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.

Download Full-text

Genome-wide detection of short tandem repeat expansions by long-read sequencing

BMC Bioinformatics ◽

10.1186/s12859-020-03876-w ◽

2020 ◽

Vol 21 (S21) ◽

Author(s):

Qian Liu ◽

Yao Tong ◽

Kai Wang

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Human Diseases ◽

Spinocerebellar Ataxia Type ◽

Human Populations ◽

Normal Ranges ◽

Long Read ◽

Repeat Expansions ◽

Genomic Scale ◽

Short Tandem

Abstract Background Short tandem repeat (STR), or “microsatellite”, is a tract of DNA in which a specific motif (typically < 10 base pairs) is repeated multiple times. STRs are abundant throughout the human genome, and specific repeat expansions may be associated with human diseases. Long-read sequencing coupled with bioinformatics tools enables the estimation of repeat counts for STRs. However, with the exception of a few well-known disease-relevant STRs, normal ranges of repeat counts for most STRs in human populations are not well known, preventing the prioritization of STRs that may be associated with human diseases. Results In this study, we extend a computational tool RepeatHMM to infer normal ranges of 432,604 STRs using 21 long-read sequencing datasets on human genomes, and build a genomic-scale database called RepeatHMM-DB with normal repeat ranges for these STRs. Evaluation on 13 well-known repeats show that the inferred repeat ranges provide good estimation to repeat ranges reported in literature from population-scale studies. This database, together with a repeat expansion estimation tool such as RepeatHMM, enables genomic-scale scanning of repeat regions in newly sequenced genomes to identify disease-relevant repeat expansions. As a case study of using RepeatHMM-DB, we evaluate the CAG repeats of ATXN3 for 20 patients with spinocerebellar ataxia type 3 (SCA3) and 5 unaffected individuals, and correctly classify each individual. Conclusions In summary, RepeatHMM-DB can facilitate prioritization and identification of disease-relevant STRs from whole-genome long-read sequencing data on patients with undiagnosed diseases. RepeatHMM-DB is incorporated into RepeatHMM and is available at https://github.com/WGLab/RepeatHMM.

Download Full-text

Diagnostics of short tandem repeat expansion variants using massively parallel sequencing and componential tools

European Journal of Human Genetics ◽

10.1038/s41431-018-0302-4 ◽

2018 ◽

Vol 27 (3) ◽

pp. 400-407 ◽

Cited By ~ 3

Author(s):

Rick H. de Leeuw ◽

Dominique Garnier ◽

Rosemarie M. J. M. Kroon ◽

Corinne G. C. Horlings ◽

Emile de Meijer ◽

...

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Massively Parallel Sequencing ◽

Repeat Expansion ◽

Massively Parallel ◽

Parallel Sequencing ◽

Short Tandem

Download Full-text

Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

10.1101/2021.09.27.21263187 ◽

2021 ◽

Author(s):

Igor Stevanovski ◽

Sanjog R. Chintalaphani ◽

Hasindu Gamaarachchi ◽

James M. Ferguson ◽

Sandy S. Pineda ◽

...

Keyword(s):

Tandem Repeat ◽

Tandem Repeats ◽

Fragile X ◽

Genetic Diagnosis ◽

Neuromuscular Diseases ◽

Nanopore Sequencing ◽

Molecular Tests ◽

Genetic Landscape ◽

Long Read ◽

Short Tandem

ABSTRACTShort-tandem repeat (STR) expansions are an important class of pathogenic genetic variants. Over forty neurological and neuromuscular diseases are caused by STR expansions, with 37 different genes implicated to date. Here we describe the use of programmable targeted long-read sequencing with Oxford Nanopore’s ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single, simple assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of expanded and non-expanded STR sites. In doing so, the assay correctly diagnoses all individuals in a cohort of patients (n = 27) with various neurogenetic diseases, including Huntington’s disease, fragile X syndrome and cerebellar ataxia (CANVAS) and others. Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing, and identifies non-canonical STR motif conformations and internal sequence interruptions. Even in our relatively small cohort, we observe a wide diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of STR expansion disorders. Finally, we show how the flexible inclusion of pharmacogenomics (PGx) genes as secondary ReadUntil targets can identify clinically actionable PGx genotypes to further inform patient care, at no extra cost. Our study addresses the need for improved techniques for genetic diagnosis of STR expansion disorders and illustrates the broad utility of programmable long-read sequencing for clinical genomics.One sentence summaryThis study describes the development and validation of a programmable targeted nanopore sequencing assay for parallel genetic diagnosis of all known pathogenic short-tandem repeats (STRs) in a single, simple test.

Download Full-text

Correction to: Genome-wide sequencing as a first-tier screening test for short tandem repeat expansions

Genome Medicine ◽

10.1186/s13073-021-00961-4 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Indhu-Shree Rajan-Babu ◽

Junran J. Peng ◽

Readman Chiu ◽

Patricia Birch ◽

Madeline Couse ◽

...

