Sequence composition changes in short tandem repeats: heterogeneity, detection, mechanisms and clinical implications.

Short tandem repeats (STRs) are a class of repetitive elements, composed of tandem arrays of 1-6 base pair sequence motifs, that comprise a substantial fraction of the human genome. STR expansions can cause a wide range of neurological and neuromuscular conditions, known as repeat expansion disorders, whose age of onset, severity, penetrance and/or clinical phenotype are influenced by the length of the repeats and their sequence composition. The presence of non-canonical motifs, depending on the type, frequency and position within the repeat tract, can alter clinical outcomes by modifying somatic and intergenerational repeat stability, gene expression and mutant transcript-mediated and/or protein-mediated toxicities. Here, we review the diverse structural conformations of repeat expansions, technological advances for the characterization of changes in sequence composition, their clinical correlations and the impact on disease mechanisms.

A novel online genomic counseling and variant interpretation certificate: Learning design, learning analytics, and evaluation.

In this paper we describe the analysis, planning, design, development, implementation and evaluation of a new online Graduate Certificate in Genomic Counselling and Variant Interpretation (GCGCVI) at The University of British Columbia (UBC). Genetic counselling is now a prerequisite for diagnostic genomic testing in many countries, demanding that genetic counselling practitioners have up-to-the-moment genomic counselling skills and knowledge. Current practitioners reported a desire for more training in this rapidly developing field: our international survey revealed substantial interest in online continuing education addressing themes such as testing and clinical bioinformatics, applied variant interpretation, evidence-based genomic counselling, and other emerging genomic topics. However, our market analysis found no post-graduate program globally that offered such training. To fill this gap, our oversight team of genetic counsellors and geneticists therefore guided development of curriculum and materials, and online learning specialists developed rigorous interactive asynchronous online graduate courses through collaboration with subject matter experts, following best practices in online learning design. Since launch in September 2020, we have gathered learner feedback using surveys and focus groups, and we have used learning analytics to understand how learners engaged with each other and with course materials. Together, these have helped us understand learner behaviour and guide the continuous process of design improvement to support the learning goals of this audience of professional learners. Our courses have been reviewed and approved by the UBC Faculty of Medicine, UBC Senate, and the Province of British Columbia Ministries of Advanced Education and Health, and assessed by the National Society of Genetic Counselors (NSGC, USA) and the Canadian Association of Genetic Counsellors (CAGC) to enable learners to receive North American continuing education credits. To date, 151 individuals from 18 countries have succeeded in one or more course and 43 have completed the entire certificate.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel with linked multi-marker haplotype analysis of 13 highly polymorphic microsatellite markers located within 1 Mb of the FMR1 CGG repeat, and the AMELX/Y dimorphism for gender identification. The assay was optimised and validated on single lymphoblasts isolated from fragile X reference cell lines, and applied to a simulated PGD case and a clinical in vitro fertilisation (IVF)-PGD case. In the simulated PGD case, definitive diagnosis of the expected results was achieved for all 'embryos'. In the clinical IVF-PGD case, delivery of a healthy baby girl was achieved after transfer of an expansion-negative blastocyst. FMR1 TP-PCR reliably detects presence of expansion mutations and obviates reliance on informative normal alleles for determining expansion status in female embryos. Together with multi-marker haplotyping and gender determination, misdiagnosis and diagnostic ambiguity due to allele dropout is minimised, and couple-specific assay customisation can be avoided.

Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

A comprehensive tandem repeat catalog of the human genome.

With the increasing availability of long-read sequencing data, high-quality human genome assemblies, and software for fully characterizing tandem repeats, genome-wide genotyping of tandem repeat loci on a population scale becomes more feasible. Such efforts not only expand our knowledge of the tandem repeat landscape in the human genome but also enhance our ability to differentiate pathogenic tandem repeat mutations from benign polymorphisms. To this end, we analyzed 272 genomes assembled using datasets from three public initiatives that employed different long-read sequencing technologies. Here, we report a catalog of over 18 million tandem repeat loci, many of which were previously unannotated. Some of these loci are highly polymorphic, and many of them reside within coding sequences.

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.

Triplet-Primed PCR Assays for Accurate Screening of FMR1 CGG Repeat Expansion and Genotype Verification.

Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (>200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor- and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for melting curve analysis Basic Protocol 2: Melting curve analysis of direct triplet-primed PCR amplicons on the Rotor-Gene Q MD × 5plex high-resolution melt platform Alternate Protocol: Melting curve analysis of direct triplet-primed PCR amplicons on the LightCycler 480 system Basic Protocol 3: Generation of direct triplet-primed PCR melting curve analysis profiles Basic Protocol 4: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for capillary electrophoresis Basic Protocol 5: Generation of control FMR1 plasmids for direct triplet-primed PCR melting curve analysis Basic Protocol 6: Sanger sequencing assay to verify FMR1 CGG repeat size and structure of plasmid DNA controls.

