Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced ...pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a “proof of concept” for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.
The identification of cancer-associated long noncoding RNAs (lncRNAs) and the investigation of their molecular and biological functions are important to understand the molecular biology of cancer and ...its progression. Although the functions of lncRNAs and the mechanisms regulating their expression are largely unknown, recent studies are beginning to unravel their importance in human health and disease. Here, we report that a number of lncRNAs are differentially expressed in melanoma cell lines in comparison to melanocytes and keratinocyte controls. One of these lncRNAs, SPRY4-IT1 (GenBank accession ID AK024556), is derived from an intron of the SPRY4 gene and is predicted to contain several long hairpins in its secondary structure. RNA-FISH analysis showed that SPRY4-IT1 is predominantly localized in the cytoplasm of melanoma cells, and SPRY4-IT1 RNAi knockdown results in defects in cell growth, differentiation, and higher rates of apoptosis in melanoma cell lines. Differential expression of both SPRY4 and SPRY4-IT1 was also detected in vivo, in 30 distinct patient samples, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic melanoma. The elevated expression of SPRY4-IT1 in melanoma cells compared to melanocytes, its accumulation in cell cytoplasm, and effects on cell dynamics, including increased rate of wound closure on SPRY4-IT1 overexpression, suggest that the higher expression of SPRY4-IT1 may have an important role in the molecular etiology of human melanoma.
During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. ...Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.
RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse ...sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼7 d.
Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is ...poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed that all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a MA ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modeled variants result in strongly attenuated stretch-activated currents when expressed in naive cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after 4 years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.
ObjectiveDespite concern regarding high rates of mental illness and suicide amongst the medical profession, the link between working hours and doctors’ mental health remains unclear. This study ...examines the relationship between average weekly working hours and junior doctors’ (JDs’) mental health in Australia.Design and participantsA randomly selected sample of 42 942 Australian doctors were invited to take part in an anonymous Beyondblue National Mental Health Survey in 2013, of whom 12 252 doctors provided valid data (response rate approximately 27%). The sample of interest comprised 2706 full-time graduate medical trainees in various specialties, at either intern, prevocational or vocational training stage. Consultants and retired doctors were excluded.Outcome measuresMain outcomes of interest were caseness of common mental disorder (CMD) (assessed using a cut-off of 4 as a threshold on total General Health Questionnaire-28 score), presence of suicidal ideation (SI) (assessed with a single item) and average weekly working hours. Logistic regression modelling was used to account for the impact of age, gender, stage of training, location of work, specialty, marital status and whether JDs had trained outside Australia.ResultsJDs reported working an average of 50.1 hours per week (SD=13.4). JDs who worked over 55 hours a week were more than twice as likely to report CMD (adjusted OR=2.05; 95% CI 1.62 to 2.59, p<0.001) and SI (adjusted OR=2.00; 95% CI 1.42 to 2.81, p<0.001) compared to those working 40–44 hours per week.ConclusionsOur results show that around one in four JDs are currently working hours that are associated with a doubling of their risk of common mental health problems and SI. These findings suggest that management of working hours represents an important focus for workplaces to improve the mental health of medical trainees.
A variety of methods have been developed to demultiplex pooled samples in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or sample genotypes prior to ...pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled samples. scSplit also enables mapping clusters to original samples. Using simulated, merged, and pooled multi-individual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to samples without external genotype information and is available at: https://github.com/jon-xu/scSplit.
To examine the perceptions of health professionals regarding the gaps in mental health service provision in Australia and their need for assistance in managing patients with mental illness.
A total ...of 570 health professionals participated in an anonymous online survey in January 2018 that assessed: i) health professionals’ current levels of need for assistance in the management of patients with mental health conditions; and ii) perceived gaps in the mental health care system, and how these can be addressed. Data were analysed using a mixed‐methods approach.
Of those surveyed, 71.2% of health professionals and 77.3% of general practitioners reported that they required assistance in managing their patients with at least one stage of care for at least one type of mental disorder. Qualitative analyses revealed eight major themes in health professionals’ perceptions of gaps in mental health service provision, including affordability and accessibility, the problems with crisis‐driven care and the ‘missing middle’.
Overall, the results of this study provide a concerning insight into the substantial gaps in mental health care within the Australian system.
The results of this study add weight to ongoing calls for reform of and increased investment in the Australian mental health care system.
Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially ...regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 5' end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.
Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we ...report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.
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