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10-period Al 0.57 Ga 0.43 N/Al 0.38 Ga 0.62 N multi-quantum wells (MQWs) were grown on a relaxed Al 0.58 Ga 0.42 N buffer on AlN templates on sapphire. The threading dislocations and V-pits were characterized and their origin is discussed. The influence of V-pits on the structural quality of the MQWs and on optical emission at 280 nm was analyzed. It was observed that near-surface V-pits were always associated with grain boundaries consisting of edge threading dislocations originating from the AlN/Al 2 O 3 interface. Although the high density of V-pits disrupted MQWs growth, it did not affect the internal quantum efficiency which was measured to be ~1% at room temperature even when V-pit density was increased from 7×10 7 cm −2 to 2×10 9 cm −2 . The results help to understand the origin, propagation and influences of the typical defects in AlGaN MQWs grown on AlN/Al 2 O 3 templates which may lead to further improvement of the performance of DUV devices. [ABSTRACT FROM AUTHOR]

We demonstrate a method to reduce the mode volume of optical micro/nanocavities by positioning an opaque microtip in close proximity of the structures. This concept is used to blueshift the resonance of an active photonic crystal nanocavity by up to 16 nm. This tuning range is shown to be about 10 times larger than the redshift achieved with a bare dielectric microtip of the same size and shape. By imagining materials or multilayered devices with the ability to become transparent and opaque under external control, the blue and redshifts of the resonance would become possible with a single perturbing device. [ABSTRACT FROM AUTHOR]

Heterozygous mutations in SLC40A1, encoding a multi-pass membrane protein of the major facilitator superfamily known as ferroportin 1 (FPN1), are responsible for two distinct hereditary iron-overload diseases: ferroportin disease, which is associated with reduced FPN1 activity (i.e., decrease in cellular iron export), and SLC40A1-related hemochromatosis, which is associated with abnormally high FPN1 activity (i.e., resistance to hepcidin). Here, we report three SLC40A1 missense variants with opposite functional consequences. In cultured cells, the p.Arg40Gln and p.Ser47Phe substitutions partially reduced the ability of FPN1 to export iron and also partially reduced its sensitivity to hepcidin. The p.Ala350Val substitution had more profound effects, resulting in low FPN1 iron egress and weak FPN1/hepcidin interaction. Structural analyses helped to differentiate the first two substitutions, which are predicted to cause local instabilities, and the third, which is thought to prevent critical rigid-body movements that are essential to the iron transport cycle. The phenotypic traits observed in a total of 12 affected individuals are highly suggestive of ferroportin disease. Our findings dismantle the classical dualism of FPN1 loss versus gain of function, highlight some specific and unexpected functions of FPN1 transmembrane helices in the molecular mechanism of iron export and its regulation by hepcidin, and extend the spectrum of rare genetic variants that may cause ferroportin disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on protein stability and its conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in membrane bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366, and Arg371) that are located at the membrane-cytosol interface and consistently interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward-facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid polar heads. This results in a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt-bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis., Results: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity., Conclusions: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing., (© 2024. The Author(s).)

