Tandem Repeat Expansions Research Articles

Genomic architecture of autism spectrum disorder in Qatar: The BARAKA-Qatar Study

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impaired social and communication skills, restricted interests, and repetitive behaviors. The prevalence of ASD among children in Qatar was recently estimated to be 1.1%, though the genetic architecture underlying ASD both in Qatar and the greater Middle East has been largely unexplored. Here, we describe the first genomic data release from the BARAKA-Qatar Study—a nationwide program building a broadly consented biorepository of individuals with ASD and their families available for sample and data sharing and multi-omics research.MethodsIn this first release, we present a comprehensive analysis of whole-genome sequencing (WGS) data of the first 100 families (372 individuals), investigating the genetic architecture, including single-nucleotide variants (SNVs), copy number variants (CNVs), tandem repeat expansions (TREs), as well as mitochondrial DNA variants (mtDNA) segregating with ASD in local families.ResultsOverall, we identify potentially pathogenic variants in known genes or regions in 27 out of 100 families (27%), of which 11 variants (40.7%) were classified as pathogenic or likely-pathogenic based on American College of Medical Genetics (ACMG) guidelines. Dominant variants, including de novo and inherited, contributed to 15 (55.6%) of these families, consisting of SNVs/indels (66.7%), CNVs (13.3%), TREs (13.3%), and mtDNA variants (6.7%). Moreover, homozygous variants were found in 7 families (25.9%), with a sixfold increase in homozygous burden in consanguineous versus non-consanguineous families (13.6% and 1.8%, respectively). Furthermore, 28 novel ASD candidate genes were identified in 20 families, 23 of which had recurrent hits in MSSNG and SSC cohorts.ConclusionsThis study illustrates the value of ASD studies in under-represented populations and the importance of WGS as a comprehensive tool for establishing a molecular diagnosis for families with ASD. Moreover, it uncovers a significant role for recessive variation in ASD architecture in consanguineous settings and provides a unique resource of Middle Eastern genomes for future research to the global ASD community.

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.

The role of tandem repeat expansions in brain disorders.

The human genome contains numerous genetic polymorphisms contributing to different health and disease outcomes. Tandem repeat (TR) loci are highly polymorphic yet under-investigated in large genomic studies, which has prompted research efforts to identify novel variations and gain a deeper understanding of their role in human biology and disease outcomes. We summarize the current understanding of TRs and their implications for human health and disease, including an overview of the challenges encountered when conducting TR analyses and potential solutions to overcome these challenges. By shedding light on these issues, this article aims to contribute to a better understanding of the impact of TRs on the development of new disease treatments.

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.

Chromosome-level genomes of two armyworms, Mythimna separata and Mythimna loreyi, provide insights into the biosynthesis and reception of sex pheromones.

Mythimna separata and Mythimna loreyi are global pests of gramineous cereals, heavily controlled with synthetic insecticides. Here, we generated two high-quality chromosome-level genome assemblies for M. separata (688 Mb) and M. loreyi (683 Mb). Our analysis identified Z and W chromosomes, with few genes and abundant transposable elements (TEs) found on the W chromosome. We also observed a recent explosion of long interspersed nuclear elements (LINEs), which contributed to the larger genomes of Mythimna. The two armyworms diverged ~10.5 MYA, with only three chromosomes have intrachromosomal rearrangements. Additionally, we observed a tandem repeat expansion of α-amylase genes in Mythimna, which may promote the digestion of carbohydrates and exacerbate their damage to crops. Furthermore, we inferred the sex pheromone biosynthesis pathway for M. separata, M. loreyi and Spodoptera frugiperda. We discovered that M. loreyi and S. frugiperda synthesized the same major constituents of sex pheromones through different pathways. Specifically, the double bonds in the dominant sex pheromone components of S. frugiperda were generated by Δ9- and Δ11-desaturase, while they were generated by Δ11-desaturase and chain-shortening reactions in M. loreyi. We also identified pheromone receptor (PR) genes and inferred their corresponding components. These findings provide a better understanding of sex pheromone communication and promote the development of a new pest control strategy involving pheromone traps, which are more effective and environmentally friendly than current strategies.

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.

Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

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.

Sequencing through hyperexpanded Friedreich's ataxia-GAA repeats by nanopore technology: implications in genotype-phenotype correlation.

