Integrative diagnosis of patients with unresolved neuromuscular diseases and establishment of their iPSC

Abstract

Rare diseases collectively as a group are not that rare. They affect the lives of nearly 5% of the general population which translates to over 350 million patients worldwide. It is estimated that 80% of rare diseases have a genetic component. However, despite the advancement in next generation sequencing, genomics centric efforts only manage to solve around 35% of cases with the rest remaining undiagnosed. The challenges faced by rare disease families on the path to a conclusive diagnosis have been termed as 'diagnostic odyssey'. The absence of a confirmed diagnosis can significantly impact both patients and their families. It may lead to patients missing treatment opportunities for precision medicine and facing lifelong consequences. More recently, RNA sequencing (RNA-seq) has emerged as a complementary assay to help improve the diagnostic yield (by up to 35 %). It enables the detection of perturbations in the transcriptome. This includes aberrant splicing events, outlying genes and isoform usage, and allelic imbalance at heterozygotic sites. Altogether this information can implicate candidate disease-causal genetic variants and thus simplify disease causality inference. However, a particular challenge in establishing RNA-seq as a routine diagnostic tool for rare diseases lies in the availability of disease relevant tissue for transcriptomic profiling. Neuromuscular disorders overcome this challenge, as the collection of muscle tissue via needle biopsy for histopathological evaluation is a key diagnostic step for challenging cases. This study takes advantage of the availability of muscle tissue from unsolved patients in an effort to help reach a confirmed diagnosis. To this end, RNA-seq was conducted on these samples and the data was co-analyzed with familial DNA sequencing data. The results of the multi-omics effort were interpreted for each patient in light of their specific clinical information. Many large genes play a pivotal role in normal muscle function. The RNA-Seq data allowed the precise detection of perturbed isoforms at base pair resolution in these genes. This is particularly meaningful in assessing the impact of loss-of-function mutations which can either downregulate the whole gene or particular isoforms. Disease relevant inferences from omics data were validated in independent assays. Through this effort we were able to diagnose patients with mutations in TTN, COL6A1, COL6A2, DMD, TBCK and POMT1. In addition, we identified viable candidate mutations in PLEC in a still unsolved case. For future work, we have narrowed our focus to a subset of TTN metatranscript-only mutant patients with a unique isoform usage pattern. Patient-derived induced pluripotent stem cells (iPSCs) have been established for these patients and control donors. This will provide an opportunity to examine the mechanical abnormalities in skeletal and cardiac tissue through in vitro differentiation of these cells. Overall, this thesis presents the results of 9 diagnosed patients who had previously failed to receive a confirmed diagnosis.