Abnormal expansion of nucleotide repeats was first identified 34 years ago as a unique mutational mechanism. It is now linked to numerous neurogenetic disorders, several of which discovered only recently. The identification of these expansions has led to various classifications based on clinical presentation, repeat nature and genomic location (coding or non-coding regions). Precise diagnosis of these conditions relies on molecular testing, currently performed on a gene-by-gene basis. Their analysis remains challenging, especially for long expansions. We evaluated CRISPR-Cas9-mediated target enrichment coupled to Oxford Nanopore Technologies (ONT) long read sequencing, to accelerate and improve the time-consuming molecular diagnosis of repeat expansion disorders. We simultaneously targeted nine loci involved in 10 repeat expansion disorders in a single capture panel, including
FMR1
,
HTT
,
DMPK
,
CNBP/ZNF9
,
ATXN2
,
JPH3
,
FXN
,
C9ORF72
and
RFC1
, covering a broad range of repeat types, sizes and diagnostic needs. Results were compared with standard routine testing methods. ONT sequencing using Flongle flow cells yielded results consistent with standard techniques for most loci, particularly for non-complex repeats. However, limitations were observed for structurally complex regions such as
RFC1
, and inter-run variability required the aggregation of multiple Flongle runs per sample to achieve robust genotyping. These findings highlight both the potential and current limitations of CRISPR-Cas9-enriched ONT sequencing for multiplex diagnosis of repeat expansion disorders in a clinical setting. The approach deserves further development, particularly optimisation of protocols, inclusion of larger sample sizes, and comparison with alternative technologies.
P. Fergelot, C. Boury, B. Penaud et al.· Scientific Reports· 0 citations
NFIC-related disorder represents a novel neurodevelopmental syndrome characterized by intellectual disability and macrocephaly, highlighting the importance of NFIC dosage supporting a mirror-syndrome model.
Nathalie Vanden Eynde, L. Hérissant, E. Landais et al.· Clinical Genetics· 0 citations
Variants in BRSK2, encoding brain specific kinase-2, have recently been associated with an autosomal dominant neurodevelopmental disorder (NDD). We have assembled 52 cases with heterozygous BRSK2 variants and variable neurodevelopmental phenotypes with frequent neuropsychiatric and behavioral symptoms. The variant spectrum included 15 different truncating variants, seven (potential) splice variants, three structural variants, and 12 different missense variants. Of the missense variants, seven were in the kinase domain, and the others in the UBA and the KA1 domain or outside domains. Variants occurred de novo in 19 cases and were inherited in 18. We utilized Drosophila melanogaster as a model and assessed viability and performed climbing and bang sensitivity assays upon knockdown of the fly orthologue sff or upon overexpression of wildtype or mutant human BRSK2. Pan-neuronal knockdown of sff resulted in impaired locomotor behavior and seizure susceptibility. Ubiquitous or pan-neuronal overexpression of human wildtype BRSK2 in Drosophila resulted in lethality or locomotor impairment, respectively, indicating toxicity. Overexpressing mutant BRSK2 did not or incompletely affect viability and locomotor behavior for six of seven tested kinase domain missense variants and one KA1 domain variant, indicating a (partial) loss-of-function effect. Interestingly, overexpressing BRSK2 with the remaining missense variant from the kinase domain and the two most C-terminal missense variants resulted in possible gain of function. Our findings further delineate the clinical and molecular spectrum of BRSK2-associated NDD and provide further insights into the role of BRSK2/sff in nervous system function and dysfunction.
Palak Singhal, Tzung-Chien Hsieh, Nadja Ehmke et al.· European Journal of Human Ge...· 0 citations