Linked-read sequencing for detecting short tandem repeat expansions.

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.

High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes.

Methylated FMR1 full-mutation expansions cause fragile X syndrome. FMR1 premutation carriers are susceptible to other late-onset conditions, and women with premutation are at risk of transmitting a fully expanded FMR1 allele to offspring. Identification of individuals with actionable FMR1 genotypes (full-mutation males and females, and premutation females at risk for primary ovarian insufficiency and/or having fragile X-affected offspring) can enable timely access to intervention services and genetic counseling. This study presents a rapid, first-tier test based on melting curve analysis of methylation-specific triplet-primed PCR amplicons (msTP-PCR MCA) for concurrent detection of FMR1 CGG-repeat expansions and their methylation status. The msTP-PCR MCA assay was optimized on 20 fragile X reference samples, and its performance was evaluated on 111 peripheral blood-derived DNA samples from patients who have undergone prior molecular testing with PCR and/or Southern blot analysis. The msTP-PCR MCA assay detected all samples with a methylated FMR1 CGG-repeat expansion, and had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 92.06%, 91.1%, and 100%, respectively. The msTP-PCR MCA assay identified premutation/full-mutation mosaicism down to 1%, detected skewed inactivation in females with FMR1 expansions, and enabled selective identification of all individuals with an actionable FMR1 genotype. The msTP-PCR MCA assay may aid in fragile X screening of at-risk populations and newborns and voluntary carrier screening of women of reproductive age.

REViewer: haplotype-resolved visualization of read alignments in and around tandem repeats.

BACKGROUND: Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads. RESULTS: We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR. CONCLUSIONS: Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

Straglr: discovering and genotyping tandem repeat expansions using whole genome long-read sequences.

Tandem repeat (TR) expansion is the underlying cause of over 40 neurological disorders. Long-read sequencing offers an exciting avenue over conventional technologies for detecting TR expansions. Here, we present Straglr, a robust software tool for both targeted genotyping and novel expansion detection from long-read alignments. We benchmark Straglr using various simulations, targeted genotyping data of cell lines carrying expansions of known diseases, and whole genome sequencing data with chromosome-scale assembly. Our results suggest that Straglr may be useful for investigating disease-associated TR expansions using long-read sequencing.

Genome-wide sequencing as a first-tier screening test for short tandem repeat expansions.

BACKGROUND: Screening for short tandem repeat (STR) expansions in next-generation sequencing data can enable diagnosis, optimal clinical management/treatment, and accurate genetic counseling of patients with repeat expansion disorders. We aimed to develop an efficient computational workflow for reliable detection of STR expansions in next-generation sequencing data and demonstrate its clinical utility. METHODS: We characterized the performance of eight STR analysis methods (lobSTR, HipSTR, RepeatSeq, ExpansionHunter, TREDPARSE, GangSTR, STRetch, and exSTRa) on next-generation sequencing datasets of samples with known disease-causing full-mutation STR expansions and genomes simulated to harbor repeat expansions at selected loci and optimized their sensitivity. We then used a machine learning decision tree classifier to identify an optimal combination of methods for full-mutation detection. In Burrows-Wheeler Aligner (BWA)-aligned genomes, the ensemble approach of using ExpansionHunter, STRetch, and exSTRa performed the best (precision = 82%, recall = 100%, F1-score = 90%). We applied this pipeline to screen 301 families of children with suspected genetic disorders. RESULTS: We identified 10 individuals with full-mutations in the AR, ATXN1, ATXN8, DMPK, FXN, or HTT disease STR locus in the analyzed families. Additional candidates identified in our analysis include two probands with borderline ATXN2 expansions between the established repeat size range for reduced-penetrance and full-penetrance full-mutation and seven individuals with FMR1 CGG repeats in the intermediate/premutation repeat size range. In 67 probands with a prior negative clinical PCR test for the FMR1, FXN, or DMPK disease STR locus, or the spinocerebellar ataxia disease STR panel, our pipeline did not falsely identify aberrant expansion. We performed clinical PCR tests on seven (out of 10) full-mutation samples identified by our pipeline and confirmed the expansion status in all, showing absolute concordance between our bioinformatics and molecular findings. CONCLUSIONS: We have successfully demonstrated the application of a well-optimized bioinformatics pipeline that promotes the utility of genome-wide sequencing as a first-tier screening test to detect expansions of known disease STRs. Interrogating clinical next-generation sequencing data for pathogenic STR expansions using our ensemble pipeline can improve diagnostic yield and enhance clinical outcomes for patients with repeat expansion disorders.

Triplet-Repeat Primed PCR and Capillary Electrophoresis for Characterizing the Fragile X Mental Retardation 1 CGG Repeat Hyperexpansions.

Fragile X mental retardation 1 (FMR1) CGG repeat expansions cause fragile X syndrome-the leading monogenic form of intellectual disability-and increase the risk for fragile X-associated tremor ataxia syndrome and fragile X-associated primary ovarian insufficiency. Southern blot (SB) analysis is the current gold standard test for FMR1 molecular diagnosis. Several polymerase chain reaction (PCR)-based methods are now available for sizing FMR1 CGG repeat expansions. These methods offer higher diagnostic sensitivity and specificity compared to SB analysis, significantly reduce the turnaround time and increase throughput. In this chapter, we describe a triplet-repeat primed PCR protocol that employs capillary electrophoresis to resolve the derived amplicon products, enabling precise determination of the FMR1 genotypes in both males and females and characterization of the CGG repeat structure.

Validation of a commercially available test that enables the quantification of the numbers of CGG trinucleotide repeat expansion in FMR1 gene.

In the present study, we evaluated a commercially available TP-PCR-based assay, the FastFraXTM FMR1 Sizing kit, as a test in quantifying the number of CGG repeats in the FMR1 gene. Based on testing with well characterized DNA samples from Coriell, the kit yielded size results within 3 repeats of those obtained by common consensus (n = 14), with the exception of one allele. Furthermore, based on data obtained using all Coriell samples with or without common consensus (n = 29), the Sizing kit was 97.5% in agreement with existing approaches. Additionally, the kit generated consistent size information in repeatability and reproducibility studies (CV 0.39% to 3.42%). Clinical performance was established with 198 archived clinical samples, yielding results of 100% sensitivity (95% CI, 91.03% to 100%) and 100% specificity (95% CI, 97.64% to 100%) in categorizing patient samples into the respective normal, intermediate, premutation and full mutation genotypes.

Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders.

Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)n expansion screening in newborns, women of reproductive age and high-risk populations.

Defining the Performance Parameters of a Rapid Screening Tool for FMR1 CGG-Repeat Expansions Based on Direct Triplet-Primed PCR and Melt Curve Analysis.

Population-based screening for CGG-repeat expansions in the fragile X mental retardation 1 (FMR1) gene that cause fragile X syndrome can now be performed more cost-effectively and simply by combining direct triplet-primed PCR (dTP-PCR) with melting curve analysis (MCA). We have now performed a detailed technical validation to define the operational parameters for achieving robust and reliable performance of the FMR1 dTP-PCR MCA assay. We compared the assay's performance on 2 real-time PCR platforms and determined its analytic sensitivity and specificity. We also assessed the assay's performance on DNA isolated from different sources, the effect of differences in CGG-repeat length and AGG-interruption pattern on melt peak temperature (Tm), and the effect of common substances found in DNA solutions on Tms. The assay performed well in distinguishing normal from expansion-carrying samples. The assay had detection sensitivity down to 1 ng and an analytical specificity beyond 150 ng. In addition to peripheral blood DNA, analysis could also be performed on DNA from saliva, buccal swabs, and dried blood spots. Salt increased Tms, glycogen contamination had minimal effect, whereas AGG interruptions lowered Tms. The FMR1 dTP-PCR MCA screening assay is highly sensitive and specific, performs well using DNA from different sources, and is robust and reproducible when reagent concentrations are maintained across all tested samples.

Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis of Fragile X Mental Retardation 1-related disorders.

AIM: Molecular diagnosis of fragile X syndrome demands assessment of fragile X mental retardation 1 (FMR1) CGG repeat size and methylation status, while predicting disease transmission risk requires determination of AGG interruption pattern. There is currently no single assay that provides all three categories of information. We describe a single-tube methylation-specific triplet-primed PCR assay for concurrently assessing methylation state, repeat size and structure of CGG repeat(s). METHODS: Differentially labeled primers specific for methylated and unmethylated FMR1 alleles were used to amplify bisulfite-modified DNA, followed by capillary electrophoresis. Twenty-four reference DNAs and 107 patient samples were analyzed to evaluate assay performance. RESULTS: Repeat size, AGG interruption pattern and methylation state were correctly identified in all tested samples. The assay also detected skewed X-inactivation when present in females, and somatic mosaicism in fragile X males. CONCLUSION: When used in a molecular diagnostic setting, this novel assay could significantly minimize the need to reflex patient samples for Southern analysis.