Δ70 mutations result in defective rate-related accumulation of I_Ks current.

<p>A series of 100 pulses mimicked action potential trains at three rates: 60, 120, or 150 per minute. On the voltage protocol, the grey stripe indicates the 10 ms over which the current measured was averaged and plotted on the graphs. The insets for B, C, and D show the Y-axis magnified and the points plotted without error bars. X- and Y-axes labels are the same as the main graph. A) Cells transfected with KCNQ1 (Q1) and wild-type KCNE1 (E1); depolarization length 325 ms. B) Cells transfected as in A, but depolarization length of voltage protocol adjusted as follows: 60 pulses/min-325 ms; 120 pulses/min-175 ms; 150 pulses/min-145 ms. C) Cells transfected with Q1 and E1-D76N; depolarization length 325 ms. D) Cells transfected with Q1 and E1-Δ70; depolarization length 325 ms. n = 6.</p

Figure 5

<p>A) Co-immunoprecipitation of wild-type and mutant KCNE1 with KCNQ1. KCNQ1 (Q1) was expressed with KCNE1 (E1), E1-D76N, or E1-Δ70. KCNQ1 was immunoprecipitated with goat anti-KCNQ1 antibody (Santa Cruz), and western blot analysis performed with mouse anti-FLAG antibody (Sigma). The asterisks (*) indicate a row of nonspecific bands that appeared in every lane, including control lanes. For controls, lane 1 shows results from untransfected cells, lanes 2–4 from cells transfected with KCNQ1 and KCNE1 (WT, D76N, or Δ70 as indicated) and pulled down with control IgG. B) Digital subtraction of Q1 current from mixed Q1-E1 current (E1-Δ70 mutant). Left tracing shows the currents obtained from a cell transfected with a 1∶1 ratio of KCNQ1∶KCNE1-Δ70 plasmids. Middle tracing shows a pure KCNQ1 current scaled to match the amplitude of the initial rapid current deflection seen in the mixed current to the left. Right side tracing shows the resulting digital subtraction of the two currents resulting in a current with slow sigmoidal activation characteristic of I_Ks. C) Voltage clamp tracing from cells transfected with KCNE1-Δ70 plasmid alone demonstrating that the mutant KCNE1 did not induce any other conductances on its own.</p

Figure 1

<p>A) Schematic of KCNQ1 subunit and KCNE1, showing location of KCNE1 mutations. B) Current traces of activation protocol, with protocol in inset.</p

Deactivation rates are accelerated by both KCNE1-D76N and KCNE1-Δ70.

<p>A) Current traces, with deactivation protocol in inset, and B) rates of deactivation plotted against voltage from CHO cells transiently expressing KCNQ1 (Q1) either without KCNE1 (E1), or with wild-type E1, E1-D76N, or E1-Δ70. Asterisks (*) indicate significant change between wild-type E1 and both E1 mutations. n = 6. C) Co-immunoprecitation experiment of C-termini KCNE1 with full length KCNQ1. Full length KCNQ1 was expressed with KCNE1 C-terminus (KCNE1-CT), either wild-type or with D76E, D76N, or D76A point mutations. Immunoprecipitation was performed with goat anti-KCNQ1 antibody, and immunoblot with rabbit anti-FLAG antibody (Santa Cruz). For controls, lane 1 shows results from untransfected cells, lane 2 from KCNQ1 alone, lane 3 from KCNE1 wild type (D76) alone, and lane 4 from KCNQ1 and KCNE1 with control antibody. The different KCNE1 forms are designated by the single amino acid letter at the 76^th position.</p

Development of a Targeted Multi-Disorder High-Throughput Sequencing Assay for the Effective Identification of Disease-Causing Variants

<div><p>Background</p><p>While next generation sequencing (NGS) is a useful tool for the identification of genetic variants to aid diagnosis and support therapy decision, high sequencing costs have limited its application within routine clinical care, especially in economically depressed areas. To investigate the utility of a multi-disease NGS based genetic test, we designed a custom sequencing assay targeting over thirty disease-associated areas including cardiac disorders, intellectual disabilities, hearing loss, collagenopathies, muscular dystrophy, Ashkenazi Jewish genetic disorders, and complex Mendelian disorders. We focused on these specific areas based on the interest of our collaborative clinical team, suggesting these diseases being the ones in need for the development of a sequencing-screening assay.</p><p>Results</p><p>We targeted all coding, untranslated regions (UTR) and flanking intronic regions of 650 known disease-associated genes using the Roche-NimbleGen EZ SeqCapV3 capture system and sequenced on the Illumina HiSeq 2500 Rapid Run platform. Eight controls with known variants and one HapMap sample were first sequenced to assess the performance of the panel. Subsequently, as a proof of principle and to explore the possible utility of our test, we analyzed test disease subjects (n = 16). Eight had known Mendelian disorders and eight had complex pediatric diseases. In addition to assess whether copy number variation may be of utility as a companion assay relative to these specific disease areas, we used the Affymetrix Genome-Wide SNP Array 6.0 to analyze the same samples.</p><p>Conclusion</p><p>We identified potentially disease-associated variants: 22 missense, 4 nonsense, 1 frameshift, and 1 splice variants (16 previously identified, 12 novel among dbSNP and 15 novel among NHLBI Exome Variant Server). We found multi-disease targeted high-throughput sequencing to be a cost efficient approach in detecting disease-associated variants to aid diagnosis.</p></div

Summary of sequencing coverage and detected variants for test cohort.

<p>* indicates samples that were multiplexed together.</p><p>TG471.002 was added to another lane for logistic reasons.</p><p>Summary of sequencing coverage and detected variants for test cohort.</p

Cost comparison of target sequencing panel Einstein_v1 versus Whole Exome Sequencing.

<p>* The number of SNVs/InDels identified was based on samples used in the current analysis (n = 2 for WES and matching target sequencing).</p><p>** Based on estimated $1,400/lane 150 bp pair end sequencing on Illumina 2500.</p><p>Cost comparison of target sequencing panel Einstein_v1 versus Whole Exome Sequencing.</p