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…
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…
Expression of mutant Ataxin-1 with an abnormally expanded polyglutamine domain is necessary for the onset and progression of spinocerebellar ataxia type 1 (SCA1). Understanding how Ataxin-1 expression is regulated in the human brain could inspire novel molecular therapies for this fatal, dominantly inherited neurodegenerative disease. Previous studies have shown that the ATXN1 3’UTR plays a key role in regulating the Ataxin-1 cellular pool via diverse post-transcriptional mechanisms. Here we show that elements within the ATXN1 5’UTR also participate in the regulation of Ataxin-1 expression. PCR and PacBio sequencing analysis of cDNA obtained from control and SCA1 human brain samples revealed…
Alzheimer’s disease (AD) is a leading cause of mortality in the elderly. While the coding change of APOE-e4 is a key risk factor for late-onset AD and has been believed to be the only risk factor in the APOE locus, it does not fully explain the risk effect conferred by the locus. Here, we report the identification of AD causal variants in PVRL2 and APOC1 regions in proximity to APOE and define common risk haplotypes independent of APOE-e4 coding change. These risk haplotypes are associated with changes of AD-related endophenotypes including cognitive performance, and altered expression of APOE and its…
Michael Lutz, from the Duke University Medical Center, discussed a recently published software tool that can now be used in a pipeline with SMRT Sequencing data to find structural variant biomarkers for neurodegenerative diseases with a focus on Alzheimer’s disease, ALS, and Lewy body dementia. His team is particularly interested in short sequence repeats and short tandem repeats, which have already been implicated in neurodegenerative disease.
At AGBT 2017, Lars Paulin from the University of Helsinki presented this poster on whole genome sequencing of the virus responsible for progressive multifocal leukoencephalopathy, a rare and dangerous brain infection. His team used long amplicon analysis to resolve the whole virus genome from three patient samples, pooled them for SMRT Sequencing, and identified variants and rearrangements. This work represents the first time the viral genome was sequenced from patients.
Over 40% of male and ~16% of female carriers of a premutation FMR1 allele (55-200 CGG repeats) will develop fragile X-associated tremor/ataxia syndrome, an adult onset neurodegenerative disorder, while about 20% of female carriers will develop fragile X-associated primary ovarian insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown whether all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to…
Regulating fluctuating endogenous nitric oxide (NO) levels is necessary for proper physiological functions. Aberrant NO pathways are implicated in a number of neurological disorders, including Alzheimer’s disease (AD) and Parkinson’s disease. The mechanism of NO in oxidative and nitrosative stress with pathological consequences involves reactions with reactive oxygen species (e.g., superoxide) to form the highly reactive peroxynitrite, hydrogen peroxide, hypochloride ions and hydroxyl radical. NO levels are typically regulated by endogenous nitric oxide synthases (NOS), and inflammatory iNOS is implicated in the pathogenesis of neurodegenerative diseases, in which elevated NO mediates axonal degeneration and activates cyclooxygenases to provoke neuroinflammation. NO…
FMR1 premutation carriers (55-200 CGG repeats) are at risk for developing Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder. Approximately 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI), in addition to a number of clinical problems affecting premutation carriers throughout their life span. Marked elevation in FMR1 mRNA levels have been observed with premutation alleles resulting in RNA toxicity, the leading molecular mechanism proposed for the FMR1 associated disorders observed in premutation carriers. The FMR1 gene undergoes alternative splicing and we have recently reported that the relative abundance of all FMR1 mRNA isoforms is…
Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation specific antibodies. We used mutational scanning by cell-surface display to screen 1,341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single molecule real time gene sequencing was used to quantify enrichment of mutants, returning on average 26,000 high quality full-length reads for each screened population. Relative enrichment of mutants correlated to the magnitude of the change in binding affinity. Mutations that diminished binding of…
A large, non-coding ATTCT repeat expansion causes the neurodegenerative disorder, spinocerebellar ataxia type 10 (SCA10). In a subset of SCA10 patients, interruption motifs are present at the 5′ end of the expansion and strongly correlate with epileptic seizures. Thus, interruption motifs are a predictor of the epileptic phenotype and are hypothesized to act as a phenotypic modifier in SCA10. Yet, the exact internal sequence structure of SCA10 expansions remains unknown due to limitations in current technologies for sequencing across long extended tracts of tandem nucleotide repeats. We used the third generation sequencing technology, Single Molecule Real Time (SMRT) sequencing, to…
X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA…
Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion…