Showing papers by "Emmanuelle Masson published in 2020"

Role of the Common PRSS1-PRSS2 Haplotype in Alcoholic and Non-Alcoholic Chronic Pancreatitis: Meta- and Re-Analyses

Abstract: The association between a common PRSS1-PRSS2 haplotype and alcoholic chronic pancreatitis (ACP), which was revealed by the first genome-wide association study of chronic pancreatitis (CP), has been consistently replicated. However, the association with non-ACP (NACP) has been controversial. Herein, we sought to clarify this basic issue by means of an allele-based meta-analysis of currently available studies. We then used studies informative for genotype distribution to explore the biological mechanisms underlying the association data and to test for gene-environment interaction between the risk haplotype and alcohol consumption by means of a re-analysis. A literature search was conducted to identify eligible studies. A meta-analysis was performed using the Review Manager software. The association between the risk genotypes and NACP or ACP was tested for the best-fitting genetic model. Gene-environment interaction was estimated by both case-only and multinomial approaches. Five and eight studies were employed for the meta-analysis of ACP and NACP findings, respectively. The risk allele was significantly associated with both ACP (pooled odds ratio (OR) 1.67, 95% confidence interval (CI) 1.56-1.78; p < 0.00001) and NACP (pooled OR 1.28, 95% CI 1.17-1.40; p < 0.00001). Consistent with a dosage effect of the risk allele on PRSS1/PRSS2 mRNA expression in human pancreatic tissue, both ACP and NACP association data were best explained by an additive genetic model. Finally, the risk haplotype was found to interact synergistically with alcohol consumption.

The Experimentally Obtained Functional Impact Assessments of 5' Splice Site GT'GC Variants Differ Markedly from Those Predicted.

Abstract: Introduction: 5' splice site GT>GC or +2T>C variants have been frequently reported to cause human genetic disease and are routinely scored as pathogenic splicing mutations. However, we have recently demonstrated that such variants in human disease genes may not invariably be pathogenic. Moreover, we found that no splicing prediction tools appear to be capable of reliably distinguishing those +2T>C variants that generate wild-type transcripts from those that do not. Methodology: Herein, we evaluated the performance of a novel deep learning-based tool, SpliceAI, in the context of three datasets of +2T>C variants, all of which had been characterized functionally in terms of their impact on pre-mRNA splicing. The first two datasets refer to our recently described “in vivo” dataset of 45 known disease-causing +2T>C variants and the “in vitro” dataset of 103 +2T>C substitutions subjected to full-length gene splicing assay. The third dataset comprised 12 BRCA1 +2T>C variants that were recently analyzed by saturation genome editing. Results: Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of these variants (and smaller datasets of +2T>A and +2T>G variants) revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue was that the impact of those +2T>C (and +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to an almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Conclusion: Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

Most unambiguous loss-of-function CPA1 mutations are unlikely to predispose to chronic pancreatitis.

Abstract: We have read with interest the recent publication of Hegyi and Sahin-Toth1 reporting that chronic pancreatitis (CP)-predisposing CPA1 mutations function through the misfolding pathway rather than through loss of CPA1 protein/activity. Herein, we explore an additional insight gleaned from this study beyond those discussed in an accompanying Editorial.2 In the original study reporting the association of CPA1 variants with CP, all unambiguous loss-of-function (LoF) variants (eg, nonsense mutations) were lumped together with missense mutations that functionally impaired the CPA1 protein.3 However, unlike missense mutations, unambiguous LoF variants often result in transcripts that contain premature termination codons (PTC) and are thus prone to nonsense-mediated RNA decay (NMD). NMD detects and degrades PTC-containing transcripts, thereby preventing the accumulation of truncated proteins.4 5 This implies that most unambiguous LoF CPA1 variants would not be able to elicit endoplasmic reticulum (ER) stress and hence, in light of the Hegyi and Sahin-Toth study, will not predispose …

5' splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity.

