Search

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first processing-specific human disorder to be described.HighlightsMutations in RMRP lncRNA impair pre-rRNA processing and T cell activationPatient derived fibroblasts show impaired pre-rRNA processingCells with the most common disease-linked mutation have specific processing defectsCytoplasmic ribosomes and intact RNase MRP complexes are also reduced in these cells

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His8, the C-terminus with His8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

Tangye and collaborators use a series of mutants to elucidate the pathways required to generate distinct subsets of human effector CD4+ T cells., Naive CD4+ T cells differentiate into specific effector subsets—Th1, Th2, Th17, and T follicular helper (Tfh)—that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4+ T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10–secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4+ T cell effector function in the settings of infection, vaccination, or immune dysregulation.

Background Unilateral lung agenesis is an uncommon congenital abnormality, with a lack of reported accurate incidence estimates. Prognosis is also uncertain, with older literature reporting poor outcomes. Methods The North of England register of congenital anomalies (Northern Congenital Abnormality Survey) records cases of congenital anomalies to mothers' resident in the region. We used the register to identify all patients with congenital lung agenesis born between 2004 and 2013 to calculate an accurate incidence estimate and report clinical outcomes with contemporary management. Results Four patients with congenital lung agenesis were born during the study period, giving an estimated incidence in the North of England of 1.22 per 100,000 live births (95% confidence interval, 0.33–3.11). Two patients had associated congenital heart disease requiring corrective surgery, and one had musculoskeletal anomalies. All four patients are alive and well without a regular oxygen requirement. Conclusion Contrary to previous reports, the medium term outcomes in our patients have been good, even when lung agenesis is associated with other congenital anomalies. Long-term prognosis with modern management remains unknown, and the potential for the development of pulmonary hypertension remains a concern. Birth Defects Research 109:857–859, 2017. © 2017 Wiley Periodicals, Inc.

Introduction Congenital lung agenesis is a rare abnormality which has been reported to have an estimated incidence of 1 in 15,000 pregnancies and to carry a poor prognosis, especially when associated with congenital heart anomalies. (1) However, precise incidence data has previously been unavailable and clinical management has improved in recent years with an unknown impact on outcomes. Methods We used the North of England register of congenital anomalies (NorCAS) cross-referenced with clinical data from our regional paediatric respiratory centre to calculate the first known accurate incidence estimate for this condition. Detailed clinical and outcome data were then collected with informed consent from the families of affected infants. Results The incidence of lung agenesis was 0.12 per 100,000 live births (95% confidence interval 0.03–0.31). Four cases were identified with a median age at follow-up of 5 years. Lung agenesis was associated with complex congenital heart disease (complete atrioventricular septal defect with left atrial isomerism) in one case, and with aortic coarctation and atrial septal defect in another. Both these patients had their heart defects successfully repaired. The third patient had a normal heart but musculoskeletal problems, while lung agenesis was the only anomaly in the fourth patient. All four patients are well and not on home oxygen. Conclusions We reviewed the course of the four patients with lung agenesis born in the region between 2004 and 2013, and report that medium term outcomes have been good, even when associated with congenital heart disease and other anomalies. This information will provide a useful starting point when counselling parents whose unborn baby has an antenatal diagnosis of lung agenesis. We aim to follow-up these patients to report long-term outcomes as these remain unknown and there is concern about the potential for the development of late onset pulmonary hypertension.1 Reference Muensterer O, Abellar R, Otterburn D, et al. Pulmonary agenesis and associated pulmonary hypertension: A case report and review on variability, therapy, and outcome. Eur J Pediatr Surg Rep 2015;3:33–9.

Summary Background: How to improve patient safety remains an intractable problem, despite large investment and some successes. Context: Academics have argued that the root of the problem is a lack of a comprehensive ‘safety culture’ in hospitals. Other safety-critical industries such as commercial aviation invest heavily in staff training to develop such a culture, but comparable programmes are almost entirely absent from the health care sector. Innovation: In rock climbing and many other dangerous activities, the ‘buddy system’ is used to ensure that safety systems are adhered to despite adverse circumstances. This system involves two or more people using simple checks and clear communication to prevent problems causing harm. Using this system as an example could provide a simple, original and entertaining way of introducing medical students to the idea that human factors are central to ensuring patient safety. Implications: Teaching the buddy system may improve understanding and acceptance of other patient safety initiatives, and could also be used by junior doctors as a tool to improve the safety of their practice.

SummaryProteasomes and lysosomes constitute the major cellular systems that catabolize proteins to recycle free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPPII) is a large cytosolic proteolytic complex that functions in tandem with the proteasome-ubiquitin protein degradation pathway. We found that autosomal recessive TPP2 mutations cause recurrent infections, autoimmunity, and neurodevelopmental delay in humans. We show that a major function of TPPII in mammalian cells is to maintain amino acid levels and that TPPII-deficient cells compensate by increasing lysosome number and proteolytic activity. However, the overabundant lysosomes derange cellular metabolism by consuming the key glycolytic enzyme hexokinase-2 through chaperone-mediated autophagy. This reduces glycolysis and impairs the production of effector cytokines, including IFN-γ and IL-1β. Thus, TPPII controls the balance between intracellular amino acid availability, lysosome number, and glycolysis, which is vital for adaptive and innate immunity and neurodevelopmental health.

ICOS encodes the Inducible T-cell Co-Stimulator (ICOS). Deficiency of this receptor in humans causes a common variable immunodeficiency (CVID) characterised by an absence of class-switched memory B cells and hypogammaglobulinemia. Three pathogenic mutations in ICOS have been described to date in a total of 13 cases. Here we report a novel homozygous 10 base pair frameshift deletion in exon 2 discovered by whole exome sequencing of two siblings from a family of Pakistani origin. Both patients presented in early childhood with diarrhea, colitis and transaminitis and one showed defective handling of human herpesvirus 6. Activated patient CD3+CD4+ T lymphocytes demonstrated a complete absence of ICOS expression and, consistent with previous reports, we detected a reduction in circulating T follicular helper cells. Findings in this kindred emphasise the phenotypic variability of ICOS deficiency and, in particular, the variably impaired antiviral immunity that is a poorly understood facet of this rare disorder. Electronic supplementary material The online version of this article (doi:10.1007/s10875-015-0193-x) contains supplementary material, which is available to authorized users.