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Auto Tag: Repeat expansions

April 21, 2020  |  

Long-read sequencing for rare human genetic diseases.

During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.

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April 21, 2020  |  

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.

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April 21, 2020  |  

High satellite repeat turnover in great apes studied with short- and long-read technologies.

Satellite repeats are a structural component of centromeres and telomeres, and in some instances their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats of unit sizes up to 50?bp in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads. The density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: (1) the (AATGG)n repeat (critical for heat shock response) and its derivatives; and (2) subtelomeric 32-mers involved in telomeric metabolism. Using the densities of abundant repeats, individuals could be classified into species. However clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males vs. females; using Y chromosome assemblies or FIuorescent In Situ Hybridization, we validated their location on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59?kb in length and consisted of perfect repeats interspersed with other similar sequences. Our results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Profiling the genome-wide landscape of tandem repeat expansions.

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. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020  |  

Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.

Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.

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April 21, 2020  |  

Genetic Variation, Comparative Genomics, and the Diagnosis of Disease.

The discovery of mutations associated with human genetic dis- ease is an exercise in comparative genomics (see Glossary). Although there are many different strategies and approaches, the central premise is that affected persons harbor a significant excess of pathogenic DNA variants as com- pared with a group of unaffected persons (controls) that is either clinically defined1 or established by surveying large swaths of the general population.2 The more exclu- sive the variant is to the disease, the greater its penetrance, the larger its effect size, and the more relevant it becomes to both disease diagnosis and future therapeutic investigation. The most popular approach used by researchers in human genetics is the case–control design, but there are others that can be used to track variants and disease in a family context or that consider the probability of different classes of mutations based on evolutionary patterns of divergence or de novo mutational change.3,4 Although the approaches may be straightforward, the discovery of patho- genic variation and its mechanism of action often is less trivial, and decades of research can be required in order to identify the variants underlying both mendelian and complex genetic traits.

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April 21, 2020  |  

A 12-kb structural variation in progressive myoclonic epilepsy was newly identified by long-read whole-genome sequencing.

We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions and three insertions) on the genes tagged with autosomal recessive phenotypes. Among them, a 12.4-kb deletion involving the CLN6 gene was the top candidate because its homozygous abnormalities cause neuronal ceroid lipofuscinosis. This deletion included the initiation codon and was found in a GC-rich region containing multiple repetitive elements. These results indicate the presence of a causal variant in a difficult-to-sequence region and suggest that such variants that remain enigmatic after the application of current whole-exome sequencing technology could be uncovered by unbiased application of long-read whole-genome sequencing.

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April 21, 2020  |  

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees.We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions.Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees.We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies. © Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

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April 21, 2020  |  

Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases.

Long-read sequencing technology is now capable of reading single-molecule DNA with an average read length of more than 10?kb, fully enabling the coverage of large structural variations (SVs). This advantage may pave the way for the detection of unprecedented SVs as well as repeat expansions. Pathogenic SVs of only known genes used to be selectively analyzed based on prior knowledge of target DNA sequence. The unbiased application of long-read whole-genome sequencing (WGS) for the detection of pathogenic SVs has just begun. Here, we apply PacBio SMRT sequencing in a Japanese family with benign adult familial myoclonus epilepsy (BAFME). Our SV selection of low-coverage WGS data (7×) narrowed down the candidates to only six SVs in a 7.16-Mb region of the BAFME1 locus and correctly determined an approximately 4.6-kb SAMD12 intronic repeat insertion, which is causal of BAFME1. These results indicate that long-read WGS is potentially useful for evaluating all of the known SVs in a genome and identifying new disease-causing SVs in combination with other genetic methods to resolve the genetic causes of currently unexplained diseases.

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April 21, 2020  |  

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

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.

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April 21, 2020  |  

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

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April 21, 2020  |  

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test.We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs.Robust identification of CNVs by GS is possible within a clinical testing environment.

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April 21, 2020  |  

Recompleting the Caenorhabditis elegans genome.

Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted =53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology. © 2019 Yoshimura et al.; Published by Cold Spring Harbor Laboratory Press.

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April 21, 2020  |  

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50?bp) and 27,622 SVs (=50?bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.

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