Loss of Function Mutations in NNT Are Associated With Left Ventricular Noncompaction

Abstract

Background: Left ventricular noncompaction (LVNC) is an autosomal-dominant, genetically heterogeneous cardiomyopathy with variable severity, which may co-occur with cardiac hypertrophy.

Methods and results: Here, we generated whole exome sequence data from multiple members from 5 families with LVNC. In 4 of 5 families, the candidate causative mutation segregates with disease in known LVNC genes MYH7 and TPM1. Subsequent sequencing of MYH7 in a larger LVNC cohort identified 7 novel likely disease causing variants. In the fifth family, we identified a frameshift mutation in NNT, a nuclear-encoded mitochondrial protein, not implicated previously in human cardiomyopathies. Resequencing of NNT in additional LVNC families identified a second likely pathogenic missense allele. Suppression of nnt in zebrafish caused early ventricular malformation and contractility defects, probably driven by altered cardiomyocyte proliferation. In vivo complementation studies showed that mutant human NNT failed to rescue nnt morpholino-induced heart dysfunction, indicating a probable haploinsufficiency mechanism.

Conclusions: Together, our data expand the genetic spectrum of LVNC and demonstrate how the intersection of whole exome sequence with in vivo functional studies can accelerate the identification of genes that drive human genetic disorders.

Keywords: genetics; genomics; human; mutation; nonisolated left ventricular noncompaction.

Figure 1

Genetic and Functional evaluation of NNT in LVNC. A. Schematic of the human NNT locus on human chromosome 5 (top) showing non-coding exons (white boxes), and coding exons (blue boxes). Double slash indicates large introns (> 5kb). A schematic of NNT protein is shown below with functional domains Alanine dehydrogenase/PNT, N-terminal domain (AlaDh_PNT_N, shown in green), Alanine dehydrogenase/PNT, C-terminal domain (AlaDh_PNT_N, shown in purple) and twelve transmembrane domains (orange). The two mutations identified in LVNC families are shown with asterisks. B. Schematic of NNT function in mitochondria to regenerate NADPH in the cell; NADPH is then used to regenerate Thioredoxin Reductase 2 (TrxR-2) and glutathione reductase (GR). C. Lateral view of representative 3 dpf control (top) and nnt-a/b morphant (bottom) larvae showing cardiac edema (arrow). D. In vivo complementation assay of NNT p.D277Y; quantification of cardiac edema in 3 dpf morphant zebrafish larvae (5 ng nnt-a/b MOs) with and without addition of 200 pg human wild type NNT RNA (WT), and mutant RNA (p.D277Y). Statistically significant differences were calculated with χ² tests and p<0.0001 are indicated (*); n=46–51 embryos/injection batch, repeated three times with masked scoring.

Figure 2

nnt-a/nnt-b morphants display contractile dysfunction. Representative live ventral views of 2 dpf cmlc2:GFP larvae at ventricular systole (top panels) and ventricular diastole (bottom panels). Ventricles in control larvae expand normally as the atrium contracts (compare white dashed circle to violet dashed circle in the bottom inset); ventricles in nnt-a/nnt-b morphants fail to expand during atrial contraction (5 ng each MO injected/embryo). Dashed box corresponds to the magnified image to the right of each large panel; L, left; R, right; A, atrium; V, ventricle; scale bars, 100 μm. nnt-a/nnt-b morphants also display brachycardia (mean 91.5 vs. 66.8 beats/minute, controls vs. morphants; p<0.0001; student’s t-test; n=10 larvae/injection, repeated twice; see Supplemental movies).

Figure 3

Suppression of nnt-a/nnt-b in zebrafish results in altered cardiomyocyte proliferation. A. Representative maximum intensity projections of cardiac sections from control and nnt-a/nnt-b morphants (5 ng each MO injected/embryo) at 2 dpf and 3 dpf were used to monitor non-proliferating (cmlc2:mCherry-zCdt1, red, G1 phase of the cell cycle) and proliferating (cmlc2:Venus-hGeminin, green, S/G2/M phase of the cell cycle) cardiomyocyte counts. Cardiomyocytes with only the green signal were counted as proliferating (exemplified by 2 dpf nnt-a/nnt-b or 3 dpf control panels). Scale bars, 50 μm; arrowheads point to the ventricle (V); or atrium (A). B. Quantification of non-proliferating cardiomyocytes as indicated by red⁺green⁻ or red⁺green⁺ cells. C. Quantification of proliferating cardiomyocytes as indicated by red⁻green⁺ cells. p<0.0001; student’s t-test; n=12 larvae/injection batch, repeated once with similar results. Error bars represent standard error of the mean (sem).