Inferring disease course from differential exon usage in the wide titinopathy spectrum

Abstract

Objective: Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging.

Methods: In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE.

Results: We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case.

Interpretation: This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing.

Figure 1

Exon usage of “false” canonical exons. Although exons 13,133, 149, and 156 are traditionally described as canonical exons included in postnatally expressed skeletal muscle isoforms, our analysis shows that all of them have a variable PSI score. AH, postnatal heart; AM, postnatal muscles; FH, fetal heart; FM, fetal muscles.

Figure 2

TTN exon usage. (A) Heat map showing the PSI index score of TTN exons in skeletal and cardiac muscles in prenatal and postnatal samples. (B) PSI graphical representation in prenatal (red) and postnatal (green) skeletal muscles. (C) PSI scores of the mutated exon in postnatal (x‐axis) and fetal (y‐axis) skeletal muscles correlate with the clinical phenotype and the disease course, showing the three clusters: pre‐ or perinatal death (P1, P2, and P3 in blue); improving disease course (P5–P8 in pink); and worsening disease course (P9–P11 in dark gray).