Short Tandem Repeat Expansions Research Articles

An Update on the Adult-Onset Hereditary Cerebellar Ataxias: Novel Genetic Causes and New Diagnostic Approaches.

The hereditary cerebellar ataxias (HCAs) are rare, progressive neurologic disorders caused by variants in many different genes. Inheritance may follow autosomal dominant, autosomal recessive, X-linked or mitochondrial patterns. The list of genes associated with adult-onset cerebellar ataxia is continuously growing, with several new genes discovered in the last few years. This includes short-tandem repeat (STR) expansions in RFC1, causing cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), FGF14-GAA causing spinocerebellar ataxia type 27B (SCA27B), and THAP11. In addition, the genetic basis for SCA4, has recently been identified as a STR expansion in ZFHX3. Given the large and growing number of genes, and different gene variant types, the approach to diagnostic testing for adult-onset HCA can be complex. Testing methods include targeted evaluation of STR expansions (e.g. SCAs, Friedreich ataxia, fragile X-associated tremor/ataxia syndrome, dentatorubral-pallidoluysian atrophy), next generation sequencing for conventional variants, which may include targeted gene panels, whole exome, or whole genome sequencing, followed by various potential additional tests. This review proposes a diagnostic approach for clinical testing, highlights the challenges with current testing technologies, and discusses future advances which may overcome these limitations. Implementing long-read sequencing has the potential to transform the diagnostic approach in HCA, with the overall aim to improve the diagnostic yield.

Short tandem repeat expansions in cortical layer-specific genes implicate in phenotypic severity and adaptability of autism spectrum disorder.

Short tandem repeats (STRs) are repetitive DNA sequences and highly mutable in various human disorders. While the involvement of STRs in various genetic disorders has been extensively studied, their role in autism spectrum disorder (ASD) remains largely unexplored. In this study, we aimed to investigate genetic association of STR expansions with ASD using whole genome sequencing (WGS) and identify risk loci associated with ASD phenotypes. We analyzed WGS data of 634 ASD families and performed genome-wide evaluation for 12,929 STR loci. We found rare STR expansions that exceeded normal repeat lengths in autism cases compared to unaffected controls. By integrating single cell RNA and ATAC sequencing datasets of human postmortem brains, we prioritized STR loci in genes specifically expressed in cortical development stages. A deep learning method was used to predict functionality of ASD-associated STR loci. In ASD cases, rare STR expansions predominantly occurred in early cortical layer-specific genes involved in neurodevelopment, highlighting the cellular specificity of STR-associated genes in ASD risk. Leveraging deep learning prediction models, we demonstrated that these STR expansions disrupted the regulatory activity of enhancers and promoters, suggesting a potential mechanism through which they contribute to ASD pathogenesis. We found that individuals with ASD-associated STR expansions exhibited more severe ASD phenotypes and diminished adaptability compared to non-carriers. Short tandem repeat expansions in cortical layer-specific genes are associated with ASD and could potentially be a risk genetic factor for ASD. Our study is the first to show evidence of STR expansion associated with ASD in an under-investigated population.

Evaluation of Automated Magnetic Bead-Based DNA Extraction for Detection of Short Tandem Repeat Expansions With Nanopore Sequencing.

Long-read technologies such as nanopore sequencing provide new opportunities to detect short tandem repeat expansions. Therefore, a DNA extraction method is necessary that minimizes DNA fragmentation and hence allows the identification of large repeat expansions. In this study, an automated magnetic bead-based DNA extraction method and the required EDTA blood storage conditions as well as DNA and sequencing quality were evaluated for their suitability for repeat expansion detection with nanopore sequencing. DNA was extracted from EDTA blood, and subsequently, its concentration, purity, and integrity were assessed. DNA was then subjected to nanopore sequencing, and quality metrics of the obtained sequencing data were evaluated. DNA extracted from fresh EDTA blood as well as from cooled or frozen EDTA blood revealed high DNA integrity whereas storage at room temperature over 7 days had detrimental effects. After nanopore sequencing, the read length N50 values of approximately 9 kb were obtained, and based on adaptive sampling of samples with a known repeat expansion, repeat expansions up to 10 kb could be detected. The automated magnetic bead-based DNA extraction was sufficient to detect short tandem repeat expansions, omitting the need for high-molecular-weight DNA extraction methods. Therefore, DNA should be extracted either from fresh blood or from blood stored in cooled or frozen conditions. Consequently, this study may help other laboratories to evaluate their DNA extraction method regarding the suitability for detecting repeat expansions with nanopore sequencing.

