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Acta Neuropathologica Communications May 2021Short tandem repeat (STR) expansion disorders are an important cause of human neurological disease. They have an established role in more than 40 different phenotypes... (Review)
Review
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.
Topics: Animals; DNA Repeat Expansion; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Nervous System Diseases; Sequence Analysis, DNA
PubMed: 34034831
DOI: 10.1186/s40478-021-01201-x -
Genome Medicine Mar 2021We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center...
BACKGROUND
We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting.
METHODS
Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams.
RESULTS
Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange.
CONCLUSIONS
Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.
Topics: Cohort Studies; DNA Copy Number Variations; Delivery of Health Care; Genetic Heterogeneity; Genomics; High-Throughput Nucleotide Sequencing; Humans; Information Dissemination; Inheritance Patterns; Microsatellite Repeats; Mutation; Rare Diseases; Sweden; Uniparental Disomy; Whole Genome Sequencing
PubMed: 33726816
DOI: 10.1186/s13073-021-00855-5 -
International Journal of Molecular... Jul 2019Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy... (Review)
Review
Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant (DM1) and (DM2) genes results in the synthesis of CUG and CCUG repeat expansion (CUG, CCUG) RNAs, respectively. These CUG and CCUG RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.
Topics: Alternative Splicing; Animals; Cell Nucleus; Humans; Microsatellite Repeats; Muscle, Skeletal; Myotonic Dystrophy; RNA; Ribonucleoproteins; Trinucleotide Repeat Expansion
PubMed: 31323950
DOI: 10.3390/ijms20133365 -
Science Advances Mar 2022More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use...
More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. 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 assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort ( = 37) including patients with various neurogenetic diseases ( = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.
Topics: Alleles; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Nanopore Sequencing; Sequence Analysis, DNA
PubMed: 35245110
DOI: 10.1126/sciadv.abm5386 -
Microbiology Spectrum Oct 2022Candida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To...
Candida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To take adequate measures during an outbreak, it is essential to understand the transmission route, which requires isolate genotyping. In 2019, a short tandem repeat (STR) genotyping analysis was developed for C. auris. To determine the discriminatory power of this method, we performed STR analysis of 171 isolates with known whole-genome sequencing (WGS) data using Illumina reads, and we compared their resolutions. We found that STR analysis separated the 171 isolates into four clades (clades I to IV), as was also seen with WGS analysis. Then, to improve the separation of isolates in clade IV, the STR assay was optimized by the addition of 2 STR markers. With this improved STR assay, a total of 32 different genotypes were identified, while all isolates with differences of >50 single-nucleotide polymorphisms (SNPs) were separated by at least 1 STR marker. Altogether, we optimized and validated the C. auris STR panel for clades I to IV and established its discriminatory power, compared to WGS SNP analysis using Illumina reads. The emerging fungal pathogen Candida auris poses a threat to public health, mainly causing outbreaks in intensive care units. Genotyping is essential for investigating potential outbreaks and preventing further spread. Previously, we developed a STR genotyping scheme for rapid and high-resolution genotyping, and WGS SNP outcomes for some isolates were compared to STR data. Here, we compared WGS SNP and STR outcomes for a larger sample cohort. Also, we optimized the resolution of this typing scheme with the addition of 2 STR markers. Altogether, we validated and optimized this rapid, reliable, and high-resolution typing scheme for C. auris.
Topics: Humans; Candida; Candidiasis; Antifungal Agents; Candida auris; Microbial Sensitivity Tests; Microsatellite Repeats
PubMed: 36190407
DOI: 10.1128/spectrum.02645-22 -
Genes Dec 2021Forensic investigation is important to analyze evidence and facilitate the search for key individuals, such as suspects and victims in a criminal case. The forensic use...
Forensic investigation is important to analyze evidence and facilitate the search for key individuals, such as suspects and victims in a criminal case. The forensic use of genomic DNA has increased with the development of DNA sequencing technology, thereby enabling additional analysis during criminal investigations when additional legal evidence is required. In this study, we used next-generation sequencing to facilitate the generation of complementary data in order to analyze human evidence obtained through short tandem repeat (STR) analysis. We examined the applicability and potential of analyzing microbial genome communities. Microbiological supplementation information was confirmed for two of four failed STR samples. Additionally, the accuracy of the gargle sample was confirmed to be as high as 100% and was highly likely to be classified as a body fluid sample. Our experimental method confirmed that anthropological and microbiological evidence can be obtained by performing two experiments with one extraction. We discuss the advantages and disadvantages of using these techniques, explore prospects in the forensic field, and highlight suggestions for future research.
