ABCC9-related Intellectual disability Myopathy Syndrome is a KATP channelopathy with loss-of-function mutations in ABCC9

Mutations in genes encoding KATP channel subunits have been reported for pancreatic disorders and Cantú syndrome. Here, we report a syndrome in six patients from two families with a consistent phenotype of mild intellectual disability, similar facies, myopathy, and cerebral white matter hyperintensities, with cardiac systolic dysfunction present in the two oldest patients. Patients are homozygous for a splice-site mutation in ABCC9 (c.1320 + 1 G > A), which encodes the sulfonylurea receptor 2 (SUR2) subunit of KATP channels. This mutation results in an in-frame deletion of exon 8, which results in non-functional KATP channels in recombinant assays. SUR2 loss-of-function causes fatigability and cardiac dysfunction in mice, and reduced activity, cardiac dysfunction and ventricular enlargement in zebrafish. We term this channelopathy resulting from loss-of-function of SUR2-containing KATP channels ABCC9-related Intellectual disability Myopathy Syndrome (AIMS). The phenotype differs from Cantú syndrome, which is caused by gain-of-function ABCC9 mutations, reflecting the opposing consequences of KATP loss- versus gain-of-function.


Mouse model
Behavioral, emotionality and cognitive testing of SUR2-Stop mice 1-hour locomotor activity, exploratory behavior and sensorimotor battery: Baseline locomotor activity was assessed using a transparent polystyrene enclosure (47.6 x 25.4 x 20.6 cm) and computerized photobeam instrumentation as described previously 1,2 . Total ambulatory time and vertical rearings were taken as measures of activity whilst the time spent in the center of the field (33 x 11 cm) and the edges of the field (5.5 cm from walls) were used as indices of emotionality. The next day, mice were subjected to a series of sensorimotor tests selected to assess coordination, balance (ledge, platform, pole and inclined screen tests), strength (screen tests), and initiation of movement (walking initiation), as described previously 1,3 . For the balance tests, mice were placed on a Plexiglass ledge (0.75 cm wide, 30 cm elevation) or a small circular platform (3 cm in diameter, 47 cm elevation) and the time the mice remaining on the ledge/platform was recorded. For the pole test mice were placed headupwards with forepaws on top of a textured rod (8 mm diameter, 55 cm height) and the time taken for the mouse to turn and descend the pole was recorded. The screen tests were conducted by placing a mouse head-downwards upon a mesh screen (16 squares per 10 cm, elevated 47 cm and angled at 60 or 90o), the time the mouse took to turn and climb to the top of the screen was recorded. The times of two trials for each test were averaged for analysis.
Morris Water Maze Navigation: The day after the sensorimotor battery we employed the MWM using a computerized tracking system (ANY-maze; Stoelting) to assess spatial learning and memory as described previously 1,2 . Cued trials were performed in which a visible platform was variably placed in the water maze with visible cues. Four trials were conducted per day (60 s maximum time) for two consecutive days with the platform being moved to different locations for each new trial using a 30 min inter-trial interval (ITI) and with limited, distal spatial cues being present to limit spatial learning. The time, distance and swimming speed for mice to find the platform was recorded across four blocks of trials (two trials/block). Three days later, place trials were initiated in which the platform was submerged and hidden but remained in a constant location, to determine spatial learning. Mice were required to learn the single location of a submerged platform in relation to spatial cues. Place trial data were recorded from over five blocks of trials (four trials/block), in which each block included the performance level for each of five consecutive days of testing. Finally, a probe trial wherein a mouse was released into the water maze where the platform had been removed was administered ~ 1 h after the final Place Trial (on day 5 of place trial testing). The time spent in the various pool quadrants, including the target quadrant (where the platform had previously been placed), was recorded.
Object recognition test: Elevated Plus Maze: Anxiety-like behavior was measured using the Elevated Plus Maze as previously described 2,4 . The black Plexiglass EPM apparatus consisted of two opposed open arms (without walls) and two opposed closed (walled) arms (36 x 6.1 x 15 cm) which extended in a + shape from a central square platform (5.5 x 5.5 cm). Behavior in the maze was recorded using an automated, computerized recording set up (Kinder Scientific). Beam break data was recorded and analyzed using MotorMonitor software (Kinder Scientific), distance traveled, time spent in each area and entries into open and closed arms were recorded. Test sessions were performed in a dimly lit room (lighting with 13-watt blacklight bulbs; Ecobulbs, Feit) where each session began by placing a mouse in the center of the maze allowing for free exploration of the maze. Each session lasted 5 min and the mice were tested over 3 consecutive days.

Statistical analyses
Data was analyzed as previously reported 5 . Numerical data were presented as mean ± SEM. ANOVA models were used to test for statistical significance. Repeated-measures ANOVA (rmANOVA) models containing two between-subjects variables (genotype and sex) and once within-subject (repeated measures) variable (e.g. blocks of trials) were typically used to analyze the MWM and EPM data. The Huynh-Fledt adjustment of α levels was used for all within-subject effects containing more than two levels to protect against violations of sphericity/compound symmetry assumptions underlying rmANOVA models. We used oneway ANOVA models to test for between-group differences in the 1 h locomotor activity and sensorimotor tests.

Whole-embryo brightfield imaging and measurement of interorbital distance and body length
In vivo phenotypic assessment for whole-embryo imaging were carried out on a Leica M165FC stereomicroscope (Leica Microsystems) with transmitted light. Images were captured with a DFC420 digital microscope camera (Leica Microsystems). Images were applied to measure the distance between the convex tip of the eyes (interorbital distance) in 5 dpf larvae using ImageJ (NIH). To account for variations in size periorbital distance was normalized to overall larval body length. Body length was measured from the tip of the head to the end of the trunk (before the caudal fin).

AFOG staining
Adult zebrafish hearts were dissected and fixed in 4% paraformaldehyde (in Phosphate buffer with 4% sucrose) at 4°C for 4 hours, incubated in PO4+30% sucrose at 4°C overnight and subjected to embeding in tissue freezing medium (Leica) and sectioning at 10 μm intervals. Acid Fuchsin Orange-G (AFOG) staining was performed as described previously 6 . Image acquisition was conducted using a Leica DM4000 B LED upright automated microscope.

SUR2-STOP fish show no abnormalities in myofiber structure.
Immunohistochemistry on sagittal sections of adult hearts of WT (A) and SUR2-STOP fish (A') with antibody against to tropomyosin (red); nuclei are stained with DAPI (blue). To assess structure of myofibers four different areas were assessed: center of ventricle, apex, ventricular border and atrium. Areas are indicated in boxes. (B-E) and (B′-E′) Higher magnification views of the boxed areas in A and A'. Arrowheads indicate stained tropomyosin. Sample size, WT, n=6, SUR2-STOP, n=6. Scale bars, 500 m in A and A'; 100 m in B-E and B'-E'.

Coverage statistics
Supplementary Table 3 Supplementary