Cost-Effective Cas9-Mediated Targeted Sequencing of Spinocerebellar Ataxia Repeat Expansions

Hereditary repeat diseases are caused by an abnormal expansion of short tandem repeats in the genome. Among them, spinocerebellar ataxia (SCA) is a heterogeneous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. This study developed a cost-effective SCA repeat panel [Flongle SCA repeat panel sequencing (FLO-SCAp)] using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, Flongle. This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) values of coverage and on-target rate were 39.5 (12 to 72) and 11.6% (7.5% to 16.5%), respectively. This approach was validated by comparing repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing. A high correlation was observed between FLO-SCAp and short-read whole-genome sequencing when the repeat length was ≤250 bp (r=0.98; P<0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.

Optical Genome Mapping Enables Detection and Accurate Sizing of RFC1 Repeat Expansions.

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

Detection and discovery of repeat expansions in ataxia enabled by next-generation sequencing: present and future.

Hereditary cerebellar ataxias are a heterogenous group of progressive neurological disorders that are disproportionately caused by repeat expansions (REs) of short tandem repeats (STRs). Genetic diagnosis for RE disorders such as ataxias are difficult as the current gold standard for diagnosis is repeat-primed PCR assays or Southern blots, neither of which are scalable nor readily available for all STR loci. In the last five years, significant advances have been made in our ability to detect STRs and REs in short-read sequencing data, especially whole-genome sequencing. Given the increasing reliance of genomics in diagnosis of rare diseases, the use of established RE detection pipelines for RE disorders is now a highly feasible and practical first-step alternative to molecular testing methods. In addition, many new pathogenic REs have been discovered in recent years by utilising WGS data. Collectively, genomes are an important resource/platform for further advancements in both the discovery and diagnosis of REs that cause ataxia and will lead to much needed improvement in diagnostic rates for patients with hereditary ataxia.

R-LOOPs on Short Tandem Repeat Expansion Disorders in Neurodegenerative Diseases.

Expansions of short tandem repeats (STRs) have been found to be present in more than 50 diseases and have a close connection with neurodegenerative diseases. Transcriptional silencing and R-LOOP formation, RNA-mediated sequestration of RNA-binding proteins (RBPs), gain-of-function (GOF) proteins containing expanded repeats, and repeat-associated non-AUG (RAN) translation of toxic repeat peptides are some potential molecular mechanisms underlying STR expansion disorders. R-LOOP, a byproduct of transcription, is a three-stranded nucleic acid structure with abnormal accumulation that participates in the pathogenesis of STR expansion disorders by inducing DNA damage and genome instability. R-LOOPs can engender a series of DNA damage, such as DNA double-strand breaks (DSBs), single-strand breaks (SSBs), DNA recombination, or mutations in the DNA replication, transcription, or repair processes. In this review, we provide an in-depth discussion of recent advancements in R-LOOP and systematically elaborate on its genetic destabilizing effects in several neurodegenerative diseases. These molecular mechanisms will provide novel targets for drug design and therapeutic upgrading of these devastating diseases.

Genome sequencing with comprehensive variant calling identifies structural variants and repeat expansions in a large fraction of individuals with ataxia and/or neuromuscular disorders.

