Motivation:Long-read RNA sequencing has the potential to accurately quantify transcriptomes and reveal the isoform diversity of disease-causing genes. However, despite the recent advances in analysis tools for transcript discovery, long-read RNA sequencing data is still challenging to analyse, due to the detection of hundreds or even thousands of novel transcripts per gene. Results:Here, we introduce PSQAN, a workflow to help researchers prioritize high-confidence and potentially biologically relevant transcripts associated with candidate genes and make transcript characterization results more interpretable. PSQAN performs a gene-based analysis on characterized transcripts generated by SQANTI3 and TALON. PSQAN re-groups transcripts into easily interpretable categories to facilitate their prioritization, allows transcript-level expression thresholds, and generates visualizations to determine optimal expression thresholds. Overall, we demonstrate that PSQAN is a useful tool which enables users to identify known and novel transcripts of potential biological importance. Availability and implementation:PSQAN is an analysis workflow implemented in Snakemake and R and is licensed under the GNU General Public License version 3. The source code and documentation of this tool is available at https://github.com/sid-sethi/PSQAN.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson’s disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1 . We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1 . Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1 . We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1 . These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function—yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation (head-bob, hb) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function.

Improvements in functional genomic annotation have led to a critical mass of neurogenetic discoveries. This is exemplified in hereditary ataxia, a heterogeneous group of disorders characterised by incoordination from cerebellar dysfunction. Associated pathogenic variants in more than 300 genes have been described, leading to a detailed genetic classification partitioned by age-of-onset. Despite these advances, up to 75% of patients with ataxia remain molecularly undiagnosed even following whole genome sequencing, as exemplified in the 100 000 Genomes Project. This study aimed to understand whether we can improve our knowledge of the genetic architecture of hereditary ataxia by leveraging functional genomic annotations, and as a result, generate insights and strategies that raise the diagnostic yield. To achieve these aims, we used publicly-available multi-omics data to generate 294 genic features, capturing information relating to a gene's structure, genetic variation, tissue-specific, cell-type-specific and temporal expression, as well as protein products of a gene. We studied these features across genes typically causing childhood-onset, adult-onset or both types of disease first individually, then collectively. This led to the generation of testable hypotheses which we investigated using whole genome sequencing data from up to 2182 individuals presenting with ataxia and 6658 non-neurological probands recruited in the 100 000 Genomes Project. Using this approach, we demonstrated a high short tandem repeat (STR) density within childhood-onset genes suggesting that we may be missing pathogenic repeat expansions within this cohort. This was verified in both childhood- and adult-onset ataxia patients from the 100 000 Genomes Project who were unexpectedly found to have a trend for higher repeat sizes even at naturally-occurring STRs within known ataxia genes, implying a role for STRs in pathogenesis. Using unsupervised analysis, we found significant similarities in genomic annotation across the gene panels, which suggested adult- and childhood-onset patients should be screened using a common diagnostic gene set. We tested this within the 100 000 Genomes Project by assessing the burden of pathogenic variants among childhood-onset genes in adult-onset patients and vice versa. This demonstrated a significantly higher burden of rare, potentially pathogenic variants in conventional childhood-onset genes among individuals with adult-onset ataxia. Our analysis has implications for the current clinical practice in genetic testing for hereditary ataxia. We suggest that the diagnostic rate for hereditary ataxia could be increased by removing the age-of-onset partition, and through a modified screening for repeat expansions in naturally-occurring STRs within known ataxia-associated genes, in effect treating these regions as candidate pathogenic loci.

There is growing evidence for the importance of 3’ untranslated region (3’UTR) dependent regulatory processes. However, our current human 3’UTR catalogue is incomplete. Here, we develop a machine learning-based framework, leveraging both genomic and tissue-specific transcriptomic features to predict previously unannotated 3’UTRs. We identify unannotated 3’UTRs associated with 1,563 genes across 39 human tissues, with the greatest abundance found in the brain. These unannotated 3’UTRs are significantly enriched for RNA binding protein (RBP) motifs and exhibit high human lineage-specificity. We find that brain-specific unannotated 3’UTRs are enriched for the binding motifs of important neuronal RBPs such as TARDBP and RBFOX1 , and their associated genes are involved in synaptic function. Our data is shared through an online resource F3UTER ( https://astx.shinyapps.io/F3UTER/ ). Overall, our data improves 3’UTR annotation and provides additional insights into the mRNA-RBP interactome in the human brain, with implications for our understanding of neurological and neurodevelopmental diseases.