Topics: Adult; Bacteria; DNA Fingerprinting; DNA, Bacterial; Female; High-Throughput Nucleotide Sequencing; Humans; Male; Microbiota; Microsatellite Repeats; Young Adult
PubMed: 35052425
DOI: 10.3390/genes13010085 -
Genome Biology Aug 2018Short tandem repeat (STR) expansions have been identified as the causal DNA mutation in dozens of Mendelian diseases. Most existing tools for detecting STR variation...
Short tandem repeat (STR) expansions have been identified as the causal DNA mutation in dozens of Mendelian diseases. Most existing tools for detecting STR variation with short reads do so within the read length and so are unable to detect the majority of pathogenic expansions. Here we present STRetch, a new genome-wide method to scan for STR expansions at all loci across the human genome. We demonstrate the use of STRetch for detecting STR expansions using short-read whole-genome sequencing data at known pathogenic loci as well as novel STR loci. STRetch is open source software, available from github.com/Oshlack/STRetch .
Topics: Alleles; Chromosomes, Human; DNA Repeat Expansion; Genetic Loci; Genome, Human; Humans; Microsatellite Repeats; Polymerase Chain Reaction; Software
PubMed: 30129428
DOI: 10.1186/s13059-018-1505-2 -
Disease Models & Mechanisms Oct 2023Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple...
Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple passages (often indefinitely), allowing multiple experiments to be performed. However, over time, cell line identity and purity can be compromised by human errors. Cross-contamination from other cell lines and complete misidentification are both possible. Routine cell line authentication is a necessary preventive measure and has become a requirement for many funding applications and publications. Short tandem repeat (STR) profiling is the most common method for cell line authentication and is usually carried out using standard polymerase chain reaction-capillary electrophoresis analysis (STR-CE). Here, we evaluated next-generation sequencing (NGS)-based STR profiling of human and mouse cell lines at 18 and 15 loci, respectively, in a high-throughput format. Using the Python program STRight, we demonstrate that NGS-based analysis (STR-NGS) is superior to standard STR-CE in terms of the ability to report the sequence context of repeat motifs, sensitivity and flexible multiplexing capability. STR-NGS is thus a valuable alternative for cell line authentication.
Topics: Mice; Animals; Humans; Cell Line Authentication; Microsatellite Repeats; Cell Line; High-Throughput Nucleotide Sequencing
PubMed: 37712227
DOI: 10.1242/dmm.050150 -
Immunity, Inflammation and Disease Sep 2020Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human...
INTRODUCTION
Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice.
METHODS
In this study, we compared the use of CD34 HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR).
RESULTS
It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdc IL2rγ /SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model.
CONCLUSION
From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells.
Topics: Animals; Female; Hematopoietic Stem Cell Transplantation; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred NOD; Microsatellite Repeats; Technology
PubMed: 32525618
DOI: 10.1002/iid3.317 -
Scientific Reports Jun 2022Detection of short tandem repeat (STR) expansions with standard short-read sequencing is challenging due to the difficulty in mapping multicopy repeat sequences. In this...
Detection of short tandem repeat (STR) expansions with standard short-read sequencing is challenging due to the difficulty in mapping multicopy repeat sequences. In this study, we explored how the long-range sequence information of barcode linked-read sequencing (BLRS) can be leveraged to improve repeat-read detection. We also devised a novel algorithm using BLRS barcodes for distance estimation and evaluated its application for STR genotyping. Both approaches were designed for genotyping large expansions (> 1 kb) that cannot be sized accurately by existing methods. Using simulated and experimental data of genomes with STR expansions from multiple BLRS platforms, we validated the utility of barcode and phasing information in attaining better STR genotypes compared to standard short-read sequencing. Although the coverage bias of extremely GC-rich STRs is an important limitation of BLRS, BLRS is an effective strategy for genotyping many other STR loci.
Topics: Algorithms; High-Throughput Nucleotide Sequencing; Microsatellite Repeats; Sequence Analysis, DNA
PubMed: 35672336
DOI: 10.1038/s41598-022-13024-4