Advanced Genomic Tools in Translational Research of Neurodevelopmental Disorders

Sammanfattning: Neurodevelopmental disorders (NDDs) affect >4.7% of individuals world-wide. Most cases are expected to have an underlying genetic cause. However, only 36–40% of those affected get a genetic diagnosis through exome sequencing. In the last decades, a technological tsunami of advanced genomic tools has emerged with promising benefits over standard techniques, including identification of low frequency variants, large structural changes, repeats, haplotypes and methylation. The aim of this thesis was to improve diagnostics and functional characterizations of NDDs in translational research by using advanced genomic tools, and compare the results to those of standard strategies.In Paper I, we presented a novel strategy to investigate the parental origin, gonadal mosaicism and recurrence risk of two NDDs using single-molecule long-read sequencing and Droplet Digital PCR. The recurrence risk was estimated to be 37–40% in one NDD family, and <1% in another. Thus, the results were very different from the standard recurrence risk of 1% used in healthcare. In Paper II, we have developed and applied a novel method to investigate and quantify XCI using CRISPR-Cas9 enrichment followed by single-molecule long-read sequencing. The novel method has benefits over the standard PCR-based method when quantification of XCI is necessary, i.e., in samples with expression patterns <100:0. In Paper III, we presented two novel in vitro methods using single-molecule long-read sequencing to investigate the efficiency and accuracy of the CRISPR-Cas9 technology prior to a cell experiment. The novel methods illuminated Cas9 cleavage sites that would have been difficult to predict using standard computational in silico predictions or Digenome-seq and CIRCLE-Seq. In Paper IV, we presented an in-frame deletion in RBMX as the genetic cause to Gustavson syndrome in a five-generation family. The variant was detected with genome sequencing techniques, but not with standard exome sequencing. Extensive molecular characterization of Gustavson syndrome revealed a variant association to RNA processing and potentially reduced binding affinity to SH3-domains.In conclusion, this thesis has demonstrated the advantages of using advanced genomic tools over standard strategies in diagnostics and functional characterizations of NDDs. The results are useful in translational research of NDDs, by providing the affected families with a diagnosis, prognosis and improved care. The results will potentially lead to a better understanding of gene functions, disease mechanisms and therapy development.