Advancing Neurogenetic Diagnoses through Long-Read Sequencing – NRGEN-NGS3

Hereditary neurological diseases present significant clinical challenges due to overlapping clinical symptoms and extensive genetic diversity, often resulting in complex diagnostic journeys for patients. One common mutational mechanism involves nucleotide repeat expansions, whose prevalence has been steadily increasing. These expansions must be considered in the presence of several clinical entry points dominated by progressive cognitive disorders and/or abnormal movements.
Current diagnostic approaches rely on algorithms tailored to specific clinical presentations, requiring sequential genetic analyses of targeted loci across multiple consultations and dispatching samples to various local and international molecular genetic laboratories. The recent advancement of long-read sequencing technology from Oxford Nanopore offers a promising and innovative avenue for investigating nucleotide repeat expansions. Our project has then 2 main objectives, to enhance the genetic diagnosis of such mutational events and to identify novel candidate genes or repetitive sequences in neurogenetic conditions.
Our first objective aims to utilize this breakthrough technology for diagnostic purposes across all known loci housing nucleotide repeats involved in human diseases within a single experiment. Our approach involves utilizing an enrichment technology via CRISPR-CAS9 and comparing it to other strategies, including the cutting-edge optical genome mapping (Bionano) and the adaptive bioinformatic sampling. These methodologies will be applied to 120 carriers of established repeat expansions and are based on robust preliminary results. Additionally, our project will also help better define the threshold and composition of large repeats leveraging the main advantage offered by the long-read sequencing to allow full analysis of these repeat sequences.
Our second objective relies on the fact that a substantial portion of patients still lack a molecular diagnosis in neurogenetics. We aim to uncover new abnormal repeat expansions using the same technologies in a separate cohort of patients affected by candidate neurological conditions for these repeats, where genomic data have not revealed mutations or expansions in known genes. We will combine long-read sequencing with analysis of the methylation and a transcriptomic approach to improve our capacity to pinpoint novel causative genes/repeats. Validation of their involvement in the pathological process will involve genetic and in vitro cell biology analyses, including segregation analysis, replication in other cohorts, and manipulation of gene expression levels (over or down regulation of expression), in order to link the genetic anomaly to the phenotype. The consortium has already secured the samples from 38 families in this quest.
This project addresses critical needs in clinical practice by enhancing diagnosis and uncovering novel repeat-associated genes. It also promises to advance our understanding of genomic repeat expansions, providing insights into complex neurological conditions and facilitating progress in neurodegeneration research. The consortium, consisting of three teams with strong expertise in neurogenetics, has already identified 25 causative genes in the past and possesses the necessary skills for all aspects of the project, including long-read sequencing, methylation studies, optical genome mapping, transcriptomics, bioinformatics and cell biology. The 3 teams are also heavily involved in disseminating scientific results to patients and non-professional organizations.