Gain-of-Function Mutations in the K_ATP Channel (KCNJ11) Impair Coordinated Hand-Eye Tracking

<div><p>Background</p><p>Gain-of-function mutations in the ATP-sensitive potassium channel can cause permanent neonatal diabetes mellitus (PNDM) or neonatal diabetes accompanied by a constellation of neurological symptoms (iDEND syndrome). Studies of a mouse model of iDEND syndrome revealed that cerebellar Purkinje cell electrical activity was impaired and that the mice exhibited poor motor coordination. In this study, we probed the hand-eye coordination of PNDM and iDEND patients using visual tracking tasks to see if poor motor coordination is also a feature of the human disease.</p><p>Methods</p><p>Control participants (n = 14), patients with iDEND syndrome (n = 6 or 7), and patients with PNDM (n = 7) completed three computer-based tasks in which a moving target was tracked with a joystick-controlled cursor. Patients with PNDM and iDEND were being treated with sulphonylurea drugs at the time of testing.</p><p>Results</p><p>No differences were seen between PNDM patients and controls. Patients with iDEND syndrome were significantly less accurate than controls in two of the three tasks. The greatest differences were seen when iDEND patients tracked blanked targets, i.e. when predictive tracking was required. In this task, iDEND patients incurred more discrepancy errors (p = 0.009) and more velocity errors (p  = 0.009) than controls.</p><p>Conclusions</p><p>These results identify impaired hand-eye coordination as a new clinical feature of iDEND. The aetiology of this feature is likely to involve cerebellar dysfunction. The data further suggest that sulphonylurea doses that control the diabetes of these patients may be insufficient to fully correct their neurological symptoms.</p></div

Representative traces from tracking tasks completed by a control participant, and patients with PNDM and iDEND.

<p>Panels show the superimposition of 5 rightward (positive slope) and 5 leftward (negative slope) tracks from a control subject (A), a PNDM patient (R201 H mutation) (B), and an iDEND patient (V59 M mutation) (C). The person who provided the traces shown in (A) was the matched control of the iDEND patient whose traces are shown in (C). Participants tracked targets that moved at constant velocity (first column), variable velocity – accelerating and decelerating smoothly (second column), and constant velocity but with the visual presentation of the target blanked during the period indicated by the grey bar (third column). Blue lines indicate the movement of the target. Green lines indicate the movement of the cursor, which was controlled by the participant.</p

Tracking performance of patients with PNDM and iDEND on three different tasks.

<p>Scatter plots showing the performance of participants who tracked: a target moving at constant velocity (linear tracking) (A); a target moving with variable velocity (sinusoidal tracking) (B); and a target moving at constant velocity which was blanked during the middle third of each sweep (C). In each case the left panel shows discrepancy errors and the right panel velocity errors. (A) Data for PNDM (n = 7) and iDEND (n = 7) patients and their matched control, as indicated. There were no significant differences in the two error types between the groups (Kruskal-Wallis tests; p = 0.06 for discrepancy errors, p = 0.07 for velocity errors). (B) Data for PNDM (n = 7) and iDEND (n = 7) patients and their matched controls, as indicated. Patients with iDEND incurred significantly higher discrepancy errors than controls, but PNDM patients were not affected. **, p<0.01, post-hoc Mann-Whitney U-test. There were no significant differences in the velocity errors between the groups (Kruskal-Wallis test). (C) Data for PNDM (n = 7) and iDEND patients (n = 6) and their matched controls, as indicated. The target was blanked during the middle third of each sweep. Patients with iDEND were significantly less accurate than controls but PNDM patients were not different. **, p<0.01, post-hoc Mann-Whitney U-tests. In all figures the red bars indicate the median error and the blue arrows indicate data points for the iDEND patient shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062646#pone-0062646-g001" target="_blank">Figure 1</a>.</p

Comparison of visually-guided and blanked tracking performance of patients with PNDM and iDEND.

<p>Scatter plots showing discrepancy errors of iDEND patients (A, n = 6) and PNDM (B, n = 7) patients and their matched controls (Ctr), as indicated, on the linear tracking task with target blanking. The visually guided (first and last) and blanked (middle) segments of this task were analysed separately. Red bars indicate the median error. Discrepancy errors of iDEND patients were significantly higher than controls in segments 2 and 3. **, p<0.01 post-hoc Mann-Whitney U-tests. There were no differences between PNDM patients and controls in any of the three segments (Kruskal-Wallis test).</p

Overview of potential m6A sites in obesity-related genes with changed expression in FTO-4 mice.

<p>Pie chart of obesity-related genes whose expression is changed in the indicated tissues of FTO-4 mice. Dark grey, m6A sites in their mRNA transcripts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Meyer1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Dominissini1" target="_blank">[24]</a>. Light grey, no m6A sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Meyer1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Dominissini1" target="_blank">[24]</a>. The number of genes involved is indicated: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone-0097162-t002" target="_blank">Table 2</a> for gene names.</p

Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.</p

Effect of FTO overexpression on m6A levels in RNA.

<p>FTO mRNA expression in wild-type (black bar) and FTO-4 (white bar) MEFs as the fold change compared to wild-type (A). m6A levels measured as a percentage of adenosine levels for mRNA and total RNA by LC/MS in wild-type (black bar) and FTO-4 (white bar) MEFs (B). Significance was tested using Student's t-tests to compare FTO-4 to WT data. **p<0.01.</p

Top 10 genes that showed the highest amount of overexpression in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of overexpression in FTO-4 mice per tissue.</p

Differences in gene expression between WT and FTO-4 mice based on microarray data.

<p>Expression of the indicated genes in the cerebellum (A), hypothalamus (B), WAT (C) and gastrocnemius (D) of wildtype (WT, black bars) and FTO-4 (white bars) mice. Data are expressed as the fold change compared to wildtype. Significance was tested using Student's t-test to compare FTO-4 to WT. ***p<0.001, **p<0.01, *p<0.05.</p

qPCR analysis of the effect of m6A on DNA amplification.

<p>qPCR analysis using different concentrations of the DNA templates ACTB (A), HPRT1 (B) and HSPA8 (C) containing m6A (open squares) or unmethylated adenosine (black circles) revealed that m6A does not interfere with DNA amplification.</p