G1. Recruiting, biobanking and deep clinical phenotyping in presymptomatic and symptomatic RND patients. Our special emphasis will be the enrolment of affected individuals with unique (ultra) rare neurological phenotypes of unknown genetic basis, molecularly undiagnosed patients with myopathies,
amyotrophic lateral sclerosis and inherited neuropathies, ataxias and spastic paraplegias, neurodevelopmental disorders and complex neurological phenotypes, as well as carriers of TTR p.Glu89Gln causing inherited amyloidosis. We aim to obtain biomaterials from the study participants,
store them and make them available for genomic, transcriptomic and biomarker studies. Furthermore, we plan to longitudinally follow the patients and integrate their clinical findings into database of Biobank of Molecular Medicine Centre and patient registry of Medical University-Sofia, and existing international registries.

G2. Genomics studies to increase the number of known gene loci for the most heterogeneous RND groups. We will apply the latest advances in the next-generation sequencing and bioinformatics technologies. Both whole exome (WES) and whole genome (WGS) sequencing will allow untargeted genetic analyses leading to identification of causal mutations, mostly single nucleotide variants (SNVs) and small indels. Due to the “short reads”-nature of these technologies, however, it will be extremely challenging to detect large genomic rearrangements and nucleotide expansions/contractions of repetitive regions that might account for the missing heritability in our RND cohorts. There is growing evidence that the extent and complexity of these ‘structural variants’ (SVs) have been immensely underestimated and that SVs could be a major player in disease pathogenesis. Therefore, in selected WES/WGS-negative cases, we will apply long-read third-generation sequencing as a new powerful gene
discovery tool.

G3. Elucidation of pathogenesis using transcriptomics and cell biological studies. Transcriptomics (deep RNAseq) will allow detecting quantitative differences in gene expression that will augment the genomics
studies to identify promoter and enhancer variants, to validate the functional impact of SVs, and to point to misregulated cellular pathways. The differential expression analysis will be performed in patient cells, including fibroblasts, PBMCs or muscle/nerve biopsy specimens. In specific cases, patient fibroblasts will be transdifferentiated into induced neurons (iNeurons) to account for tissue-specific effects. When the identified variants are in protein-coding gene candidates, we will also study protein expression using immunoblotting and immunostaining techniques.

G4. Discovery of diagnostic and prognostic biomarkers for transthyretin amyloidosis (ATTR). The possibility to monitor the onset of ATTR is of particular importance for the Bulgarian medical community. ATTR is a genetic polyneuropathy amenable to personalized therapy. Notably, it is substantially more common among Bulgarian patients, as compared to its European prevalence, making Bulgaria an endemic region with 360 patients from 130 families identified so far. Moreover, there is significant variability in phenotype expression among carriers of the TTR p.Glu89Gln. We will perform
transcriptome analysis of 160 deeply phenotyped p.Glu89Gln carriers in order to identify molecular signatures that have the potential to become early diagnostic and prognostic biomarkers for this disease.