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Screening Test ◽

Genome Wide ◽

Repeat Expansions ◽

Short Tandem

Download Full-text

Enhanced detection of nucleotide repeat mRNA with hybridization chain reaction

10.1101/2021.01.06.425640 ◽

2021 ◽

Author(s):

Mary Rebecca Glineburg ◽

Yuan Zhang ◽

Elizabeth M Tank ◽

Sami Barmada ◽

Peter Todd

Keyword(s):

Fragile X ◽

Data Interpretation ◽

Repeat Expansion ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Cgg Repeat ◽

Hexanucleotide Repeat ◽

Rna Foci ◽

Repeat Expansions ◽

The Impact

RNAs derived from expanded nucleotide repeats form detectable foci in patient cells and these foci are thought to contribute to disease pathogenesis. The most widely used method for detecting RNA foci is fluorescence in situ hybridization (FISH). However, FISH is prone to low sensitivity and photo-bleaching that can complicate data interpretation. Here we applied hybridization chain reaction (HCR) as an alternative approach to repeat RNA foci detection of GC-rich repeats in two neurodegenerative disorders: GGGGCC (G4C2) hexanucleotide repeat expansions in C9orf72 that cause amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD) and CGG repeat expansions in FMR1 that cause Fragile X-associated tremor/ataxia syndrome. We found that HCR of both G4C2 and CGG repeats has comparable specificity to traditional FISH, but is >40x more sensitive and shows repeat-length dependence in its intensity. HCR is better than FISH at detecting both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. We used HCR to determine the impact of integrated stress response (ISR) activation on RNA foci number and distribution. G4C2 repeat RNA did not readily co-localize with the stress granule marker G3BP1, but ISR induction increased both the number of detectible nuclear RNA foci and the nuclear/cytoplasmic foci ratio in patient fibroblasts and patient derived neurons. Taken together, these data suggest that HCR can be a useful tool for detecting repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.

Download Full-text

Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing

Nature Biotechnology ◽

10.1038/s41587-019-0293-x ◽

2019 ◽

Vol 37 (12) ◽

pp. 1478-1481 ◽

Cited By ~ 18

Author(s):

Pay Giesselmann ◽

Björn Brändl ◽

Etienne Raimondeau ◽

Rebecca Bowen ◽

Christian Rohrandt ◽

...

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Nanopore Sequencing ◽

Methylation State ◽

Repeat Expansions ◽

Short Tandem

Download Full-text

Faculty Opinions recommendation of Glutaminase deficiency caused by short tandem repeat expansion in GLS.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.735525724.793574026 ◽

2020 ◽

Author(s):

Guy Rouleau

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Repeat Expansion ◽

Short Tandem

Download Full-text

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion inGLS

New England Journal of Medicine ◽

10.1056/nejmc1907427 ◽

2019 ◽

Vol 381 (12) ◽

pp. 1185-1185 ◽

Cited By ~ 2

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Repeat Expansion ◽

Short Tandem

Download Full-text

Development of a multiplex PCR short tandem repeat typing scheme for Candida krusei

Journal of Clinical Microbiology ◽

10.1128/jcm.02032-21 ◽

2021 ◽

Author(s):

Merlijn H.I. van Haren ◽

Theun de Groot ◽

Bram Spruijtenburg ◽

Kusum Jain ◽

Anuradha Chowdhary ◽

...

Keyword(s):

Multiplex Pcr ◽

Tandem Repeat ◽

Short Tandem Repeat ◽

Cost Effective ◽

Candida Krusei ◽

Str Typing ◽

Str Markers ◽

Typing Scheme ◽

Snp Calling ◽

Short Tandem

Candida krusei is a human pathogenic yeast that can cause candidemia with the lowest 90-day survival rate in comparison to other Candida species. Infections occur frequently in immunocompromised patients and several C. krusei outbreaks in health care facilities have been described. Here, we developed a short tandem repeat (STR) typing scheme for C. krusei to allow for fast and cost-effective genotyping of an outbreak and compared identified relatedness of ten isolates to SNP calling from whole-genome sequencing (WGS). From a selection of 14 novel STR markers, six were used to develop two multiplex PCRs. Additionally, three previously reported markers were selected for a third multiplex PCR. In total, 119 C. krusei isolates were typed using these nine markers and 79 different genotypes were found. STR typing correlated well with WGS SNP typing, as isolates with the same STR genotype varied by 8 and 19 SNPs, while isolates that differed in all STR markers varied at least tens of thousands of SNPs. The STR typing assay was found to be specific for C. krusei , stable in 100 subcloned generations, and comparable to SNP calling by WGS. In summary, this newly developed C. krusei STR typing scheme is a fast, reliable, easy-to-interpret and cost-effective method compared to other typing methods. Moreover, the two newly developed multiplexes showed the same discriminatory power as all nine markers combined, indicating that multiplexes M3-1 and M9 are sufficient to type C. krusei .

Download Full-text