Oligogenic inheritance of a human heart disease involving a genetic modifier

Abstract

Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single-nucleotide variants in the MKL2, MYH7, and NKX2-5 genes. The MYH7 and MKL2 variants were inherited from the affected, asymptomatic father and the rare NKX2-5 variant (minor allele frequency, 0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell-derived cardiomyocytes provided histologic and molecular evidence for the NKX2-5 variant's contribution as a genetic modifier.

Fig. 1. Presentation of familial case of LVNC.

(A) Four-chamber echocardiography view showing the left atria (LA), right atria (RA), left ventricle (LV) and right ventricle (RV). Unaffected at left (43), index patient with LVNC at right. Yellow arrowheads indicate abnormal hypertrabeculation of the dilated LV. mo=month. (B) Transverse section of heart from sibling of index patient who had fetal demise. Higher magnification of the LV is at right. Scale bar = 6mm. (C) Pedigree showing inheritance pattern of LVNC in this family. Light grey indicates individuals whose cardiac status was not determined (ND) and white indicates unaffected individuals. Orange indicates the asymptomatic adult individual and purple indicates childhood-onset LVNC.

Fig. 2. Genotypic analysis of LVNC family by exome sequencing.

(A) Workflow for analysis of sequence variants inherited from the father is shown in blue on the left and from the unaffected mother in red on the right. Inheritance pattern for variants of interest is shown in pedigree on lower right. (B) Conservation of each amino acid residue across species is shown. The variant of interest is highlighted in yellow. Predicted effects of variants on protein function based on Combined Annotation Dependent Depletion (CADD), Sift, Polyphen-2 and FATHMM programs. Damaging (D), benign (B), tolerated (T) and not determined (ND). Single nucleotide variant (SNV); Minor allele frequency (MAF).

Fig. 3. Functional Evaluation of Disease-Associated SNVs in Mice.

(A) Left lateral images of E9.5 embryos from a representative Myh7^L387F/+ x Myh7^L387F/+ cross. h, head; lv, left ventricle; a, atrium. Scale bar = 5mm. (B) H&E stained transverse histologic sections of E13.5 embryos with indicated NKX2–5 genotypes. Width of compact layer indicated by black bar in higher magnification views of boxed areas. Top scale=500μM. Bottom scale=50μM. (C) Quantification (μM) of LV free wall thickness and (D) apical wall thickness from E13.5 mice collected from three litters of mice. P-value was calculated using a t-test. (E) Short axis view of LV in systole viewed by echocardiography of P4 mice with indicated Mkl2 genotypes. m, myocardium signified by dark area; e, endocardium indicated by arrowheads. (F) Average pixel intensity (PI) and (G) minimum PI within the LV of P4 mouse hearts calculated from three litters of mice. P-value was calculated using a t-test.

Fig. 4. Mkl2^Q664H/+Myh7^L387F/+NKX2–5^A118S/+ Compound Heterozygous Mice Recapitulate Features of Human LVNC.

(A) Left: representative histologic sections in four-chambered views of P3 mice. Scale bar = 200 mM. Right: higher magnification of the apical region of the same hearts shown on left. Scale bar = 50 mM. Immunohistochemistry with green indicating wheat germ agglutinin (cell membrane) and red marking endocardium (endomucin). (B) Quantification of LV free wall thickness, (C) apical wall thickness and (D) Trabeculation based on fractal dimension analysis. * P< 0.05, ** P< 0.01, ns=not significant. P-values calculated with one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Mean indicated by horizontal bar. Data collected from ten litters of mice. (E) Heatmap of all genes differentially expressed between WT and Mkl2^Q664H/+Myh7^L387F/+ or Mkl2^Q664H/+Myh7^L387F/+NKX2–5^A118S/+ mice. Key at bottom indicates log₂ fold change. (F) Heatmap of selected genes chosen from E. Heat map generated as in E.

Fig. 5. Human iPSC-derived Cardiomyocytes Exhibit features of LVNC.

(A) Pedigree of family members included in hiPSC-studies. (B) Brightfield images of hiPSC-derived cardiomyocytes from three family members as indicated. Scale = 100 mM. Orange arrowheads indicate abnormal aggregation. (C) Quantification of cellular aggregation. Y axis is log scale. n=2. (D) Heatmap depicting log₂ fold change of cardiomyocyte maturation-related genes in asymptomatic (2) and symptomatic (3) LVNC hiPSC-derived cardiomyocytes, compared to unaffected (1). Key at bottom. Full list can be found in Table S6. (E) Box plot illustrating the distance from genes expressed at high levels in the individual labeled in red to the closest NKX2–5 ChIP-seq peak. Peak distances > 2 megabase pairs (Mbp) were excluded from the graph. P-values calculated using Wilcoxon rank-sum test with Bonferroni correction.