An update on the neurological short tandem repeat expansion disorders and the emergence of long-read sequencing diagnostics

Abstract

Background: Short tandem repeat (STR) expansion disorders are an important cause of human neurological disease. They have an established role in more than 40 different phenotypes including the myotonic dystrophies, Fragile X syndrome, Huntington's disease, the hereditary cerebellar ataxias, amyotrophic lateral sclerosis and frontotemporal dementia.

Main body: STR expansions are difficult to detect and may explain unsolved diseases, as highlighted by recent findings including: the discovery of a biallelic intronic 'AAGGG' repeat in RFC1 as the cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS); and the finding of 'CGG' repeat expansions in NOTCH2NLC as the cause of neuronal intranuclear inclusion disease and a range of clinical phenotypes. However, established laboratory techniques for diagnosis of repeat expansions (repeat-primed PCR and Southern blot) are cumbersome, low-throughput and poorly suited to parallel analysis of multiple gene regions. While next generation sequencing (NGS) has been increasingly used, established short-read NGS platforms (e.g., Illumina) are unable to genotype large and/or complex repeat expansions. Long-read sequencing platforms recently developed by Oxford Nanopore Technology and Pacific Biosciences promise to overcome these limitations to deliver enhanced diagnosis of repeat expansion disorders in a rapid and cost-effective fashion.

Conclusion: We anticipate that long-read sequencing will rapidly transform the detection of short tandem repeat expansion disorders for both clinical diagnosis and gene discovery.

Keywords: Clinical; Diagnosis; Disease; Expansion; Genetics; Long-read; Neurological; Repeats; Sequencing; Tandem.

Fig. 1

Healthy and pathogenic ranges in neurological short tandem repeat expansion disorders. Box plot indicates the range of observed sizes for the pathogenic STR in known neurological STR expansion disorders (see Table 1). For each disorder, the range of STR sizes observed among unaffected individuals is shown in black, and the sizes observed in affected individuals is shown in pink

Fig. 2

Rate of discovery of neurological short tandem repeat expansions. Bar plot indicates the number of new pathogenic STR expansion discoveries published each year during the period 1990–2021 (see Table 1 for references to original publications for each gene)

Fig. 3

Current molecular diagnostic methods. Flow chart shows an example of two current diagnostic methods for diagnosing STR expansions: Southern blot and repeat-primed PCR. The sample analysis shown in both diagnostic methods was taken from a patient with Friedrich’s ataxia with a heterozygous ‘GAA’ expansion in the FXN gene (approximately 90 and 900 repeats). The RP-PCR graph shows the characteristic tailing/stuttering pattern of expanded alleles caused by the repeat-primed probes binding to more sites within the STR expansion. For sizing, Southern blot is performed. The larger 900-repeat ‘GAA’ allele cannot be seen using the Southern blot sizing ladder shown above

Fig. 4

NGS and Long-read sequencing for diagnosing short tandem repeat expansions. Flow chart shows the use of short-read NGS and two long-read sequencing methods for genotyping STR expansions: PacBio single-molecule real-time (SMRT) sequencing and Oxford Nanopore Technology (ONT) long-read sequencing. The alignment of reads to the genome can be seen for all three methods; short-reads are ‘tiled’ together to estimate the repeat size and sequence, while long reads easily span repeat and flanking regions. Nanopore sequencing high error rates can be overcome via sufficient coverage