Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.

Noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and an overlapping disease.

Sequencing-based studies have identified novel risk genes associated with severe epilepsies and revealed an excess of rare deleterious variation in less-severe forms of epilepsy. To identify the shared and distinct ultra-rare genetic risk factors for different types of epilepsies, we performed a whole-exome sequencing (WES) analysis of 9,170 epilepsy-affected individuals and 8,436 controls of European ancestry. We focused on three phenotypic groups: severe developmental and epileptic encephalopathies (DEEs), genetic generalized epilepsy (GGE), and non-acquired focal epilepsy (NAFE). We observed that compared to controls, individuals with any type of epilepsy carried an excess of ultra-rare, deleterious variants in constrained genes and in genes previously associated with epilepsy; we saw the strongest enrichment in individuals with DEEs and the least strong in individuals with NAFE. Moreover, we found that inhibitory GABAA receptor genes were enriched for missense variants across all three classes of epilepsy, whereas no enrichment was seen in excitatory receptor genes. The larger gene groups for the GABAergic pathway or cation channels also showed a significant mutational burden in DEEs and GGE. Although no single gene surpassed exome-wide significance among individuals with GGE or NAFE, highly constrained genes and genes encoding ion channels were among the lead associations; such genes included CACNA1G, EEF1A2, and GABRG2 for GGE and LGI1, TRIM3, and GABRG2 for NAFE. Our study, the largest epilepsy WES study to date, confirms a convergence in the genetics of severe and less-severe epilepsies associated with ultra-rare coding variation, and it highlights a ubiquitous role for GABAergic inhibition in epilepsy etiology.

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Ultra-Rare Genetic Variation in the Epilepsies: A Whole-Exome Sequencing Study of 17,606 Individuals.

SUMMARY We describe a novel computational method for genotyping repeats using sequence graphs. This method addresses the long-standing need to accurately genotype medically important loci containing repeats adjacent to other variants or imperfect DNA repeats such as polyalanine repeats. Here we introduce a new version of our repeat genotyping software, ExpansionHunter, that uses this method to perform targeted genotyping of a broad class of such loci. AVAILABILITY AND IMPLEMENTATION ExpansionHunter is implemented in C++ and is available under the Apache License Version 2.0. The source code, documentation, and Linux/macOS binaries are available at https://github.com/Illumina/ExpansionHunter/. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

/pdf/expansionhunter-a-sequence-graph-based-tool-to-analyze-3w5xufshpx.pdf

ExpansionHunter: A sequence-graph based tool to analyze variation in short tandem repeat regions.

Tandem repeat (TR) expansions have been implicated in dozens of genetic diseases, including Huntington's Disease, Fragile X Syndrome, and hereditary ataxias. Furthermore, TRs have recently been implicated in a range of complex traits, including gene expression and cancer risk. While the human genome harbors hundreds of thousands of TRs, analysis of TR expansions has been mainly limited to known pathogenic loci. A major challenge is that expanded repeats are beyond the read length of most next-generation sequencing (NGS) datasets and are not profiled by existing genome-wide tools. We present GangSTR, a novel algorithm for genome-wide genotyping of both short and expanded TRs. GangSTR extracts information from paired-end reads into a unified model to estimate maximum likelihood TR lengths. We validate GangSTR on real and simulated data and show that GangSTR outperforms alternative methods in both accuracy and speed. We apply GangSTR to a deeply sequenced trio to profile the landscape of TR expansions in a healthy family and validate novel expansions using orthogonal technologies. Our analysis reveals that healthy individuals harbor dozens of long TR alleles not captured by current genome-wide methods. GangSTR will likely enable discovery of novel disease-associated variants not currently accessible from NGS.

/pdf/profiling-the-genome-wide-landscape-of-tandem-repeat-2s8k2yebou.pdf

Profiling the genome-wide landscape of tandem repeat expansions

Tandem DNA repeats vary in the size and sequence of each unit (motif). When expanded, these tandem DNA repeats have been associated with more than 40 monogenic disorders1. Their involvement in disorders with complex genetics is largely unknown, as is the extent of their heterogeneity. Here we investigated the genome-wide characteristics of tandem repeats that had motifs with a length of 2–20 base pairs in 17,231 genomes of families containing individuals with autism spectrum disorder (ASD)2,3 and population control individuals4. We found extensive polymorphism in the size and sequence of motifs. Many of the tandem repeat loci that we detected correlated with cytogenetic fragile sites. At 2,588 loci, gene-associated expansions of tandem repeats that were rare among population control individuals were significantly more prevalent among individuals with ASD than their siblings without ASD, particularly in exons and near splice junctions, and in genes related to the development of the nervous system and cardiovascular system or muscle. Rare tandem repeat expansions had a prevalence of 23.3% in children with ASD compared with 20.7% in children without ASD, which suggests that tandem repeat expansions make a collective contribution to the risk of ASD of 2.6%. These rare tandem repeat expansions included previously undescribed ASD-linked expansions in DMPK and FXN, which are associated with neuromuscular conditions, and in previously unknown loci such as FGF14 and CACNB1. Rare tandem repeat expansions were associated with lower IQ and adaptive ability. Our results show that tandem DNA repeat expansions contribute strongly to the genetic aetiology and phenotypic complexity of ASD. Genome-wide analysis of tandem DNA repeats in the genomes of individuals with autism spectrum disorder and control participants reveals a strong contribution of tandem repeat expansions to the genetic aetiology and phenotypic complexity of autism spectrum disorder.

