The importance of an integrated genotype-phenotype strategy to unravel the molecular bases of titinopathies

Abstract

Next generation sequencing (NGS) has allowed the titin gene ( TTN ) to be identified as a major contributor to neuromuscular disorders, with high clinical heterogeneity. The mechanisms underlying the phenotypic variability and the dominant or recessive pattern of inheritance are unclear. Titin is involved in the formation and stability of the sarcomeres. The effects of the different TTN variants can be harmless or pathogenic (recessive or dominant) but the interpretation is tricky because the current bioinformatics tools can not predict their functional impact effectively. Moreover, TTN variants are very frequent in the general population. The combination of deep phenotyping associated with RNA molecular analyses, western blot (WB) and functional studies is often essential for the interpretation of genetic variants in patients suspected of titinopathy. In line with the current guidelines and suggestions, we implemented for patients with skeletal myopathy and with potentially disease causing TTN variant(s) an integrated genotype-transcripts-protein-phenotype approach, associated with phenotype and variants segregation studies in relatives and confrontation with published data on titinopathies to evaluate pathogenic effects of TTN variants (even truncating ones) on titin transcripts, amount, size and functionality. We illustrate this integrated approach in four patients with recessive congenital myopathy.

Novel dominant distal titinopathy phenotype associated with copy number variation

“…Proteins solubilization from muscle biopsies and WB analyses on agarose and acrylamide gel were performed as described in [2]. Primary antibodies, anti‐titin specific for C‐terminal part M10‐1 (Rabbit; 1:1000; kindly provided by Dr Isabelle Richard 13 ), and anti‐titin N‐terminal (Mouse; 1:1000; Sigma SAB1400284, St‐Louis, MO) were incubated overnight at 4°C in Odyssey ® blocking buffer in PBS.…”

“…mRNAs extraction from muscle biopsy, RT‐PCR and Sanger sequencing, and RNAseq experiments were performed as previously described 2 . Primers used for cDNA sequencing are indicated in Table S1.…”

“…The mode of inheritance is either autosomal dominant or recessive. The combination of deep phenotyping associated with RNA molecular analyses, western blot (WB), and functional studies is often essential for the interpretation of TTN variants 2–5 . Autosomal dominant TTN mutations cause two skeletal muscle disorders.…”

Le Consortium Titine France : mieux comprendre pour mieux diagnostiquer

“…Les discussions toujours très riches permettent de développer des programmes de recherche. Par exemple, le Dr Mireille Cossée, a mis en place une étude financée en grande partie par l'AFM-Téléthon dont le but est « l'Amélioration du DIAgnostic et l'étude de corrélation phénotype-génotype chez des patients MYOpathes suspects de TITinopathie en errance diagnostique » [3]. Les inclusions des patients, initialement loco-régionales, ont pu être étendues au niveau national grâce au travail collaboratif issu du consortium.…”

An Integrated Clinical-Biological Approach to Identify Interindividual Variability and Atypical Phenotype-Genotype Correlations in Myopathies: Experience on A Cohort of 156 Families

“…This procedure was particularly important to characterize pathogenicity in complex genes, such as TTN and NEB. We identified exon skipping in transcripts carrying the splicing variant, and also activation of exonic or intronic cryptic splice sites, leading to aberrant transcripts (Figure 2, and [13]). We could perform variants segregation analyses in 58 families.…”