Long-read Genome Sequencing for the Molecular Diagnosis of Dystonia

Dystonia is a motor disorder characterized by abnormal movements and/or postures. It is caused by involuntary, intermittent, or sustained muscle contractions. Dystonia can affect all body segments, leading to highly variable and often severe functional impairment with a significant burden on healthcare systems.

The familial nature of some generalized dystonias was recognized as early as the 1940s. With the development and increased availability of high-throughput DNA sequencing technologies since the early 2010s, there has been an exponential rise in the discovery of genes associated with neurological phenotypes in which dystonia is either the main manifestation or part of a broader clinical picture. Dystonia is now known to be associated with all modes of inheritance, including autosomal dominant forms (e.g., TOR1A, KMT2B), autosomal recessive forms often presenting with complex phenotypes, X-linked dominant (e.g., WDR45) and recessive (e.g., TAF1) forms, and even mitochondrial inheritance.

To date, over 100 genes have been linked to dystonic phenotypes, highlighting the significant genetic heterogeneity of this condition. Many genetically determined dystonic syndromes are associated with structural variants (SVs) or copy number variations (CNVs), which are difficult to detect using routine short-read sequencing technologies.

Molecular diagnosis is crucial in the management of dystonia. It provides a definitive and precise diagnosis, bringing an end to the diagnostic odyssey for patients and their families. It also enables accurate genetic counseling, offering information on inheritance risks and the potential for other family members to be affected. In some cases, a molecular diagnosis directly influences therapeutic decisions. For example, pathogenic variants in GNAO1 are associated with early-onset dystonic syndromes that respond well to deep brain stimulation of the globus pallidus. Specific symptomatic treatments have been proposed for certain dystonias, such as levodopa in dopa-responsive dystonia. At the population level, identifying new genes or molecular causes can reveal novel pathogenic mechanisms and facilitate the development of animal or cellular models, paving the way for future gene therapies.

Over the past decade, the growing availability of short-read DNA sequencing technologies has enabled the simultaneous analysis of numerous dystonia-associated genes. In routine diagnostics, short-read sequencing involves fragmenting DNA into small pieces (usually a few hundred base pairs), which are then sequenced from both ends.

This approach has limitations, particularly for detecting repeat expansions that exceed the size of the sequenced fragments. These limitations may partly explain why a relatively small proportion of dystonia cases receive a molecular diagnosis compared to other neurological disorders like cerebellar ataxias or hereditary spastic paraplegias. Overall, more than 70% of dystonia patients sequenced via short-read WGS remain without a molecular diagnosis, despite strong clinical indicators of a genetic etiology.

To overcome these limitations, long-read DNA sequencing technologies have been developed. Unlike short-read methods, long-read sequencing preserves native DNA fragment sizes, which can span thousands of bases. One such approach (Oxford Nanopore) sequences DNA based on ionic current changes as the molecule passes through a nanometer-scale pore. Long-read sequencing offers several advantages. Like short-read methods, it detects small variants (SNVs and indels). However, it also excels at identifying structural variants, CNVs, methylation changes, and especially repeat expansions-key elements often missed by standard methods.

In neurogenetics, long-read sequencing has enabled the accurate detection and quantification of known repeat disorders (e.g., C9orf72 hexanucleotide expansions, RFC1), as well as the discovery of new disease-causing repeat expansions (e.g., GGC repeats in NOTCH2NLC).

It also allows phasing of alleles (cis or trans configurations) without requiring family segregation studies, which is particularly useful in recessive conditions.

Given the current diagnostic limitations of short-read sequencing, the emergence of newly identified repeat expansions, and the demonstrated advantages of long-read sequencing, the investigators hypothesize that a significant proportion of patients with undiagnosed dystonia could benefit from whole-genome long-read sequencing (lrWGS).

Such an approach could, in a single test, overcome many of the key diagnostic blind spots of short-read methods, particularly for detecting repeat expansions and structural variants. This would not only end the diagnostic odyssey for many patients, but also enable appropriate genetic counseling, access to reproductive options such as prenatal or preimplantation diagnosis, and even influence treatment strategies.