Neuromuscular disorders (NMDs) have a heterogeneous etiology. A genetic diagnosis is key to personalized healthcare and access to targeted treatment for the affected individuals. In this study, 861 patients with NMDs were analyzed with genome sequencing and comprehensive variant calling including single nucleotide variants, small insertions/deletions (SNVs/INDELs), and structural variants (SVs) in a panel of 895 NMD genes, as well as short tandem repeat expansions (STRs) at 28 loci. In addition, for unsolved cases with an unspecific clinical presentation, the analysis of a panel with OMIM disease genes was added. In the cohort, 27% (232/861) of the patients harbored pathogenic variants, of which STRs and SVs accounted for one-third of the patients (71/232). The variants were found in 107 different NMD genes. Furthermore, 18 pediatric patients harbored pathogenic variants in non-NMD genes. Our results highlight that for children with unspecific hypotonia, a genome-wide analysis rather than a disease-based gene panel should be considered as a diagnostic approach. More importantly, our results clearly show that it is crucial to include STR- and SV-analyses in the diagnostics of patients with neuromuscular disorders.

Short tandem repeat expansions in sporadic amyotrophic lateral sclerosis and frontotemporal dementia

Pathogenic short tandem repeat (STR) expansions cause over 20 neurodegenerative diseases. To determine the contribution of STRs in sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), we used ExpansionHunter, REviewer, and polymerase chain reaction validation to assess 21 neurodegenerative disease-associated STRs in whole-genome sequencing data from 608 patients with sporadic ALS, 68 patients with sporadic FTD, and 4703 matched controls. We also propose a data-derived outlier detection method for defining allele thresholds in rare STRs. Excluding C9orf72 repeat expansions, 17.6% of clinically diagnosed ALS and FTD cases had at least one expanded STR allele reported to be pathogenic or intermediate for another neurodegenerative disease. We identified and validated 162 disease-relevant STR expansions in C9orf72 (ALS/FTD), ATXN1 [spinal cerebellar ataxia type 1 (SCA1)], ATXN2 (SCA2), ATXN8 (SCA8), TBP (SCA17), HTT (Huntington's disease), DMPK [myotonic dystrophy type 1 (DM1)], CNBP (DM2), and FMR1 (fragile-X disorders). Our findings suggest clinical and pathological pleiotropy of neurodegenerative disease genes and highlight their importance in ALS and FTD.

A novel quantitative trait locus implicates Msh3 in the propensity for genome-wide short tandem repeat expansions in mice.

Short tandem repeats (STRs) are a class of rapidly mutating genetic elements typically characterized by repeated units of 1-6 bp. We leveraged whole-genome sequencing data for 152 recombinant inbred (RI) strains from the BXD family of mice to map loci that modulate genome-wide patterns of new mutations arising during parent-to-offspring transmission at STRs. We defined quantitative phenotypes describing the numbers and types of germline STR mutations in each strain and performed quantitative trait locus (QTL) analyses for each of these phenotypes. We identified a locus on Chromosome 13 at which strains inheriting the C57BL/6J (B) haplotype have a higher rate of STR expansions than those inheriting the DBA/2J (D) haplotype. The strongest candidate gene in this locus is Msh3, a known modifier of STR stability in cancer and at pathogenic repeat expansions in mice and humans, as well as a current drug target against Huntington's disease. The D haplotype at this locus harbors a cluster of variants near the 5' end of Msh3, including multiple missense variants near the DNA mismatch recognition domain. In contrast, the B haplotype contains a unique retrotransposon insertion. The rate of expansion covaries positively with Msh3 expression-with higher expression from the B haplotype. Finally, detailed analysis of mutation patterns showed that strains carrying the B allele have higher expansion rates, but slightly lower overall total mutation rates, compared with those with the D allele, particularly at tetranucleotide repeats. Our results suggest an important role for inherited variants in Msh3 in modulating genome-wide patterns of germline mutations at STRs.

ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

Implementing WGS-based rare inherited disease diagnostics in the Stockholm healthcare region

In healthcare clinical genetics is transitioning to clinical genomics and especially rare disease diagnostics is increasingly done through panel, exome and whole-genome sequencing. At Karolinska we have formed Genomic Medicine Center Karolinska Rare Diseases (GMCK-RD), a joint unit between healthcare and academia, enabling large-scale genome sequencing of patients. GMCK-RD brings together experts from various medical disciplines with clinical geneticists, bioinformaticians and researchers. Through GMCK-RD, over 10,000 individuals have been analysed by clinical genome sequencing (GS) with an overall yield of 33%, providing a genetic diagnosis to >2,543 individuals. By tight collaboration in 15 multidisciplinary expert teams thousands of patients with various rare diseases have been diagnosed. Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualisation of results for clinical interpretation is carried out in Scout- a custom-developed decision support system. To facilitate the discovery of new disease genes, GMCK-RD is reporting variants to ClinVar and has joined international data sharing initiatives, including UDNI, Beacon, and MatchMaker Exchange. Altogether, we at GMCK-RD have moved healthcare in our region towards precision diagnostics and precision medicine.

Structures and conformational dynamics of DNA minidumbbells in pyrimidine-rich repeats associated with neurodegenerative diseases

Expansions of short tandem repeats (STRs) are associated with approximately 50 human neurodegenerative diseases. These pathogenic STRs are prone to form non-B DNA structure, which has been considered as one of the causative factors for repeat expansions. Minidumbbell (MDB) is a relatively new type of non-B DNA structure formed by pyrimidine-rich STRs. An MDB is composed of two tetraloops or pentaloops, exhibiting a highly compact conformation with extensive loop-loop interactions. The MDB structures have been found to form in CCTG tetranucleotide repeats associated with myotonic dystrophy type 2, ATTCT pentanucleotide repeats associated with spinocerebellar ataxia type 10, and the recently discovered ATTTT/ATTTC repeats associated with spinocerebellar ataxia type 37 and familial adult myoclonic epilepsy. In this review, we first introduce the structures and conformational dynamics of MDBs with a focus on the high-resolution structural information determined by nuclear magnetic resonance spectroscopy. Then we discuss the effects of sequence context, chemical environment, and nucleobase modification on the structure and thermostability of MDBs. Finally, we provide perspectives on further explorations of sequence criteria and biological functions of MDBs.

Triplet-primed PCR and Melting Curve Analysis for Rapid Molecular Screening of Spinocerebellar Ataxia Types 1, 2, and 3.

There are more than 40 types of spinocerebellar ataxia (SCA), most of which are caused by abnormal expansion of short tandem repeats at various gene loci. These phenotypically similar disorders require molecular testing at multiple loci by fluorescent PCR and capillary electrophoresis to identify the causative repeat expansion. We describe a simple strategy to screen for the more common SCA1, SCA2, and SCA3 by rapidly detecting the abnormal CAG repeat expansion at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Each of the three separate assays employs a plasmid DNA carrying a known repeat size to generate a threshold melt peak temperature, which effectively distinguishes expansion-positive samples from those without a repeat expansion. Samples that are screened positive based on their melt peak profiles are subjected to capillary electrophoresis for repeat sizing and genotype confirmation. These screening assays are robust and provide accurate detection of the repeat expansion while eliminating the need for fluorescent PCR and capillary electrophoresis for every sample.

P445: Short tandem repeat expansions in the diagnosis of rare Mendelian diseases by whole genome sequencing

P445: Short tandem repeat expansions in the diagnosis of rare Mendelian diseases by whole genome sequencing

P572: Detecting short tandem repeat expansions: Three-year experience with clinical whole genome sequencing (cWGS) for a rare and undiagnosed population

P572: Detecting short tandem repeat expansions: Three-year experience with clinical whole genome sequencing (cWGS) for a rare and undiagnosed population

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci

Expansions of short tandem repeats (STRs) cause many rare diseases. Expansion detection is challenging with short-read DNA sequencing data since supporting reads are often mapped incorrectly. Detection is particularly difficult for “novel” STRs, which include new motifs at known loci or STRs absent from the reference genome. We developed STRling to efficiently count k-mers to recover informative reads and call expansions at known and novel STR loci. STRling is sensitive to known STR disease loci, has a low false discovery rate, and resolves novel STR expansions to base-pair position accuracy. It is fast, scalable, open-source, and available at: github.com/quinlan-lab/STRling.

