The human mitochondrial transcriptome.

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

Client perspectives: Telehealth for mental health services.

OBJECTIVE: The COVID-19 pandemic required mental health clinicians globally to transition to the delivery of care via telehealth. This study aimed to gain an understanding of clients' satisfaction with and attitudes towards telehealth mental health services. METHOD: Seventy adults who had attended a clinic for mood and anxiety disorders, and participated in at least one telehealth consultation with a psychologist or psychiatrist, completed an anonymous online survey. RESULTS: The majority of participants (81.5%) reported satisfaction with telehealth mental health care provided during the COVID-19 pandemic. However, satisfaction overall was significantly higher amongst participants who had received both telehealth and face-to-face mental health care, compared to participants who received care via telehealth only. Advantages of telehealth care reported included convenience and increased access to mental health clinicians. However, disadvantages of telehealth care included greater difficulty developing a rapport with a clinician and expressing oneself via telehealth. CONCLUSIONS: Whilst client satisfaction with telehealth mental health care for mood and anxiety disorders is generally high, clinicians should consider the limitations of telehealth from clients' perspectives. In particular, strategies to enhance therapeutic connection during telehealth sessions may be needed, and client preferences for mode of delivery should be taken into consideration when possible.

Heterozygous Variants in the Mechanosensitive Ion Channel TMEM63A Result in Transient Hypomyelination during Infancy.

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.

De Novo Variants in WDR37 Are Associated with Epilepsy, Colobomas, Dysmorphism, Developmental Delay, Intellectual Disability, and Cerebellar Hypoplasia.

WD40 repeat-containing proteins form a large family of proteins present in all eukaryotes. Here, we identified five pediatric probands with de novo variants in WDR37, which encodes a member of the WD40 repeat protein family. Two probands shared one variant and the others have variants in nearby amino acids outside the WD40 repeats. The probands exhibited shared phenotypes of epilepsy, colobomas, facial dysmorphology reminiscent of CHARGE syndrome, developmental delay and intellectual disability, and cerebellar hypoplasia. The WDR37 protein is highly conserved in vertebrate and invertebrate model organisms and is currently not associated with a human disease. We generated a null allele of the single Drosophila ortholog to gain functional insights and replaced the coding region of the fly gene CG12333/wdr37 with GAL4. These flies are homozygous viable but display severe bang sensitivity, a phenotype associated with seizures in flies. Additionally, the mutant flies fall when climbing the walls of the vials, suggesting a defect in grip strength, and repeat the cycle of climbing and falling. Similar to wall clinging defect, mutant males often lose grip of the female abdomen during copulation. These phenotypes are rescued by using the GAL4 in the CG12333/wdr37 locus to drive the UAS-human reference WDR37 cDNA. The two variants found in three human subjects failed to rescue these phenotypes, suggesting that these alleles severely affect the function of this protein. Taken together, our data suggest that variants in WDR37 underlie a novel syndromic neurological disorder.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms.

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.

Workplace stress, common mental disorder and suicidal ideation in junior doctors.

BACKGROUND: Doctors-in-training report elevated rates of mental disorders and high levels of stress. Whilst a number of work-related sources of stress have been identified in the medical profession, it remains unclear as to the relative importance of workplace stressors for mental ill-health in junior doctors. AIMS: To examine workplace stressors reported by junior doctors and identify variables associated with adverse mental health outcomes. METHODS: Cross-sectional analysis of national 2013 survey of Australian doctors focussing on junior medical officers (N = 3053; 24.9% of total sample). Primary outcomes were caseness of common mental disorder (CMD) and suicidal ideation in the past year. RESULTS: Perceived level of conflict between study/career and family/personal responsibility (OR = 3.76, 95% CI: 2.61-5.43; P < 0.01) and sleep deprivation (OR = 2.19, 95% CI: 1.46-3.28; P < 0.01) were significantly associated with CMD, while perceived level of conflict between study/career and family/personal responsibility (OR = 3.13, 95% CI: 1.78-5.50; P < 0.01) and bullying (OR = 2.92, 95% CI: 1.42-6.03; P < 0.01) were most strongly associated with suicidal ideation in adjusted models. CONCLUSION: This study identifies modifiable workplace variables that are influential in junior doctors' mental health, and in doing so, provides meaningful evidence-informed targets for future interventions to prevent suicide and mental disorder in this population.

