Homozygous SGCB splice-site variant causes isolated dilated cardiomyopathy through sarcoglycan complex destabilization in East Asians

Figures

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  Figure 1. Aberrant splicing and altered expression of SGCB in cardiac tissue from patients with DCM. (A) Splicing rank plot showing aberrant splicing of SGCB. (B) Expression rank plot demonstrating decreased expression of SGCB. (C) Sashimi plots illustrating splicing patterns across exons 1–3 in samples carrying either the WT (WT/WT) or homozygous c.243+6T>A (Mut/Mut) SGCB allele, identified through DROP analysis. RPKM, reads per kilobase of transcript per million mapped reads. (D) Differential exon usage analysis using DEXSeq comparing WT/WT and Mut/Mut groups across all annotated SGCB exons (exons 1–6) and a pseudoexon located between exons 2 and 3. Each bar represents normalized exon-level read counts. The x axis represents exon analysis numbers assigned by DEXSeq, with canonical SGCB exon numbers annotated below to facilitate alignment with the annotated gene structure; corresponding chromosomal positions are also displayed below. Exon 2, which is significantly decreased expression in the Mut/Mut group, is highlighted in red. P4* denotes a patient harboring 2 heterozygous variants: c.243+6T>A and c.325C>T (p.Arg109*).

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  Figure 2. Pedigrees of 4 unrelated families carrying the SGCB c.243+6T>A variant. The proband in each family is indicated by a black arrow. Filled symbols, affected individuals; open symbols, unaffected; slashed open symbols, deceased unaffected individuals. Sanger sequencing chromatograms show the variant site marked with red arrows. wt, mut1, and mut2 indicate the WT allele at c.243+6, 243+6T>A, and c.325C>T, respectively. In family 4, the proband harbors compound heterozygous variants (mut1 and mut2).

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  Figure 3. Gene and protein expression of sarcoglycan subunits in cardiac tissue. (A) RNA-seq–based expression levels of SGCB, SGCA, SGCD, and SGCG in cardiac tissue samples from individuals carrying the WT (WT/WT, n = 3), homozygous c.243+6T>A (Mut/Mut), or heterozygous c.243+6T>A (WT/Mut, n = 3) allele of SGCB. For SGCB, the Mut/Mut group includes 3 samples, excluding the compound heterozygous sample (P4), which was identified as an expression outlier in Figure 1B. For SGCA, SGCD, and SGCG, the Mut/Mut group includes 4 samples, including P4. Expression values, presented as fragments per kilobase of transcript per million mapped reads (FPKM), are shown as mean ± SD. (B) Representative Western blot analysis of β-SG, α-SG, δ-SG, and γ-SG in cardiac tissue samples from the same groups (WT/WT, n = 3; Mut/Mut, n = 4; WT/Mut, n = 2). α-Actinin 2, GAPDH, and Ponceau S staining were used as loading controls. P4* indicates a compound heterozygous sample harboring the c.243+6T>A and c.325C>T variants. (C) Quantification of Western blot band intensities shown in B, performed using ImageJ. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. P values were calculated using 1-way…

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  Figure 4. Immunohistochemical analysis of sarcoglycan subunits in cardiac tissue. Cardiac sections from samples carrying the WT (WT/WT) or homozygous c.243+6T>A (Mut/Mut) allele of SGCB were stained with antibodies against β-SG, α-SG, δ-SG, and γ-SG. Representative images from 2 WT/WT and 2 Mut/Mut samples are shown. Images were acquired at ×1,000 magnification. Scale bar: 20 μm.

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  Figure 5. Muscle strength and serum CK levels in patients with SGCB and TNNT2 variants. (A) Muscle strength assessed by grip strength/BMI and (B) knee extension strength/BMI showed no significant differences between the 2 groups. (C) Serum CK levels between the groups; the normal reference range is 54–286 U/L. TNNT2 and SGCB refer to patients with the c.407G>A variant in TNNT2 (n = 4) and the c.243+6T>A variant in SGCB (n = 5), respectively. Data are presented as the mean ± SD. P values were calculated using a 2-tailed Welch’s t test.

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  Figure 6. Sarcoglycan subunit expression in hiPSC–derived skeletal myocytes. (A) qPCR analysis of hiPSCs and hiPSC-derived skeletal myocytes (hiPSC-SMs) from a control line (253G1; WT/WT) and a patient carrying the homozygous SGCB c.243+6T>A variant (Mut/Mut) (n = 3 independent experiments). Expression levels are presented as ΔCt values normalized to GAPDH; lower ΔCt values indicate higher expression. #, Only 2 samples were analyzed because 1 sample was undetermined. (B) Representative Western blot analysis of β-SG, α-SG, δ-SG, and γ-SG in hiPSCs and hiPSC-SMs from WT/WT and Mut/Mut lines. Myosin heavy chain (MyHC) was used as a differentiation marker, and GAPDH was used as a loading control. (C) Quantification of Western blot band intensities of hiPSC-SMs shown in B, performed using ImageJ (n = 3 independent experiments). Data are presented as the mean ± SD. *P < 0.05; ***P < 0.001. P values were calculated using 1-way ANOVA followed by the Tukey–Kramer test for pairwise comparisons in A and using a 2-tailed Welch’s t test in C.

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  Figure 7. Event-free survival in patients with DCM harboring biallelic pathogenic SGCB variants, without known pathogenic variants or with TTN variants. Patients with DCM were divided into 3 groups: the SGCB group (n = 12), consisting of patients with biallelic pathogenic SGCB variants (11 homozygous for c.243+6T>A and 1 compound heterozygous); the variant-negative (V–) group (n = 50), consisting of patients without pathogenic variants in any of the 40 known DCM-associated genes; and the TTN group (n = 105), consisting of patients with TTN variants classified as P/LP according to ACMG criteria. Kaplan-Meier curves illustrate survival free of the composite endpoint — cardiovascular (CV) death, HTx, or VAD implantation — using age as the timescale. P values were calculated using a univariable Cox proportional hazard model. The number of patients at risk is shown below the Kaplan-Meier curves.