The rockefeller university

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5'-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5'-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

Background: The telomere biology disorders (TBDs) include a range of multisystem diseases characterized by mucocutaneous symptoms and bone marrow failure. In dyskeratosis congenita (DKQ, the clinical features of TBDs stem from the depletion of crucial stem cell populations in highly proliferative tissues, resulting from abnormal telomerase function. Due to the wide spectrum of clinical presentations and lack of a conclusive laboratory test it may be challenging to reach a clinical diagnosis, especially if patients lack the pathognomonic clinical features of TBDs. Methods: Clinical sequencing was performed on a cohort of patients presenting with variable immune phenotypes lacking molecular diagnoses. Hypothesis-free whole-exome sequencing (WES) was selected in the absence of compelling diagnostic hints in patients with variable immunological and haematological conditions. Results: In four patients belonging to three families, we have detected five novel variants in known TBD-causing genes (DKC1, TERT and RTEL1). In addition to the molecular findings, they all presented shortened blood cell telomeres. These findings are consistent with the displayed TBD phenotypes, addressing towards the molecular diagnosis and subsequent clinical follow-up of the patients. Conclusions: Our results strongly support the utility of WES-based approaches for routine genetic diagnostics of TBD patients with heterogeneous or atypical clinical presentation who otherwise might remain undiagnosed.

Autosomal recessive CARD9 deficiency underlies life-threatening, invasive fungal infections in otherwise healthy individuals normally resistant to other infectious agents. In less than 10 years, 58 patients from 39 kindreds have been reported in 14 countries from four continents. The patients are homozygous (n = 49; 31 kindreds) or compound heterozygous (n = 9; 8 kindreds) for 22 different CARD9 mutations. Six mutations are recurrent, probably due to founder effects. Paradoxically, none of the mutant alleles has been experimentally demonstrated to be loss-of-function. CARD9 is expressed principally in myeloid cells, downstream from C-type lectin receptors that can recognize fungal components. Patients with CARD9 deficiency present impaired cytokine and chemokine production by macrophages, dendritic cells, and peripheral blood mononuclear cells and defective killing of some fungi by neutrophils in vitro. Neutrophil recruitment to sites of infection is impaired in vivo. The proportion of Th17 cells is low in most, but not all, patients tested. Up to 52 patients suffering from invasive fungal diseases (IFD) have been reported, with ages at onset of 3.5 to 52 years. Twenty of these patients also displayed superficial fungal infections. Six patients had only mucocutaneous candidiasis or superficial dermatophytosis at their last follow-up visit, at the age of 19 to 50 years. Remarkably, for 50 of the 52 patients with IFD, a single fungus was involved; only two patients had IFDs due to two different fungi. IFD recurred in 44 of 45 patients who responded to treatment, and a different fungal infection occurred in the remaining patient. Ten patients died from IFD, between the ages of 12 and 39 years, whereas another patient died at the age of 91 years, from an unrelated cause. At the most recent scheduled follow-up visit, 81% of the patients were still alive and aged from 6.5 to 75 years. Strikingly, all the causal fungi belonged to the phylum Ascomycota: commensal Candida and saprophytic Trychophyton, Aspergillus, Phialophora, Exophiala, Corynesprora, Aureobasidium, and Ochroconis. Human CARD9 is essential for protective systemic immunity to a subset of fungi from this phylum but seems to be otherwise redundant. Previously healthy patients with unexplained invasive fungal infection, at any age, should be tested for inherited CARD9 deficiency. Inherited CARD9 deficiency (OMIM #212050) is an AR PID due to mutations that may be present in a homozygous or compound heterozygous state. CARD9 is expressed principally in myeloid cells and transduces signals downstream from CLR activation by fungal ligands. Endogenous mutant CARD9 levels differ between alleles (from full-length normal protein to an absence of normal protein). The functional impacts of CARD9 mutations involve impaired cytokine production in response to fungal ligands, impaired neutrophil killing and/or recruitment to infection sites, and defects of Th17 immunity. The key clinical manifestations in patients are fungal infections, including CMC, invasive (in the CNS in particular) Candida infections, extensive/deep dermatophytosis, subcutaneous and invasive phaeohyphomycosis, and extrapulmonary aspergillosis. The clinical penetrance of CARD9 deficiency is complete, but penetrance is incomplete for each of the fungi concerned. Age at onset is highly heterogeneous, ranging from childhood to adulthood for the same fungal disease. All patients with unexplained IFD should be tested for CARD9 mutations. Familial screening and genetic counseling should be proposed. The treatment of patients with CARD9 mutations is empirical and based on antifungal therapies and the surgical removal of fungal masses. Patients with persistent/relapsing Candida infections of the CNS could be considered for adjuvant GM-CSF/G-CSF therapy. The potential value of HSCT for CARD9-deficient patients remains unclear.

