Functional investigation of two simultaneous or separately segregating DSP variants within a single family supports the theory of a dose‐dependent disease severity

Abstract Desmoplakin (DP) is an important component of desmosomes, essential in cell–cell connecting structures in stress‐bearing tissues. Over the years, many hundreds of pathogenic variants in DSP have been associated with different cutaneous and cardiac phenotypes or a combination, known as a cardiocutaneous syndrome. Of less than 5% of the reported DSP variants, the effect on the protein has been investigated. Here, we describe and have performed RNA, protein and tissue analysis in a large family where DSP c.273+5G>A/c.6687delA segregated with palmoplantar keratoderma (PPK), woolly hair and lethal cardiomyopathy, while DSP WT/c.6687delA segregated with PPK and milder cardiomyopathy. hiPSC‐derived cardiomyocytes and primary keratinocytes from carriers were obtained for analysis. Unlike the previously reported nonsense variants in the last exon of DSP that bypassed the nonsense‐mediated mRNA surveillance system leading to protein truncation, variant c.6687delA was shown to cause the loss of protein expression. Patients carrying both variants and having a considerably more severe phenotype were shown to have 70% DP protein reduction, while patients carrying only c.6687delA had 50% protein reduction and a milder phenotype. The analysis of RNA from patient cells did not show any splicing effect of the c.273+5G>A variant. However, a minigene splicing assay clearly showed alternative spliced transcripts originating from this variant. This study shows the importance of RNA and protein analyses to pinpoint the exact effect of DSP variants instead of solely relying on predictions. In addition, the particular pattern of inheritance, with simultaneous or separately segregating DSP variants within the same family, strongly supports the theory of a dose‐dependent disease severity.


| BACKG ROU N D
Desmosomes maintain cell-cell cohesion by forming dense intercellular bonds that anchor the cell's intermediate filaments to the cell membrane in skin and heart. 1 Pathogenic variants in its major constituent desmoplakin (DP; gene DSP) may give isolated cardiomyopathy, skin fragility, palmoplantar keratoderma (PPK) or woolly hair (WH), but also a combination of these, like in Carvajal syndrome. 2,3 In skin, DSP produces nearly equivalent levels of two isoforms: DPI and the shorter DPII isoform, while the heart predominantly contains DPI. 4,5 In 2000, homozygous variants causing c-terminally truncated DP were shown to cause Carvajal syndrome, 6 but also dominant lossof-function variants have been linked to a combination of mild PPK, WH and cardiomyopathy. 7 Over the years, hundreds of DSP variants have been associated with disease, yet only in a very few studies, the effect of these variants on RNA, protein and the cells/tissues involved has been investigated, leaving the genotype-phenotype correlation poorly understood. 7,8 Most studies merely rely on the use of in silico models to predict variant-outcome. Analysis of hiPSCderived cardiomyocytes as well as primary keratinocytes is hardly performed, but could be the essential stepping stone to rightfully denote the effects of DSP variants and a better understanding of the genotype-phenotype correlation.

| QUE S TIONS ADDRE SS ED
We describe a large family where members show various expression of moderate to lethal cardiomyopathy, PPK and WH. In this family, two variants in DSP (c.273+5G>A and c.6687delA) were identified.
Previously, we investigated engineered heart tissues, derived from the most severely affected proband carrying both variants in trans. 9 Here, we investigate segregation of these DSP variants among other affected relatives. We functionally assessed the consequences of these variants on RNA and protein levels in cardiomyocytes and primary keratinocytes from several affected family members and compared these data to in silico predictions, the various heart and skin disease severities and the sparse amount of experimental studies on other DSP variants.

| E XPERIMENTAL DE S IG N
Patients' written informed consent was received prior to this study.
A gene panel for inherited cardiomyopathies was performed on the proband, and segregation of variants was confirmed in other affected family members. The effects of variants were predicted using in silico prediction software. A minigene splicing assay was deployed and skin biopsies from non-lesional, upper arm skin were obtained from DSP c.273+5G>A/c.6687delA and DSP WT/c.6687delA carriers.
Unfortunately, the DSP c.273+5G>A/WT carrier was not willing to participate in the study. Biopsy-derived keratinocytes were cultured on flexible stretch plates with and without uniaxial stretching regimes.
Immunofluorescence microscopy and electron microscopy were performed on skin biopsies and cultured keratinocytes, in addition to cardiomyocytes as previously described. 9 RNA and protein were extracted and quantitatively assessed and compared with controls.
An extensive Material and Methods section can be found in the online supplements.

