Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA

Background: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ΔripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis. Results: LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein. Conclusion: These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA

Detection of long repeat expansions from PCR-free whole-genome sequence data

Identifying large expansions of short tandem repeats (STRs) such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step towards integrating WGS into precision medicine. We have developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3,001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2,786/2,789, 95% CI [0.997, 1.00]) of the wild type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples where every sample had one of eight different pathogenic repeat expansions including those associated with fragile X syndrome, Friedreich's ataxia and Huntington's disease and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions. The software is licensed under GPL v3.0 and the source code is freely available on GitHub

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition

A new class of non-coding RNAs associated with 3\u2019 untranslated regions of mRNAs

The importance of non-coding RNAs (ncRNAs) in controlling gene expression is becoming increasingly evident. However, except for some well characterized examples, such as miRNAs, Xist and Air, the function of most non-coding transcripts is still to be determined. Moreover, while small regulatory RNAs can be relatively easily classified on the basis of their length, secondary structure, and biochemical pathway, the classification of long \u201cmRNA-like\u201d ncRNAs has been problematic. Here we identify a large class of non-coding transcripts that originate within the 3\u2019UTR of at least one third of all genes in the mouse genome. We have several lines of evidence from genome-wide bioinformatic analyses (EST coverage, CAGE data, chromatin state maps of active promoters) and from invitro studies (strand-specific RT-PCR, 5\u2019RACE, Northern blot) showing that these 3\u2019UTR-associated ncRNAs (uaRNAs) can be either linked or transcribed separately to the upstream protein-coding sequences. In addition, expression profiles obtained by custom-designed microarrays on three different developmental systems (myoblast differentiation, male gonadal ridge formation, embryonic stem cell differentiation) showed that uaRNA expression is highly regulated and tissue-specific, and might be either concordant or discordant with respect to the upstream coding region depending on the cell type and on the developmental stage. This observation is confirmed by in-situ hybridization experiments, which evidenced that uaRNA and the associated coding transcript might have different subcellular locations. Our results highlight a further level of complexity at 3\u2019UTRs, suggesting the presence of new regulatory mechanisms that control gene expression during embryonic development. Our data have also important implications for the design of in-situ hybridization and microarray probes as well as for the interpretation of gene expression dat

Leukoencephalopathy due to variants in GFPT1- associated congenital myasthenic syndrome

Objective: To determine the molecular etiology of disease in 4 individuals from 2 unrelated families who presented with proximal muscle weakness and features suggestive of mitochondrial disease. Methods: Clinical information and neuroimaging were reviewed. Genome sequencing was performed on affected individuals and biological parents. Results: All affected individuals presented with muscle weakness and difficulty walking. In one family, both children had neonatal respiratory distress while the other family had 2 children with episodic deteriorations. In each family, muscle biopsy demonstrated ragged red fibers. MRI was suggestive of a mitochondrial leukoencephalopathy, with extensive deep cerebral white matter T2 hyperintense signal and selective involvement of the middle blade of the corpus callosum. Through genome sequencing, homozygous GFPT1 missense variants were identified in the affected individuals of each family. The variants detected (p.Arg14Leu and p.Thr151Lys) are absent from population databases and predicted to be damaging by in silico prediction tools. Following the genetic diagnosis, nerve conduction studies were performed and demonstrated a decremental response to repetitive nerve stimulation, confirming the diagnosis of myasthenia. Treatment with pyridostigmine was started in one family with favorable response. Conclusions: GFPT1 encodes a widely expressed protein that controls the flux of glucose into the hexosamine-biosynthesis pathway that produces precursors for glycosylation of proteins. GFPT1 variants and defects in other enzymes of this pathway have previously been associated with congenital myasthenia. These findings identify leukoencephalopathy as a previously unrecognized phenotype in GFPT1-related disease and suggest that mitochondrial dysfunction could contribute to this disorder.Guy Helman, Suvasini Sharma, Joanna Crawford, Bijoy Patra, Puneet Jain, Stephen J. Bent, Andoni Urtizberea, Ravindra K. Saran, Ryan J. Taft, Marjo S. van der Knaap, and Cas Simon

DARS-associated leukoencephalopathy can mimic a steroid-responsive neuroinflammatory disorder

Objective: To describe the expanding clinical spectrum of a recently described hereditary leukoencephalopathy, hypomyelination with brainstem and spinal cord involvement and leg spasticity, which is caused by mutations in the aspartyl tRNA-synthetase encoding gene DARS, including patients with an adolescent onset. Methods: Three patients with mutations in DARS were identified by combining MRI pattern recognition and genetic analysis. Results: One patient had the typical infantile presentation, but 2 patients with onset in late adolescence had a disease mimicking an acquired inflammatory CNS disorder. Adolescent-onset patients presented with subacute spastic paraplegia and had positive response to steroids. They had only minor focal supratentorial white matter abnormalities, but identical spinal cord changes involving dorsal columns and corticospinal tracts. Clinical presentation included subacute spastic paraplegia with partial improvement on steroids. Conclusions: Focal T2 hyperintense white matter changes on brain MRI in combination with spinal cord signal abnormalities usually suggest acquired inflammatory conditions such as multiple sclerosis, especially in the context of relapsing course and a positive response to steroid treatment. Adolescents with mutations in DARS can present with a comparable clinical picture, broadening the clinical spectrum of hypomyelination with brainstem and spinal cord involvement and leg spasticity