Introduction Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy (DCM). Within the nucleus RBM20 partially colocalises with other splice factors, binds RNA and has a major role in myocardial alternative splicing. One of the RBM20 splicing targets is the giant sarcomere protein titin. Truncating mutations in the titin gene (TTN) are known to cause late onset cardiomyopathy.

We have found in a young patient with dilated cardiomyopathy combined heterozygosity for truncating (RBM20tv/TTNtv) and missense variants in RBM20 (p.Gly603Arg and p.Glu792GlyfsTer9) and TTN (p.Glu8271Gln and p.Lys23669Ter). The patient had an aggressive disease progression with biventricular failure and need for total artificial heart implantation at the age of 34 years. Splicing analysis of the patient’s myocardium revealed a significant impact on TTN and RYR2 splicing. The patient has a positive family history of DCM with the father carrying only the TTN-variants.

Hypothesis Most of the pathogenic RBM20 mutations identified up to now are localised in the highly conserved RS-domain of the protein. The impact of RBM20tv on disease development is currently unknown. This is the first functional report on an RBM20tv+RBM20-missense mutation leading to aberrant splicing and early disease onset. It is also the first case of combined mutations in RBM20 and its splicing target TTN.

Methods

To check if the RBM20-variants have an influence on RBM20-mRNA expression qPCRs using human myocardium were performed. We analyzed TTN and RYR2 splicing in the patient carrying the RBM20- and the TTN-variants and compared it to splicing in controls and individuals carrying only the TTN-variants. Furthermore, we compared in vitro the localization of RBM20 wildtype and mutants in different cell lines using RBM20-EYFP protein chimera.

Results qPCR analysis revealed that the RBM20-variants have no influence on RBM20-mRNA expression levels in the patient carrying the TTN/RBM20-variants. Nevertheless this patient shows aberrant splicing of the RBM20 target genes TTN and RYR2. Splicing of TTN and RYR2 is comparable to controls in the patient with the TTN-variants only. Interestingly, though leading to a modified splicing pattern the RBM20 variants have no influence on the intracellular localization of the protein.

Conclusions We present here first data showing loss of function mutations in RBM20 outside of the conserved RS-domain. The RBM20 protein is localised in the nucleus and leads to aberrant splicing of RBM20 target genes. The study reveals that combined mutations in RBM20 and TTN lead to an aggressive clinical phenotype.