February 25, 2021  |  

Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly

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 rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.

February 25, 2021  |  

Long-read trio sequencing of individuals with unsolved intellectual disability

Long-read sequencing (LRS) has the potential to comprehensively identify all medically relevant genome variation, including variation commonly missed by short-read sequencing (SRS) approaches. To determine this potential, we performed LRS around 15×–40× genome coverage using the Pacific Biosciences Sequel I System for five trios. The respective probands were diagnosed with intellectual disability (ID) whose etiology remained unresolved after SRS exomes and genomes. Systematic assessment of LRS coverage showed that ~35 Mb of the human reference genome was only accessible by LRS and not SRS. Genome-wide structural variant (SV) calling yielded on average 28,292 SV calls per individual, totaling 12.9 Mb of sequence. Trio-based analyses which allowed to study segregation, showed concordance for up to 95% of these SV calls across the genome, and 80% of the LRS SV calls were not identified by SRS. De novo mutation analysis did not identify any de novo SVs, confirming that these are rare events. Because of high sequence coverage, we were also able to call single nucleotide substitutions. On average, we identified 3 million substitutions per genome, with a Mendelian inheritance concordance of up to 97%. Of these, ~100,000 were located in the ~35 Mb of the genome that was only captured by LRS. Moreover, these variants affected the coding sequence of 64 genes, including 32 known Mendelian disease genes. Our data show the potential added value of LRS compared to SRS for identifying medically relevant genome variation.

February 25, 2021  |  

Long-read genome sequencing for the diagnosis of neurodevelopmental disorders

Purpose Exome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches.Methods Here, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads.Results We identified variants and created de novo assemblies in each trio, with global metrics indicating these data sets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), de novo L1-mediated insertion in CDKL5 that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large de novo structural variants, including insertion-translocations affecting DGKB and MLLT3, which we show disrupt MLLT3 transcript levels. We consider this extensive structural variation likely pathogenic.Conclusion The breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs strongly support the value of long-read genome sequencing for understanding rare disease.Competing Interest StatementThe authors have declared no competing interest.

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