Friedreich's ataxia, an autosomal recessive disorder, is caused by tandem GAA nucleotide repeat expansions in intron 1 of the frataxin gene. The GAA repeats over 66 in number are considered as pathogenic, and commonly occurring pathogenic repeats are within a range of 600-1200. Clinically, the spectrum of features is confined mainly to neurological tissues; however, cardiomyopathy and diabetes mellitus have been reported in 60 and 30% of the subjects, respectively. The accurate detection of GAA repeat count is of utmost importance for clinical genetic correlation, and no study so far has attempted an approach that is of high-throughput nature and defines the exact sequence of GAA repeats. Largely, the method for detection of GAA repeats so far is either through the conventional polymerase chain reaction-based screening or Southern blot, which remains the gold standard method. We utilized an approach of long-range targeted amplification of FXN-GAA repeats using Oxford Nanopore Technologies MinION platform for accurate estimation of repeat length. We were able to achieve successful amplification of GAA repeats ranging from ∼120 to 1100 at ∼2600× mean coverage. The total throughput achievable through our protocol can allow for screening of up to 96 samples per flow cell in less than 24 h. The proposed method is clinically scalable and deployable for day-to-day diagnostics. In this paper, we demonstrate to resolve the genotype-phenotype correlation of Friedreich's ataxia patients with better accuracy.

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.

In Silico Identification and Characterization of Satellite DNAs in 23 Drosophila Species from the Montium Group.

Satellite DNA (satDNA) is a class of tandemly repeated non-protein coding DNA sequences which can be found in abundance in eukaryotic genomes. They can be functional, impact the genomic architecture in many ways, and their rapid evolution has consequences for species diversification. We took advantage of the recent availability of sequenced genomes from 23 Drosophila species from the montium group to study their satDNA landscape. For this purpose, we used publicly available whole-genome sequencing Illumina reads and the TAREAN (tandem repeat analyzer) pipeline. We provide the characterization of 101 non-homologous satDNA families in this group, 93 of which are described here for the first time. Their repeat units vary in size from 4 bp to 1897 bp, but most satDNAs show repeat units < 100 bp long and, among them, repeats ≤ 10 bp are the most frequent ones. The genomic contribution of the satDNAs ranges from ~1.4% to 21.6%. There is no significant correlation between satDNA content and genome sizes in the 23 species. We also found that at least one satDNA originated from an expansion of the central tandem repeats (CTRs) present inside a Helitron transposon. Finally, some satDNAs may be useful as taxonomic markers for the identification of species or subgroups within the group.

Analysis of Tandem Repeat Expansions Using Long DNA Reads.

Abnormal expansion or shortening of tandem repeats can cause a variety of genetic diseases. The use of long DNA reads has facilitated the analysis of disease-causing repeats in the human genome. Long read sequencers enable us to directly analyze repeat length and sequence content by covering whole repeats; they are therefore considered suitable for the analysis of long tandem repeats. Here, we describe an expanded repeat analysis using target sequencing data produced by the Oxford Nanopore Technologies (hereafter referred to as ONT) nanopore sequencer.

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.

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

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

Expanded‐repeat‐RNA‐mediated disease mechanisms in myotonic dystrophy

AbstractMyotonic dystrophy (DM) is the most common muscular dystrophy in adults, affecting skeletal muscle as well as cardiac and smooth muscle. Furthermore, involvement of the central nervous system, endocrine organs, and eyes is often seen, with debilitating consequences. The condition is an autosomal‐dominant inherited genetic disease caused by abnormal genomic expansion of tandem repeats. Myotonic dystrophy type 1 (DM1) results from expansion of a CTG repeat in the 3′‐untranslated region of the gene encoding dystrophia myotonica‐protein kinase (DMPK), whereas myotonic dystrophy type 2 (DM2) is caused by expansion of a CCTG repeat in the first intron of the gene encoding CCHC‐type zinc finger nucleic acid‐binding protein (CNBP). Both types of DM exhibit abnormal mRNA transcribed from the mutated gene containing expanded repeats, which exert toxic gain‐of‐function effects on various proteins involved in cellular processes such as alternative splicing, signaling pathways, and cellular senescence. The present review discusses the expanded‐repeat‐RNA‐mediated molecular pathomechanisms in DM.

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.

Recurrent repeat expansions in human cancer genomes

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3–8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Clinical genetics of schizophrenia and related neuropsychiatric disorders

Rare structural variants have turned out to be the long sought for genetic variants of (relatively) high effect size for schizophrenia. Delineating the 22q11.2 microdeletion as the first molecular subtype of schizophrenia was a milestone in schizophrenia research, foreshadowing a more general role for rare copy number variation (CNV) in schizophrenia. The 22q11.2 microdeletion has a high effect size – one in every four individuals born with this deletion develops schizophrenia – and a relatively high prevalence for a rare condition. Discovery of this human genetic high-risk model for schizophrenia has shown how genetics can change clinical management, and also provide new opportunities for animal and cellular models. Further new findings indicate a role for tandem repeat expansion, other less complex rare variants, and collective background effects of common variants in the genetics of schizophrenia. Thus, the genetic architecture of schizophrenia is taking shape, with further advances on the horizon.