Background: Fine needle aspiration biopsy (FNAB) is the gold-standard procedure for diagnosing malignant thyroid nodules. Indeterminate cytology is identified in 10% to 40% of cases, and molecular testing may guide management in this setting. Current commercial options are expensive, and are either sensitive or specific. The aim of this study was to utilize next-generation sequencing (NGS) technology to identify informative diversities in the miRNA expression profile of benign versus malignant thyroid nodules. Methods: Ex vivo FNsamples were obtained from thyroid specimens of patients who underwent thyroidectomy at a referral center. miRNA levels were determined using NGS and multiplexing technologies. Statistical analyses identified differences between normal and malignant samples and miRNA expression profiles that associate with malignancy were established. The accuracy of the miRNA signature in predicting histologic malignancy was validated using a group of patient specimens with indeterminate cytology results. Results: A total of 274 samples were obtained from 102 patients undergoing thyroidectomy. Of these samples, 71% were benign and 29% were malignant. Nineteen miRNAs were identified as statistically different between benign and malignant samples and were used to classify 35 additional nodules with indeterminate cytology (validation). The miRNA panel's sensitivity, specificity, negative and positive predictive values, and overall accuracy were 91%, 100%, 87%, 100%, and 94%, respectively. Conclusions: Using NGS technology, we identified a panel of 19 miRNAs that may be utilized to distinguish benign from malignant thyroid nodules with indeterminate cytology. Impact: Our panel may classify indeterminate thyroid nodules at higher accuracy than commercially available molecular tests. (C) 2018 AACR.

Searches for resonances decaying into pairs of jets are performed using proton-proton collision data collected at root s = 13 TeV corresponding to an integrated luminosity of up to 36 fb(-1). A low-mass search, for resonances with masses between 0.6 and 1.6 TeV, is performed based on events with dijets reconstructed at the trigger level from calorimeter information. A high-mass search, for resonances with masses above 1.6 TeV, is performed using dijets reconstructed offline with a particle-flow algorithm. The dijet mass spectrum is well described by a smooth parameterization and no evidence for the production of new particles is observed. Upper limits at 95% confidence level are reported on the production cross section for narrow resonances with masses above 0.6 TeV. In the context of specific models, the limits exclude string resonances with masses below 7.7 TeV, scalar diquarks below 7.2 TeV, axigluons and colorons below 6.1 TeV, excited quarks below 6.0 TeV, color-octet scalars below 3.4 TeV, W' bosons below 3.3 TeV, Z' bosons below 2.7 TeV, Randall-Sundrum gravitons below 1.8 TeV and in the range 1.9 to 2.5 TeV, and dark matter mediators below 2.6 TeV. The limits on both vector and axial-vector mediators, in a simplified model of interactions between quarks and dark matter particles, are presented as functions of dark matter particle mass and coupling to quarks. Searches are also presented for broad resonances, including for the first time spin-1 resonances with intrinsic widths as large as 30% of the resonance mass. The broad resonance search improves and extends the exclusions of a dark matter mediator to larger values of its mass and coupling to quarks.

To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Cross-linking and immunoprecipitation (CLIP) serves this purpose by exploiting covalent protein-RNA cross-linking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare complementary DNA (cDNA) libraries for sequencing. Here we describe the core steps of CLIP, its primary variations, and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice and discuss the mechanistic and physiologic insights that have already been gained from studies using CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms while providing important biologic and clinically relevant insights.

Auditory transduction is fast and sensitive owing to the direct detection of mechanical stimuli by hair cells, the sensory receptors of the internal ear. A study by Dionne et al. (2018) in this issue of Neuron suggests how signals propagate through tip links, the cadherin-based strands that gate mechanically sensitive channels.

The ASBMB 2018 Bert and Natalie Vallee award in Biomedical Sciences honors our work on shelterin, a protein complex that helps cells distinguish the chromosome ends from sites of DNA damage. Shelterin protects telomeres from all aspects of the DNA damage response, including ATM and ATR serine/threonine kinase signaling and several forms of double-strand break repair. Today, this six-subunit protein complex could easily be identified in one single proteomics step. But, it took us more than 15 years to piece together the entire shelterin complex, one protein at a time. Although we did a lot of things right, here I tell the story of shelterin's discovery with an emphasis on the things that I got wrong along the way.

Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1-Npl4 heterodimer. Here, we report structures of the Cdc48-Ufd1-Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn2+-finger domains to anchor the enzymatically inactive Mpr1-Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48's central pore, a position similar to the MPN domain from deubiquitinase Rpn11 in the proteasome. Our results indicate that Npl4 is unique among Cdc48 cofactors and suggest a mechanism for binding and translocation of polyubiquitinated substrates into the ATPase.