Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability

The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene

Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome

Overlap between Central and Peripheral Transcriptomes in Parkinson’s Disease but Not Alzheimer’s Disease

Most neurodegenerative disorders take decades to develop, and their early detection is challenged by confounding non-pathological ageing processes. Therefore, the discovery of genes and molecular pathways in both peripheral and brain tissues that are highly predictive of disease evolution is necessary. To find genes that influence Alzheimer’s disease (AD) and Parkinson’s disease (PD) pathogenesis, human RNA-Seq transcriptomic data from Brodmann Area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC), whole blood (WB), and peripheral blood mononuclear cells (PBMC) were analysed using a combination of differential gene expression and a random forest-based machine learning algorithm. The results suggest that there is little overlap between PD and AD, and the AD brain signature is unique mainly compared to blood-based samples. Moreover, the AD-BA9 was characterised by changes in ‘nervous system development’ with Myocyte-specific enhancer factor 2C (Mef2C), encoding a transcription factor that induces microglia activation, a prominent feature. The peripheral AD transcriptome was associated with alterations in ‘viral process’, and FYN, which has been previously shown to link amyloid-beta and tau, was the prominent feature. However, in the absence of any overlap with the central transcriptome, it is unclear whether peripheral FYN levels reflect AD severity or progression. In PD, central and peripheral signatures are characterised by anomalies in ‘exocytosis’ and specific genes related to the SNARE complex, including Vesicle-associated membrane protein 2 (VAMP2), Syntaxin 1A (STX1A), and p21-activated kinase 1 (PAK1). This is consistent with our current understanding of the physiological role of alpha-synuclein and how alpha-synuclein oligomers compromise vesicle docking and neurotransmission. Overall, the results describe distinct disease-specific pathomechanisms, both within the brain and peripherally, for the two most common neurodegenerative disorders

The gene encoding δ-hexatoxin-Hi1a is intronless.

<p>(A) Schematic of the putative δ-hexatoxin gene showing the region that each primer set (L1–L5) is designed to amplify. (B) Gels showing the PCR products obtained using each of the designed primer sets L1–L5: (i) cDNA template; (ii) gDNA template; (iii) Southern Blot. For each gel, ML denotes 1 kb molecular-weight ladder, while L1–L5 denote the primer sets A–E shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043699#pone-0043699-t001" target="_blank">Table 1</a>.</p

Architecture of gene encoding the lethal toxin from Australian funnel-web spiders.

<p>Alignment of the cDNA and gDNA sequences obtained for δ-hexatoxin-Hi1a. The nucleotide sequences are identical except at a single position (T versus C, highlighted in grey) that does not alter the encoded protein sequence. The stop codon is denoted by an asterisk. A schematic of the toxin precursor showing the signal peptide, propeptide, mature toxin, and 3’ untranslated region in yellow, blue, purple and white, respectively, is shown above the sequences. The protein sequence (i.e., a translation of the cDNA/gDNA) is shown sandwiched between the cDNA and gDNA sequences.</p

Architecture of spider-toxin genes.

<p>Intron-exon organization for genes encoding (A) μ-diguetoxin-Dc1a from the American desert spider <i>Diguetia canities </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043699#pone.0043699-Krapcho1" target="_blank">[15]</a>; (B) 24 different disulfide-rich venom peptides from the Chinese tarantulas <i>Haplopelma hainanum</i> and <i>Haplopelma huwenum </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043699#pone.0043699-Jiang1" target="_blank">[16]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043699#pone.0043699-Qiao1" target="_blank">[18]</a>. In panel (A), the colors denote exons encoding the signal peptide, propeptide, and mature toxin. In panel (B), the entire toxin prepropeptide precursor is encoded by an intronless ORF.</p