FLNC Gene Splice Mutations Cause Dilated Cardiomyopathy

Abstract

Objective: To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism.

Background: DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes.

Methods: We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model.

Results: A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure.

Conclusions: Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM.

Keywords: cardiovascular genetics; dilated cardiomyopathy; filamin C; heart failure; zebrafish.

Graphical abstract

Figure 1

Family Pedigrees TSFDC029, TSFDC031, and DNFDC057 Squares and circles indicate male and female subjects, respectively, and shading indicates a dilated cardiomyopathy phenotype. The arrowheads indicate probands. Plus and minus signs indicate carriers (+) or noncarriers (–) of the filamin C (FLNC) variant, respectively. Individuals II:2, III:4, and IV:1 in family TSFDC029 (A); individuals II:2, II:3, and III:1 in family TSFDC031 (B); and individuals II:1 and II:2 in family DNFDC057 (C) underwent whole exome sequencing, and are indicated by an ∗. Reported ages at death are indicated. (D) Electrocardiogram of subject TSFDC031 III:1 showing atrial fibrillation, which first occurred at the age of 21 years, and (E) the 2-dimensional echocardiogram images showing a mildly enlarged left ventricle with a left ventricular ejection fraction of 40% (age 36 years). LVEDD = left ventricular end-diastolic diameter; LVEDV = left ventricular end-diastolic volume; NCD = noncardiac death; SCD = sudden cardiac death.

Figure 2

Affected Individuals Carry a Mutation at the Intronic Border of Exon 43 (A) Sanger sequenced chromatogram of healthy individual II:3 from family TSFDC031 with a normal FLNC gene and an affected individual from the same family (III:1) showing the FLNC c.7251+1 G>A variant. (B) Real-time polymerase chain reaction of ribonucleic acid isolated from lymphoblastoid human cells for exons 41 to 45 spanning probe showing presence of shortened product (missing 116 nucleotides, exon 43) in an affected patient (A-III:1) and normal product in healthy relatives (H-II:3 and H-III:2). (C) Diagram of normal (upper) and aberrant (lower) splicing; thick, solid line (lower) shows exon 43 being excluded. bp = base pair; nt = nucleotide.

Figure 3

FLNC Cardiac Protein Expression Is Decreased (A) Western blot analysis shows decreased FLNC protein expression in affected individual II:1 (A-II:1) from family DNFDC057, and (B) analysis of densitometry of Western blots of affected subject A-II:1 relative to control subjects were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ± SEM (n = 2).

Figure 4

MO Knockdown of flncb in Zebrafish Showing a Cardiac Phenotype (A) Black bars: percentage of surviving embryos with severe cardiac phenotype at 72 h post-fertilization (hpf) for uninjected (n = 411), p53 morpholino (MO)-injected (n = 139), 25-N+p53 control MO-injected (n = 351), and filamin Cb (flncb)+p53 MO-injected (n = 241); *p = 1.04 × 10^-83 (for comparison of uninjected heart vs. flncb+p53 MO using chi-square testing). Gray bars: percentage of surviving embryos at 7 days for uninjected (n = 411), p53 MO-injected (n = 139), 25-N+p53 control MO-injected (n = 351), and flncb+p53 MO-injected (n = 164); †p = 1.65 × 10^-19 (for comparison of uninjected embryo vs. flncb+p53 MO using chi-square testing). (B) Representative zebrafish heart pictures for embryo uninjected and flncb MO coinjected with p53 MO zebrafish embryos at 48 hpf. The flncb+p53 MO-injected zebrafish show cardiac phenotype (pericardial edema [P]), and GFP hearts showing less heart looping, elongated atrium, pericardial edema, and a truncated ventricle. (C) Representative zebrafish heart pictures for embryo uninjected and flncb MO coinjected with p53 MO zebrafish embryos at 72 hpf. Among the flncb+p53 MO-injected zebrafish, they show cardiac phenotype, and GFP hearts further show less heart looping, elongated atrium, pericardial edema, and a truncated ventricle.