Non-Mendelian inheritance patterns and extreme deviation rates of CGG repeats in autism.

As expansions of CGG short tandem repeats (STRs) are established as the genetic etiology of many neurodevelopmental disorders, we aimed to elucidate the inheritance patterns and role of CGG STRs in autism-spectrum disorder (ASD). By genotyping 6063 CGG STR loci in a large cohort of trios and quads with an ASD-affected proband, we determined an unprecedented rate of CGG repeat length deviation across a single generation. Although the concept of repeat length being linked to deviation rate was solidified, we show how shorter STRs display greater degrees of size variation. We observed that CGG STRs did not segregate by Mendelian principles but with a bias against longer repeats, which appeared to magnify as repeat length increased. Through logistic regression, we identified 19 genes that displayed significantly higher rates and degrees of CGG STR expansion within the ASD-affected probands (P < 1 × 10-5). This study not only highlights novel repeat expansions that may play a role in ASD but also reinforces the hypothesis that CGG STRs are specifically linked to human cognition.

Long-read sequencing identified intronic (GGCCTG)n expansion in NOP56 in one SCA36 family and literature review

BackgroundSpinocerebellar ataxias (SCA) are often caused by expansions of short tandem repeats. Recent methodological advances have made repeat expansion detection with long-read sequencing (LRS) feasible. Our study investigated one family with SCA 36 and further summarized the genetic and clinical characteristics of the total of 161 patients across different ethnic groups reported worldwide. MethodsWe enrolled a pedigree of 4 patients. The proband was a 55-year-old male. And he was screened for dynamic mutations of SCA subtypes by Tri-prime PCR (TP-PCR) and capillary electrophoresis, showing NOP56 as the candidate gene. The cosegregation was conducted by screening the NOP56 gene in his daughter and further confirmed by low-coverage (∼15 ×) LRS on the Oxford Nanopore platform. ResultsThe SCA36 pedigree included a total of 4 patients. The proband showed the initial manifestation at the age of 45 years old, which was characterized by truncal ataxia. Genetic test results showed the (GGCCTG)n expansion in NOP56 gene (3/>15 and 6/>15 times respectively). To clarify the diagnosis genetically, LRS was performed in his daughter showing a large intronic insertion (chr20: 2633004 INS 7603 bp) containing (GGCCTG)n expansion of 782 units in NOP56 as the causative mutation. ConclusionsWe identified one SCA36 pedigree by combining TP-PCR with LRS. Our study suggested LRS as an effective tool for molecular diagnosis. LRS also worked as a supplementary but necessary diagnostic tool for dynamic mutation-related SCA on the basis of repeat-primed PCR as well as capillary electrophoresis.

Unexpected diagnosis of myotonic dystrophy type 2 repeat expansion by genome sequencing

Several neurological disorders, such as myotonic dystrophy are caused by expansions of short tandem repeats (STRs) which can be difficult to detect by molecular tools. Methodological advances have made repeat expansion (RE) detection with whole genome sequencing (WGS) feasible. We recruited a multi-generational family (family A) ascertained for genetic studies of autism spectrum disorder. WGS was performed on seven children from four nuclear families from family A and analyzed for REs of STRs known to cause neurological disorders. We detected an expansion of a heterozygous intronic CCTG STR in CNBP in two siblings. This STR causes myotonic dystrophy type 2 (DM2). The expansion did not segregate with the ASD phenotype. Repeat-primed PCR showed that the DM2 CCTG motif was expanded above the pathogenic threshold in both children and their mother. On subsequent examination, the mother had mild features of DM2. We show that screening of STRs in WGS datasets has diagnostic utility, both in the clinical and research domain, with potential management and genetic counseling implications.