The rockefeller university

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.

The gamma-tubulin ring complex (gamma-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human gamma-TuRC at similar to 3.8 angstrom resolution, revealing an asymmetric, cone-shaped structure. Pseudoatomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the gamma-TuRC "seam.'' We also identify an unanticipated structural bridge that includes an actin-like protein and spans the gamma-TuRC lumen. Despite its asymmetric architecture, the gamma-TuRC arranges gamma-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the gamma-TuRC subunits introduces large (>100,000 angstrom(2)) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the gamma-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.

The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases.

Deficiency of ubiquitin-specific peptidase 18 (USP18) is a severe type I interferonopathy. USP18 down-regulates type I interferon signaling by blocking the access of Janus-associated kinase 1 (JAK1) to the type I interferon receptor. The absence of USP18 results in unmitigated interferon-mediated inflammation and is lethal during the perinatal period. We describe a neonate who presented with hydrocephalus, necrotizing cellulitis, systemic inflammation, and respiratory failure. Exome sequencing identified a homozygous mutation at an essential splice site on USP18. The encoded protein was expressed but devoid of negative regulatory ability. Treatment with ruxolitinib was followed by a prompt and sustained recovery. (Funded by King Saud University and others.) A neonate with a loss-of-function mutation in USP18 and exuberant expression of interferon-stimulated genes was experimentally treated with ruxolitinib, which suppresses interferon signaling. The initiation of treatment was followed by an improvement in the child's clinical course.

Background Cytomegalovirus (CMV) can cause severe disease in children and adults with a variety of inherited or acquired T-cell immunodeficiencies, who are prone to multiple infections. It can also rarely cause disease in otherwise healthy persons. The pathogenesis of idiopathic CMV disease is unknown. Inbred mice that lack the gene encoding nitric oxide synthase 2 (Nos2) are susceptible to the related murine CMV infection. Methods We studied a previously healthy 51-year-old man from Iran who after acute CMV infection had an onset of progressive CMV disease that led to his death 29 months later. We hypothesized that the patient may have had a novel type of inborn error of immunity. Thus, we performed whole-exome sequencing and tested candidate mutant alleles experimentally. Results We found a homozygous frameshift mutation in NOS2 encoding a truncated NOS2 protein that did not produce nitric oxide, which determined that the patient had autosomal recessive NOS2 deficiency. Moreover, all NOS2 variants that we found in homozygosity in public databases encoded functional proteins, as did all other variants with an allele frequency greater than 0.001. Conclusions These findings suggest that inherited NOS2 deficiency was clinically silent in this patient until lethal infection with CMV. Moreover, NOS2 appeared to be redundant for control of other pathogens in this patient. (Funded by the National Center for Advancing Translational Sciences and others.) A 51-year-old Iranian man presented with severe CMV infection, which proved to be fatal over the next 29 months. Investigation determined that he had a homozygous frameshift mutation in the gene encoding a nonfunctional NOS2 protein.

Objectives The aim of the study was to estimate the rate of adverse reactions to live BCG Moreau vaccine, manufactured by Biomed in Poland, in severe combined immunodeficiency (SCID) patients. Material The profiles of 52 SCID patients vaccinated at birth with BCG, hospitalized in Children's Memorial Health Institute, Warsaw (CMHI), in the years 1980-2015 were compared with those of 349 BCG-vaccinated SCID patients from other countries analyzed by Beatriz E. Marciano et al. in a retrospective study (Marciano et al. J Allergy Clin Immunol. 2014;133(4):1134-1141). Results Significantly less disseminated BCG infections (10 out of 52 SCID, 19%) occurred in comparison with Marciano study-119 out of 349, 34% (p = 0.0028), with no death in patients treated with SCID anti-TB drug, except one in lethal condition. In our study, disseminated BCG infection was observed only in SCID with T-B+NK- phenotype and significantly lower NK cell counts (p = 0.0161). NK cells do not influence on the frequency of local BCG reaction. A significantly higher number of hematopoietic stem cells transplantations (HSCT) were performed in CMHI study (p = 0.0001). Anti-TB treatment with at least two medicines was provided. Conclusion The BCG Moreau vaccine produced in Poland, with well-documented genetic characteristics, seems to be safer than other BCG substrains used in other regions of the world. Importantly, NK cells seem to play a role in protecting SCID patients against disseminated BCG complications, which NK- SCID patients are more prone to. HSCT and TB therapy could be relevant due to the patients' survival and the fact that they protect against BCG infection.

The advances made in bioorthogonal chemistry and the development of chemical reporters have afforded new strategies to explore the targets and functions of specific metabolites in biology. These metabolite chemical reporters have been applied to diverse classes of bacteria including Gram-negative, Gram-positive, mycobacteria, and more complex microbiota communities. Herein we summarize the development and application of metabolite chemical reporters to study fundamental pathways in bacteria as well as microbiota mechanisms in health and disease.