The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest ...signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60:40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms.
Type 2 diabetes mellitus results from the interaction of environmental factors with a combination of genetic variants, most of which were hitherto unknown. A systematic search for these variants was ...recently made possible by the development of high-density arrays that permit the genotyping of hundreds of thousands of polymorphisms. We tested 392,935 single-nucleotide polymorphisms in a French case-control cohort. Markers with the most significant difference in genotype frequencies between cases of type 2 diabetes and controls were fast-tracked for testing in a second cohort. This identified four loci containing variants that confer type 2 diabetes risk, in addition to confirming the known association with the TCF7L2 gene. These loci include a non-synonymous polymorphism in the zinc transporter SLC30A8, which is expressed exclusively in insulin-producing beta-cells, and two linkage disequilibrium blocks that contain genes potentially involved in beta-cell development or function (IDE-KIF11-HHEX and EXT2-ALX4). These associations explain a substantial portion of disease risk and constitute proof of principle for the genome-wide approach to the elucidation of complex genetic traits.
A new study reports that two common loss-of-function mutations in the gene encoding filaggrin are important risk factors for atopic dermatitis, and interestingly, for asthma in association with ...atopic dermatitis, but not asthma in the absence of atopic dermatitis. These findings suggest the importance of the epidermal barrier in preventing sensitization to allergens.
Targeted sequencing of 103 leukemia-associated genes in leukemia cells from 841 treatment-naive patients with chronic lymphocytic leukemia (CLL) identified 89 (11%) patients as having CLL cells with ...mutations in genes encoding proteins that putatively are involved in hedgehog (Hh) signaling. Consistent with this finding, there was a significant association between the presence of these mutations and the expression of GLI1 (χ2 test, P < .0001), reflecting activation of the Hh pathway. However, we discovered that 38% of cases without identified mutations also were GLI1+. Patients with GLI1+ CLL cells had a shorter median treatment-free survival than patients with CLL cells lacking expression of GLI1 independent of IGHV mutation status. We found that GANT61, a small molecule that can inhibit GLI1, was highly cytotoxic for GLI1+ CLL cells relative to that of CLL cells without GLI1. Collectively, this study shows that a large proportion of patients have CLL cells with activated Hh signaling, which is associated with early disease progression and enhanced sensitivity to inhibition of GLI1.
•A large proportion of patients have CLL cells with activation of Hh signaling leading to expression of GLI1.•Activation of Hh signaling predicts for relatively short treatment-free survival and sensitivity to GLI1 inhibition.
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We have performed a genome-wide analysis of common genetic variation controlling differential expression of transcript isoforms in the CEU HapMap population using a comprehensive exon tiling ...microarray covering 17,897 genes. We detected 324 genes with significant associations between flanking SNPs and transcript levels. Of these, 39% reflected changes in whole gene expression and 55% reflected transcript isoform changes such as splicing variants (exon skipping, alternative splice site use, intron retention), differential 5′ UTR (initiation of transcription) use, and differential 3′ UTR (alternative polyadenylation) use. These results demonstrate that the regulatory effects of genetic variation in a normal human population are far more complex than previously observed. This extra layer of molecular diversity may account for natural phenotypic variation and disease susceptibility.
In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically ...diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.
The optimal timing between meal ingestion and simple physical activity for improving blood glucose control is unknown. This study compared the effects of physical activity on postprandial ...interstitial glucose responses when the activity was conducted either immediately before, immediately after, or 30 min after breakfast. Forty-eight adults were randomized to three separate physical activity interventions: standing still (for 30 min), walking (for 30 min), and bodyweight exercises (3 sets of 10 squats, 10 push-ups, 10 lunges, 10 sit-ups). In each intervention, 16 participants completed four trials (A to D) during which a 500 kcal mixed nutrient liquid breakfast meal was consumed. Interstitial glucose responses were recorded using continuous glucose monitoring for 2 h after the meal. The activity was completed either after the glucose monitoring period (trial A; control) or immediately before (trial B), immediately after (trial C), or 30 min after (trial D) the breakfast. Mean, coefficient of variance (CV), and area under the curve (AUC) for glucose were calculated and compared between the four trials. Walking and bodyweight exercises immediately after the meal improved mean, CV, and AUC glucose (
P
≤ 0.05 vs. control), while standing immediately after the meal only improved AUC glucose (
P
≤ 0.05 vs. control) and nearly improved mean glucose (
P
= 0.06). Mean, CV, and AUC glucose were not affected by standing, walking, or bodyweight exercise conducted immediately before, or 30 min after the meal (all
P
> 0.05 vs. control). Energy intake (diet records) and energy expenditure (Actigraph) were consistent throughout the studies and did not influence the findings. Low- to moderate-intensity activity should be implemented soon after eating to improve glucose control following breakfast. The type of activity appears less important than the timing. These findings will help optimize exercise-meal timing in general health guidelines.
ClinicalTrials.gov Identifier: NCT03730727
Obstructive upper airway pathologies are a great clinical challenge for the airway surgeon. Protection against acute obstruction is critical, but avoidance of unnecessary tracheostomy must also be ...considered. Decision-making regarding airway, although supported by some objective findings, is largely guided by subjective experience and training. This investigation aims to study the relationship between clinical respiratory distress and objective measures of airway resistance in laryngeal cancer as determined by computational fluid dynamic (CFD) and morphometric analysis.
Retrospective CT and clinical data were obtained for series of 20 cases, defined as newly diagnosed laryngeal cancer patients who required admission or urgent airway surgery, and 20 controls. Cases and controls were matched based on T-staging. Image segmentation and morphometric analysis were first performed. Computational models based on the lattice Boltzmann method were then created and used to quantify the continuous mass flow, rigid wall, and constant static pressure inlet boundary conditions.
The analysis demonstrated a significant relationship between airway resistance and acute obstruction (OR 1.018, 95% CI 1.001-1.045). Morphometric analysis similarly demonstrated a significant relationship when relating measurements based on the minimum cross-section, but not on length of stenosis. Morphometric measurements also showed significance in predicting CFD results, and their relationship demonstrated that airway pressures increase exponentially below 2.5 mm. Tumor subsite did not show a significant difference, although the glottic subgroup tended to have higher resistances.
Airway resistance analysis from CFD computation correlated with presence of acute distress requiring emergent management. Morphometric analysis showed a similar correlation, demonstrating a radiologic airway assessment technique on which future risk estimation could be performed.
4 (case-control study) Laryngoscope, 2023.
The interplay between genetic and epigenetic variation is only partially understood. One form of epigenetic variation is methylation at CpG sites, which can be measured as methylation quantitative ...trait loci (meQTL). Here we report that in a panel of lymphocytes from 1,748 individuals, methylation levels at 1,919 CpG sites are correlated with at least one distal (trans) single-nucleotide polymorphism (SNP) (P<3.2 × 10(-13); FDR<5%). These trans-meQTLs include 1,657 SNP-CpG pairs from different chromosomes and 262 pairs from the same chromosome that are >1 Mb apart. Over 90% of these pairs are replicated (FDR<5%) in at least one of two independent data sets. Genomic loci harbouring trans-meQTLs are significantly enriched (P<0.001) for long non-coding transcripts (2.2-fold), known epigenetic regulators (2.3-fold), piwi-interacting RNA clusters (3.6-fold) and curated transcription factors (4.1-fold), including zinc-finger proteins (8.75-fold). Long-range epigenetic networks uncovered by this approach may be relevant to normal and disease states.
Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining ...insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of
-strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (T
), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative T
shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR.
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