Genome-wide detection of tandem DNA repeats that are expanded in autism.

Repeat expansions cause more than 30 inherited disorders, predominantly neurogenetic. These can present with overlapping clinical phenotypes, making molecular diagnosis challenging. Single-gene or small-panel PCR-based methods can help to identify the precise genetic cause, but they can be slow and costly and often yield no result. Researchers are increasingly performing genomic analysis via whole-exome and whole-genome sequencing (WES and WGS) to diagnose genetic disorders. However, until recently, analysis protocols could not identify repeat expansions in these datasets. We developed exSTRa (expanded short tandem repeat algorithm), a method that uses either WES or WGS to identify repeat expansions. Performance of exSTRa was assessed in a simulation study. In addition, four retrospective cohorts of individuals with eleven different known repeat-expansion disorders were analyzed with exSTRa. We assessed results by comparing the findings to known disease status. Performance was also compared to three other analysis methods (ExpansionHunter, STRetch, and TREDPARSE), which were developed specifically for WGS data. Expansions in the assessed STR loci were successfully identified in WES and WGS datasets by all four methods with high specificity and sensitivity. Overall, exSTRa demonstrated more robust and superior performance for WES data than did the other three methods. We demonstrate that exSTRa can be effectively utilized as a screening tool for detecting repeat expansions in WES and WGS data, although the best performance would be produced by consensus calling, wherein at least two out of the four currently available screening methods call an expansion.

Detecting Expansions of Tandem Repeats in Cohorts Sequenced with Short-Read Sequencing Data

Short tandem repeats (STRs), also known as microsatellites, are commonly defined as consisting of tandemly repeated nucleotide motifs of 2-6 base pairs in length. STRs appear throughout the human genome, and about 239,000 are documented in the Simple Repeats Track available from the UCSC (University of California, Santa Cruz) genome browser. STRs vary in size, producing highly polymorphic markers commonly used as genetic markers. A small fraction of STRs (about 30 loci) have been associated with human disease whereby one or both alleles exceed an STR-specific threshold in size, leading to disease. Detection of repeat expansions is currently performed with polymerase chain reaction-based assays or with Southern blots for large expansions. The tests are expensive and time-consuming and are not always conclusive, leading to lengthy diagnostic journeys for patients, potentially including missed diagnoses. The advent of whole exome and whole genome sequencing has identified the genetic cause of many genetic disorders; however, analysis pipelines are focused primarily on the detection of short nucleotide variations and short insertions and deletions (indels). Until recently, repeat expansions, with the exception of the smallest expansion (SCA6), were not detectable in next-generation short-read sequencing datasets and would have been ignored in most analyses. In the last two years, four analysis methods with accompanying software (ExpansionHunter, exSTRa, STRetch, and TREDPARSE) have been released. Although a comprehensive comparative analysis of the performance of these methods across all known repeat expansions is still lacking, it is clear that these methods are a valuable addition to any existing analysis pipeline. Here, we detail how to assess short-read data for evidence of expansions, reviewing all four methods and outlining their strengths and weaknesses. Implementation of these methods should lead to increased diagnostic yield of repeat expansion disorders for known STR loci and has the potential to detect novel repeat expansions.

/pdf/recent-advances-in-the-detection-of-repeat-expansions-with-pzgzqhs6l9.pdf

Recent advances in the detection of repeat expansions with short-read next-generation sequencing

Repeat expansions cause over 30, predominantly neurogenetic, inherited disorders. These can present with overlapping clinical phenotypes, making molecular diagnosis challenging. Single gene or small panel PCR-based methods are employed to identify the precise genetic cause, but can be slow and costly, and often yield no result. Genomic analysis via whole exome and whole genome sequencing (WES and WGS) is being increasingly performed to diagnose genetic disorders. However, until recently analysis protocols could not identify repeat expansions in these datasets. A new method, called exSTRa (expanded Short Tandem Repeat algorithm) for the identification of repeat expansions using either WES or WGS was developed and performance of exSTRa was assessed in a simulation study. In addition, four retrospective cohorts of individuals with eleven different known repeat expansion disorders were analysed with the new method. Results were assessed by comparing to known disease status. Performance was also compared to three other analysis methods (ExpansionHunter, STRetch and TREDPARSE), which were developed specifically for WGS data. Expansions in the STR loci assessed were successfully identified in WES and WGS datasets by all four methods, with high specificity and sensitivity, excepting the FRAXA STR where expansions were unlikely to be detected. Overall exSTRa demonstrated more robust/superior performance for WES data in comparison to the other three methods. exSTRa can be applied to existing WES or WGS data to identify likely repeat expansions and can be used to investigate any STR of interest, by specifying location and repeat motif. We demonstrate that methods such as exSTRa can be effectively utilized as a screening tool to interrogate WES data generated with PCR-based library preparations and WGS data generated using either PCR-based or PCR-free library protocols, for repeat expansions which can then be followed up with specific diagnostic tests. exSTRa is available via GitHub (https://github.com/bahlolab/exSTRa).

https://www.biorxiv.org/content/biorxiv/early/2018/07/25/157792.full.pdf

Peter Degorski

Papers

Detecting Expansions of Tandem Repeats in Cohorts Sequenced with Short-Read Sequencing Data

Recent advances in the detection of repeat expansions with short-read next-generation sequencing

Detecting tandem repeat expansions in cohorts sequenced with short-read sequencing data