| Segregation of DSP variants in affected family members
A 52-year-old woman who underwent heart transplantation at the age of 44 because of a progressive and life-threatening cardiomyopathy was referred to a dermatologist. WH and a mild focal PPK without skin fragility ( Figure 1A; IV-2) were observed. Her similarly affected sister (IV-1) died from sudden heart failure at the age of 23 years, while several 3rd and 4th generation family members on the maternal side were diagnosed with cardiomyopathy and PPK. Next-generation sequencing with a targeted cardiac gene panel had revealed two DSP variants in the index patient: c.273+5G>A 10 and c.6687delA. 9,11 Skin biopsies were obtained after ethical approval and informed consent. The paternally derived splice site variant, c.273+5G>A, was detected in four family members, whereas the maternally derived c.6687delA variant was found in eight ( Figure 1A (Table S1). showed 50% reduction of DPI and DPII levels, while compound heterozygous DSP c.273+5G>A/c.6687delA carriers showed 70% reduction 9 (p < 0.05) (Figure 2A). In addition, cells with c.6687delA in heterozygous and compound heterozygous state showed a twofold reduction in total DSP mRNA levels (with the exception of heterozygous DSP WT/c.6687delA keratinocytes), suggesting NMD.

| Assessment of intercellular contact and differentiation behaviour of patient keratinocytes
To assess the effects of DP deficiency on the intercellular contact and differentiation behaviour of keratinocytes, we performed a keratinocyte dissociation assay (KDA) 18

| CON CLUS I ON S AND PER S PEC TIVE S
Here, we report a large family in which two DSP variants (c.6687delA Although DPI is the main isoform in the heart, the cardiac phenotype of these patients suggests that DPII may be more important in the heart than initially assumed, as full loss of DP unlikely allows human heart formation. 28 In the skin, loss of DPI also caused a more severe skin phenotype, observed as epidermolytic PPK with F I G U R E 2 DP expression in non-palmoplantar ex vivo skin and in vitro keratinocytes and hiPSC-derived cardiomyocytes. (A) Western blots of desmoplakin protein expression using different antigen maps to DP-I and DP-I&II in compound heterozygous (DSP c.273+5G>A/c.6687delA ) and heterozygous (DSP WT/c.6687delA ) carrier in comparison with control. Quantified protein levels of DP isoform-I and II, using different antibodies in cultured primary keratinocytes. 2 controls (n = 3), 1 patient of each genotype (n = 3/patient). Also, quantified the protein levels of DP isoform-I in patient pluripotent stem cell-derived cardiomyocytes. 2 controls (n = 4), 1 patient of each genotype (n = 6 vs. 4); *p < 0.05 and **p < 0.01 (1-way ANOVA, Tukey's multiple comparison compared with control keratinocytes); **p < 0.01 (1-way ANOVA, Tukey's multiple comparison compared with control cardiomyocytes). In the bars, the average percentages of available proteins are depicted. (B) IFM of antigen mapping to DP-I on ex vivo skin in comparison with the in vitro cultured keratinocytes and hiPSC-derived cardiomyocytes. Scale bars = 50 µm. (C) IFM of antigen mapping of N and C-terminal directed antibodies of DP-I&II on ex vivo skin in comparison with the in vitro cultured keratinocytes and hiPSC-derived cardiomyocytes. Scale bars = 50 µm. The population-based, quantified IFM intensity at the membrane and cytosol, is depicted in Figure S1D.  19 Furthermore, cell-sheet dissociation did not seem to correlate with DPI depletion (90% reduction in HaCaT, nor the 70% reduction reported in this study), but more so with DPII depletion (90% reduction in HaCaT, but not with the 70% reduction reported in this study). These data and ours suggest that threshold levels of DPII to induce fragmentation lie somewhere between 10% and 30% of wildtype expression. Our results further indicate that IFM expression and EM of ex vivo non-palmoplantar skin are not reliable in indicating the molecular defect, let alone to predict cardiomyopathy later in life.
Many studies have attempted to establish a genotypephenotype correlation for DSP variants. However, the effect of the vast majority of reported variants has been predicted instead of being experimentally assessed. Our data point out that the actual effect of DSP nonsense and splice-site variants on DP protein is more complex than current in silico programs can predict. The unique pathogenicity of the variants reported here has broadened the understanding of the DSP genotype-phenotype correlation in both skin and heart and should be taken into account when evaluating other nonsense or splice-site variants. In addition, the particular pattern of inheritance, with either simultaneous or separately segregating DSP variants within the same family, hereby strongly supports the theory of a dose-dependent disease severity in both the skin and heart. Analysis of variants for the effect on RNA and protein level and tissue effects in both skin and heart, preferably in 3D models, will be the stepping stone to elucidate the pathogenicity of DSP variants and better genotype-phenotype correlations in the future. Above all, these are essential in the light of advancements in more targeted protein, RNA and/or DNA based therapies.

ACK N OWLED G EM ENTS
Part of the work has been performed at the UMCG Imaging and Microscopy Center (UMIC), for which we express our gratitude.
We would like to thank Ludolf G. Boven for his technical assistance.

CO N FLI C T O F I NTE R E S T
The authors have nothing to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data can be found in the main manuscript and Appendix S1.