ABSTRACT The human genome contains numerous duplicated regions, such as parent-pseudogene pairs, causing sequencing reads to align equally well to either gene. The extent to which this ambiguity complicates transcriptomic analyses is currently unknown. This is concerning as many parent genes have been linked to disease, including GBA1, causally linked to both Parkinson’s and Gaucher disease. We find that most of the short sequencing reads that map to GBA1 , also map to its pseudogene, GBAP1 . Using long-read RNA-sequencing in human brain, where all reads mapped uniquely, we demonstrate significant differences in expression compared to short-read data. We identify novel transcripts from both GBA1 and GBAP1 , including protein-coding transcripts that are translated in vitro and detected in proteomic data, but that lack GCase activity. By combining long-read with single-nuclear RNA-sequencing to analyse brain-relevant cell types we demonstrate that transcript expression varies by brain region with cell-type-selectivity. Taken together, these results suggest a non-lysosomal function for both GBA1 and GBAP1 in brain. Finally, we demonstrate that inaccuracies in annotation are widespread among parent genes, with implications for many human diseases.

Sequestosome-1 (SQSTM1/p62) is involved in cellular processes such as autophagy and metabolic reprogramming. Mutations resulting in the loss of function of SQSTM1 lead to neurodegenerative diseases including frontotemporal dementia. The pathogenic mechanism that contributes to SQSTM1-related neurodegeneration has been linked to its role as an autophagy adaptor, but this is poorly understood, and its precise role in mitochondrial function and clearance remains to be clarified. Here, we assessed the importance of SQSTM1 in human induced pluripotent stem cell (iPSC)-derived cortical neurons through the knockout of SQSTM1. We show that SQSTM1 depletion causes altered mitochondrial gene expression and functionality, as well as autophagy flux, in iPSC-derived neurons. However, SQSTM1 is not essential for mitophagy despite having a significant impact on early PINK1-dependent mitophagy processes including PINK1 recruitment and phosphorylation of ubiquitin on depolarized mitochondria. These findings suggest that SQSTM1 is important for mitochondrial function rather than clearance.

Background Efforts to elucidate the function of enhancers in vivo are underway but their vast numbers alongside differing enhancer architectures make it difficult to determine their impact on gene activity. By systematically annotating multiple mouse tissues with super- and typical-enhancers, we have explored their relationship with gene function and phenotype. Results Though super-enhancers drive high total- and tissue-specific expression of their associated genes, we find that typical-enhancers also contribute heavily to the tissue-specific expression landscape on account of their large numbers in the genome. Unexpectedly, we demonstrate that both enhancer types are preferentially associated with relevant 'tissue-type' phenotypes and exhibit no difference in phenotype effect size or pleiotropy. Modelling regulatory data alongside molecular data, we built a predictive model to infer gene-phenotype associations and use this model to predict potentially novel disease-associated genes. Conclusion Overall our findings reveal that differing enhancer architectures have a similar impact on mammalian phenotypes whilst harbouring differing cellular and expression effects. Together, our results systematically characterise enhancers with predicted phenotypic traits endorsing the role for both types of enhancers in human disease and disorders.

ABSTRACT Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2+/Y265X) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2+/+). The Polg2+/Y265X and Polg2+/+ mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2+/Y265X kidneys was reduced compared to Polg2+/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2+/Y265X mice, compared to Polg2+/+ littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

ABSTRACT Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T) mice, when compared with wild-type (Brd4+/+) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.

In the version of this article originally published, minus signs were missing from the three β-values for BMI given in Table 1. The errors have been corrected in the HTML and PDF versions of the article.

An AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3 significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3Sci/+, we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3Sci/+ mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3Sci/+-dependent network.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N -ethyl- N -nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The brain is a functionally complex organ, the patterning and development of which are key to adult health. To help elucidate the genetic networks underlying mammalian brain patterning, we conducted detailed transcriptional profiling during embryonic development of the mouse brain. A total of 2,400 genes were identified as showing differential expression between three developmental stages. Analysis of the data identified nine gene clusters to demonstrate analogous expression profiles. A significant group of novel genes of as yet undiscovered biological function were detected as being potentially relevant to brain development and function, in addition to genes that have previously identified roles in the brain. Furthermore, analysis for genes that display asymmetric expression between the left and right brain hemispheres during development revealed 35 genes as putatively asymmetric from a combined data set. Our data constitute a valuable new resource for neuroscience and neurodevelopment, exposing possible functional associations between genes, including novel loci, and encouraging their further investigation in human neurological and behavioural disorders.