Abstract: In the human genome, most 5' splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5' splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5' splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta-analysis of reported +2C>T "pathogenic" variants together with a functional analysis of engineered +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5' splice sites in mammalian genomes.

The reversion variant (p.Arg90Leu) at the evolutionarily adaptive p.Arg90 site in CELA3B predisposes to chronic pancreatitis

Abstract: A gain-of-function missense variant in the CELA3B gene, p.Arg90Cys (c.268C>T), has recently been reported to cause pancreatitis in an extended pedigree. Herein, we sequenced the CELA3B gene in 644 genetically unexplained French chronic pancreatitis (CP) patients (all unrelated) and 566 controls. No predicted loss-of-function variants were identified. None of the six low frequency or common missense variants detected showed significant association with CP. Nor did the aggregate rare/very rare missense variants (n=14) show any significant association with CP. However, p.Arg90Leu (c.269G>T), which was found in 4 patients but no controls and affects the same amino acid as p.Arg90Cys, serves to revert p.Arg90 to the human elastase ancestral allele. Since p.Arg90Leu has previously been shown to exert a similar functional effect to p.Arg90Cys, our findings not only confirm the involvement of CELA3B in the etiology of CP but also pinpoint a new evolutionarily adaptive site in the human genome.

p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis

Abstract: Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation. Summary statement p.E152K-STIM1 variant found in pancreatitis patients leads to intracellular changes in calcium homeostasis through SERCA interaction, enabling intracellular trypsin activation and pancreatic acinar cell death.

RNA secondary structure mediated by Alu insertion as a novel disease-causing mechanism

Abstract: We have recently reported a homozygous Alu insertion variant (termed Alu_Ins) within the 39-untranslated region (39-UTR) of the SPINK1 gene as the cause of a new pediatric disease entity. Although Alu-Ins has been shown, by means of a full-length gene expression assay (FLGEA), to result in the complete loss of SPINK1 mRNA expression, the precise underlying mechanism(s) has remained elusive. Herein, we filled this knowledge gap by adopting a hypothesis-driven approach. Employing RepeatMasker, we identified two Alu elements (termed Alu1 and Alu2) within the SPINK1 locus; both are located deep within intron 3 and, most importantly, reside in the opposite orientation to Alu-Ins. Using FLGEA, we provide convincing evidence that Alu-Ins disrupts splicing by forming RNA secondary structures with Alu1 in the pre-mRNA sequence. Our findings reveal a previously undescribed disease-causing mechanism, resulting from an Alu insertion variant, which has implications for Alu detection and interpretation in human disease genes.

Role of the common PRSS1-PRSS2 haplotype in alcoholic and non-alcoholic chronic pancreatitis: meta- and re-analyses

Abstract: The association between a common PRSS1-PRSS2 haplotype and alcoholic chronic pancreatitis (ACP), which was revealed by the first genome-wide association study of chronic pancreatitis (CP), has been consistently replicated. However, the association with non-ACP (NACP) has been controversial. Herein, we sought to clarify this basic issue by means of an allele-based meta-analysis of currently available studies. We then used studies informative for genotype distribution to explore the biological mechanisms underlying the association data and to test for gene-environment interaction between the risk haplotype and alcohol consumption by means of a re-analysis. A literature search was conducted to identify eligible studies. Meta-analysis was performed using the Review Manager software. The association between the risk genotypes and NACP or ACP was tested for the best-fitting genetic model. Gene-environment interaction was estimated by both case-only and multinomial approaches. Five and eight studies were employed for the meta-analysis of ACP and NACP findings, respectively. The risk allele was significantly associated with both ACP (pooled OR 1.67, 95% CI 1.56-1.78; P<0.00001) and NACP (pooled OR 1.28, 95% CI 1.17-1.40; P<0.00001). Consistent with a dosage effect of the risk allele on PRSS1/PRSS2 mRNA expression in human pancreatic tissue, both ACP and NACP association data were best explained by an additive genetic model. Finally, the risk haplotype was found to interact synergistically with ACP.