Figure 5

Morphological Analysis Demonstrating Impaired Cardiac Function of flncb+p53 MO-Injected Zebrafish Embryos High-speed videos of hearts in 48 hpf embryos were analyzed using custom MATLAB software (Mathworks, Natick, Massachusetts) , . Embryos injected with flnc+p53 MO (n = 21) were compared with uninjected control embryos (n = 17) and control embryos injected with a scrambled MO (25-N+p53 MO; n = 11). (A) Mean reverse flow fraction (±SE) for each treatment (uninjected: 0.017, 25-N+p53 MO: 0.008, flncb+p53 MO: 0.061). Significant differences were observed for flncb+p53 MO-injected relative to control embryos by analysis of variance (ANOVA) (p = 6.1 × 10^-5). Follow-up Tukey tests indicated differences between flncb+p53 MO and uninjected embryos (p = 0.023); and flncb+p53 MO compared with 25-N+p53 MO (p = 0.007), but no difference between the control embryos (p = 0.420). (B) Mean heart rate (±SE) for each treatment (uninjected: 193.5 beats/min [bpm], 25-N+p53 MO: 181.0 bpm, flncb+p53 MO: 162.0 bpm (ANOVA; p = 2.1 × 10^-7). flncb+p53 MO decreased the heart rate at 48 hpf compared with both uninjected embryos and 25-N+p53 MO-injected embryos. (***p = 0.001, ***p = 0.005, respectively, by Tukey Test); heart rates of uninjected versus 25-N+p53 MO were not different (p = 0.118). (C) Mean stroke volume (±SE) for each treatment (uninjected: 0.125 nl, 25-N+p53 MO: 0.185 nl, flncb+p53 MO: 0.100 nl). No significant difference was detected between treatments (Kruskal-Wallis 1-way ANOVA on ranks; p = 0.482). (D) Mean cardiac output (±SE) for each treatment (uninjected: 24.4 nl/min, 25-N+p53 MO: 32.6 nl/min, flncb+p53 MO: 16.0 nl/min). No significant difference was detected between treatments (Kruskal-Wallis 1-way ANOVA on ranks; p = 0.293). For Videos 1, 2, and 3, please see the online version of this article. Abbreviations as in Figure 4.

Online Video 1 Ventral view of an uninjected control zebrafish heart at 48 hours post-fertilization. For online presentation, these supplemental videos have been reduced in resolution and modified to read at 30 frames per second.

Online Video 2 Ventral view of a 25N-MO control zebrafish heart at 48 hours post-fertilization. For online presentation, these supplemental videos have been reduced in resolution and modified to read at 30 frames per second.

Online Video 3 Ventral view of a representative flncb MO zebrafish heart at 48 hours post-fertilization. Note the increased fraction of retrograde flow through the atrioventricular junction. For online presentation, these supplemental videos have been reduced in resolution and modified to read at 30 frames per second.

Figure 6

Ultrastructural Analysis Indicates Deficiencies in Sarcomere Organization and Z-Disc Formation Transmission electron micrographs of zebrafish hearts at 72 hpf indicate that (A) wild-type embryos had assembled several consecutive sarcomeres with clearly distinct Z-discs (black arrow), whereas flncb+p53 MO hearts (B to F) show evidence of disorganized ultrastructure. (B) Most Z-discs were diffusely stained, irregular in shape (black arrowheads), or seemingly absent (white arrowheads). Insets: Cross section through myofilament bundles indicated a normal primary organization of thick and thin filaments in hexagonal lattices in flncb-depleted hearts. (C) Sarcomere arrangement in myofibrils (m) was often nonconsecutive, with (D) multiple sarcomeres sometimes adjoined to a mass of Z-disc-like material (z). (E) Small vacuoles (v) were often present between or near the plasma membranes of adjacent cardiomyocytes. These vacuoles could be large, creating a gap between adjacent cells. (F) Autophagic vesicles (a) were observed. Scale bar: 1 μmol/l. Abbreviations as in Figure 4.