8 research outputs found
Prevalence and Clinical Features of Inflammatory Bowel Diseases Associated With Monogenic Variants, Identified by Whole-Exome Sequencing in 1000 Children at a Single Center
BACKGROUND & AIMS: A proportion of infants and young children with inflammatory bowel diseases (IBDs) have subtypes associated with a single gene variant (monogenic IBD). We aimed to determine the prevalence of monogenic disease in a cohort of pediatric patients with IBD.
METHODS: We performed whole-exome sequencing analyses of blood samples from an unselected cohort of 1005 children with IBD, aged 0-18 years (median age at diagnosis, 11.96 years) at a single center in Canada and their family members (2305 samples total). Variants believed to cause IBD were validated using Sanger sequencing. Biopsies from patients were analyzed by immunofluorescence and histochemical analyses.
RESULTS: We identified 40 rare variants associated with 21 monogenic genes among 31 of the 1005 children with IBD (including 5 variants in XIAP, 3 in DOCK8, and 2 each in FOXP3, GUCY2C, and LRBA). These variants occurred in 7.8% of children younger than 6 years and 2.3% of children aged 6-18 years. Of the 17 patients with monogenic Crohn\u27s disease, 35% had abdominal pain, 24% had nonbloody loose stool, 18% had vomiting, 18% had weight loss, and 5% had intermittent bloody loose stool. The 14 patients with monogenic ulcerative colitis or IBD-unclassified received their diagnosis at a younger age, and their most predominant feature was bloody loose stool (78%). Features associated with monogenic IBD, compared to cases of IBD not associated with a single variant, were age of onset younger than 2 years (odds ratio [OR], 6.30; P = .020), family history of autoimmune disease (OR, 5.12; P = .002), extra-intestinal manifestations (OR, 15.36; P \u3c .0001), and surgery (OR, 3.42; P = .042). Seventeen patients had variants in genes that could be corrected with allogeneic hematopoietic stem cell transplantation.
CONCLUSIONS: In whole-exome sequencing analyses of more than 1000 children with IBD at a single center, we found that 3% had rare variants in genes previously associated with pediatric IBD. These were associated with different IBD phenotypes, and 1% of the patients had variants that could be potentially corrected with allogeneic hematopoietic stem cell transplantation. Monogenic IBD is rare, but should be considered in analysis of all patients with pediatric onset of IBD
Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.
Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for correcting variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations
Localization of Heat Shock Proteins HSPA6 (HSP70B') and HSPA1A (HSP70-1) in Cultured Differentiated Human Neuronal Cells Following Thermal Stress
Heat shock proteins (Hsps) are a set of highly conserved proteins that are involved in cellular repair and protective mechanisms. In order to identify stress-sensitive sites in differentiated human neuronal cells, cytoplasmic and nuclear localization of two inducible members of the HSPA (HSP70) family was investigated, namely HSPA6 (HSP70B') and HSPA1A (HSP70-1). Stably transfected SH-SY5Y neuronal cell lines were generated that express YFP-HSPA6 and YFP-HSPA1A proteins. Following differentiation, transfected SH-SY5Y cells were exposed to mild heat shock at 43째C for 20 min. Immediately following heat shock, YFP-tagged HSPA6 and HSPA1A proteins localized to centrioles in the cytoplasm and nuclear speckles that are enriched in RNA splicing factors. Subsequently, both stress proteins associated with the granular component of the nucleolus which is the site of ribosomal subunit assembly and ribosomal RNA processing. Later in the recovery period, YFP-HSPA6 protein, but not YFP-HSPA1A, localized to the periphery of nuclear speckles that are sites of RNA transcription and RNA splicing. The present results identify centrioles, nuclear speckles, and the GC component of the nucleolus as potential stress-sensitive sites in differentiated human neuronal cells. The inducible HSPA6 and HSPA1A proteins exhibit similar localization to these sites. The unique association of the HSPA6 protein with the periphery of nuclear speckles identifies this nuclear site as being stress sensitive, and suggests that the little studied HSPA6 protein may be involved in the recovery of RNA transcription and/or splicing. HSPA6 is a strictly inducible protein, whose gene is present in the human genome but not in rat and mouse. Hence current animal models of neurodegenerative diseases are lacking a potentially protective member of the HSPA family. Sequence and structural analysis revealed that HSPA6 has three additional cysteine amino acids compared to HSPA1A. One of these cysteine amino acids, located in the linker domain, is not found in any other member of the human HSPA family. Identification of stress-sensitive sites in differentiated human neuronal cells could aid the design of therapeutic approaches to protect neurons from the progression of neurodegenerative diseases.Ph.D.2016-11-30 00:00:0
Translation initiation in human immunodeficiency virus type 1 (HIV-1)
Translation of human immunodeficiency virus type 1 (HIV-1) mRNAs is entirely dependent on the host translation machinery. There are two prevailing hypotheses regarding the translation initiation mechanism in HIV-1; conventional cap-dependent ribosomal scanning mechanism (CDRSM) and cap-independent entry of the ribosome, usually through an internal ribosome entry site (IRES). The first mechanism makes use of the Kozak consensus sequence in locating the translation initiation codon, similar to the mechanism observed in human mRNAs. Therefore, a thorough understanding of the Kozak consensus and translation initiation in human would also shed light on the mechanism of translation initiation in HIV-1. The role of Kozak +4G site in translation initiation has been controversial, with the alternative hypothesis explaining the prevalence of +4G by invoking the observation that small amino acids, coded by G-starting codons, which are efficient for N-terminal methionine excision (NME), are preferred at the penultimate (second) position. Using two bioinformatics approaches we provide strong support for this alternative hypothesis and provide evidence contradicting the involvement of +4G in translation initiation.
One of the predictions of the CDRSM hypothesis is a high conservation of Kozak consensus sequence in different HIV-1 sequences. Our results presented here validate this prediction. The CDRSM hypothesis also predicts that there should be a selective pressure against ATG usage in optimal context in the HIV-1 5'-UTR to avoid their erroneous detection by the scanning ribosome, whereas the IRES-dependent mechanism in the presence of stable secondary structures, predicts no such selective pressure because these ATGs would be embedded in the secondary structures. Here we demonstrate this selective pressure in the HIV-1 5'-UTR which further supports the CDRSM hypothesis. Finally, we present evidence for strong site conservation in the 5'-UTR of HIV-1 sequences, which not only point to as yet unknown mechanisms of translation initiation, but also provide a mean to separate HIV-1 and human mRNAs. This implies that it is theoretically possible to design HIV-1-specific translation inhibition drugs
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Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.
Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations
Increased light intensity induces heat shock protein Hsp60 in coral species
The effect of increased light intensity and heat stress on heat shock protein Hsp60 was examined in two coral species using a branched coral and a laminar coral, selected for their different resistance to environmental perturbation. Transient Hsp60 induction was observed in the laminar coral following either light or thermal stress. Sustained induction was observed when these stresses were combined. The branched coral exhibited comparatively weak transient Hsp60 induction after heat stress and no detectable induction following light stress, consistent with its susceptibility to bleaching in native environments compared to the laminar coral. Our observations also demonstrate that increased light intensity and heat stress exhibited a greater negative impact on the photosynthetic capacity of environmentally sensitive branched coral than the more resistant laminar coral. This supports a correlation between stress induction of Hsp60 and (a) ability to counter perturbation of photosynthetic capacity by light and heat stress and (b) resistance to environmentally induced coral bleaching