Help-seeking for depression among Australian doctors.

BACKGROUND: Depression is common among doctors. However, concerns remain that doctors are unlikely to ask for help when symptoms of depression arise. AIMS: To determine rates and patterns of help-seeking for depression among doctors and to identify predictors of and barriers to such behaviour. METHODS: A secondary analysis was conducted on a nation-wide survey of 12 252 Australian doctors. The study sample consisted of doctors who reported having ever felt seriously depressed (n = 4154; 33.9% of total sample). Rates of help-seeking, professional help-seeking behaviours and self-reported barriers were explored. Logistic regression was used to examine the association between professional help-seeking and predetermined predictive factors. RESULTS: Sixty percent (95% confidence interval (CI): 58.5-61.5) of doctors who have ever felt seriously depressed reported some form of professional help-seeking for depression. The most common barrier to help-seeking was 'privacy/confidentiality'. Females (odds ratio (OR) = 1.74; 95% CI: 1.50-2.01; P < 0.001), locally trained doctors (OR = 1.34; 95% CI: 1.12-1.59; P = 0.001) and senior doctors (OR = 1.35; 95% CI: 1.14-1.61; P = 0.001) were more likely to seek professional help than their counterparts. Compared with general practitioners, psychiatrists (OR = 1.565; 95% CI: 1.15-2.13; P = 0.004) were more likely to seek professional help while surgeons (OR = 0.518; 95% CI: 0.37-0.72; P < 0.001) and pathologists/radiologists (OR = 0.695; 95% CI: 0.49-0.99; P = 0.043) were less likely. CONCLUSION: While it is reassuring that the majority of depressed doctors were able to seek professional help, many were not. Major barriers to professional help-seeking, particularly concerns about confidentiality and impact on career, remain a problem. Male, overseas-trained, junior doctors, surgeons and pathologists/radiologists were less likely to seek help for depression. Targeted interventions are required to increase appropriate help-seeking for depression in doctors.

'The hardest job I've ever done': a qualitative exploration of the factors affecting junior doctors' mental health and well-being during medical training in Australia.

BACKGROUND: Medical practitioners can experience considerable stress and poor mental health during their careers, with doctors in training known to be particularly vulnerable. Previous research has documented work-related factors that may play a role in the mental health status of junior doctors. However, these and additional factors, need to be explored further by considering theory-driven, social, structural and contextual issues. This qualitative study aimed to explore the experiences of junior doctors working in Australian hospitals to identify factors that impact their mental health during medical training. METHOD: Semi-structured interviews were conducted with 12 junior medical officers (JMOs) employed across six hospitals in Australia. Transcribed de-identified interviews were analysed thematically using a data-driven inductive approach. RESULTS: Four interrelated main themes were identified: i) professional hierarchies; ii) occupational stress; iii) emotional labour, and iv) taking distress home; which detail the complex affective, relational and professional experiences of JMOs. The accounts demonstrate how the social, professional and organisational dimensions of these experiences impact upon trainee's well-being and mental health, both positively and negatively. Together, the findings document the dynamic, nuanced aspects of junior doctors' experiences of medical training and practice and highlights the importance of relational connections and the workplace environment in shaping JMOs' social and emotional well-being. CONCLUSION: The current study adds to the understanding of how junior doctors navigate medical training in Australian hospitals and highlights the complexities of this experience, particularly the ways in which mental health and well-being are shaped by different elements. These findings have important implications to inform new strategies to improve JMO mental health and to leverage work and non-work contexts to better support JMOs during medical training.

Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.