Mutations in the PNLIP gene have recently been implicated in chronic pancreatitis. Several PNLIP missense variants have been reported to cause protein misfolding and endoplasmic reticulum stress although genetic evidence supporting their association with chronic pancreatitis is currently lacking. Protease-sensitive PNLIP missense variants have also been associated with early-onset chronic pancreatitis although the underlying pathological mechanism remains enigmatic. Herein, we provide new evidence to support the association of protease-sensitive PNLIP variants (but not misfolding PNLIP variants) with pancreatitis. Specifically, we identified protease-sensitive PNLIP variants in 5 of 373 probands (1.3%) with a positive family history of pancreatitis. The protease-sensitive variants, p.F300L and p.I265R, were found to segregate with the disease in three families, including one exhibiting a classical autosomal dominant inheritance pattern. Consistent with previous findings, protease-sensitive variant-positive patients were often characterized by early-onset disease and invariably experienced recurrent acute pancreatitis, although none has so far developed chronic pancreatitis., (Copyright © 2023 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Background: Prevalence, risk factors and medical management of persistent pain symptoms after critical care illness have not been thoroughly investigated., Methods: We performed a prospective multicentric study in patients with an intensive care unit (ICU) length of stay ≥ 48 h. The primary outcome was the prevalence of significant persistent pain, defined as a numeric rating scale (NRS) ≥ 3, 3 months after admission. Secondary outcomes were the prevalence of symptoms compatible with neuropathic pain (ID-pain score > 3) and the risk factors of persistent pain., Results: Eight hundred fourteen patients were included over a 10-month period in 26 centers. Patients had a mean age of 57 (± 17) years with a SAPS 2 score of 32 (± 16) (mean ± SD). The median ICU length of stay was 6 [4-12] days (median [interquartile]). At 3 months, the median intensity of pain symptoms was 2 [1-5] in the entire population, and 388 (47.7%) patients had significant pain. In this group, 34 (8.7%) patients had symptoms compatible with neuropathic pain. Female (Odds Ratio 1.5 95% CI [1.1-2.1]), prior use of anti-depressive agents (OR 2.2 95% CI [1.3-4]), prone positioning (OR 3 95% CI [1.4-6.4]) and the presence of pain symptoms on ICU discharge (NRS ≥ 3) (OR 2.4 95% CI [1.7-3.4]) were risk factors of persistent pain. Compared with sepsis, patients admitted for trauma (non neuro) (OR 3.5 95% CI [2.1-6]) were particularly at risk of persistent pain. Only 35 (11.3%) patients had specialist pain management by 3 months., Conclusions: Persistent pain symptoms were frequent in critical illness survivors and specialized management remained infrequent. Innovative approaches must be developed in the ICU to minimize the consequences of pain., Trial Registration: NCT04817696. Registered March 26, 2021., (© 2023. The Author(s).)

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome., (© 2022 SSIEM.)

Background: The American College of Medical Genetics and Genomics (ACMG)-recommended five variant classification categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) have been widely used in medical genetics. However, these guidelines are fundamentally constrained in practice owing to their focus upon Mendelian disease genes and their dichotomous classification of variants as being either causal or not. Herein, we attempt to expand the ACMG guidelines into a general variant classification framework that takes into account not only the continuum of clinical phenotypes, but also the continuum of the variants' genetic effects, and the different pathological roles of the implicated genes., Main Body: As a disease model, we employed chronic pancreatitis (CP), which manifests clinically as a spectrum from monogenic to multifactorial. Bearing in mind that any general conceptual proposal should be based upon sound data, we focused our analysis on the four most extensively studied CP genes, PRSS1, CFTR, SPINK1 and CTRC. Based upon several cross-gene and cross-variant comparisons, we first assigned the different genes to two distinct categories in terms of disease causation: CP-causing (PRSS1 and SPINK1) and CP-predisposing (CFTR and CTRC). We then employed two new classificatory categories, "predisposing" and "likely predisposing", to replace ACMG's "pathogenic" and "likely pathogenic" categories in the context of CP-predisposing genes, thereby classifying all pathologically relevant variants in these genes as "predisposing". In the case of CP-causing genes, the two new classificatory categories served to extend the five ACMG categories whilst two thresholds (allele frequency and functional) were introduced to discriminate "pathogenic" from "predisposing" variants., Conclusion: Employing CP as a disease model, we expand ACMG guidelines into a five-category classification system (predisposing, likely predisposing, uncertain significance, likely benign, and benign) and a seven-category classification system (pathogenic, likely pathogenic, predisposing, likely predisposing, uncertain significance, likely benign, and benign) in the context of disease-predisposing and disease-causing genes, respectively. Taken together, the two systems constitute a general variant classification framework that, in principle, should span the entire spectrum of variants in any disease-related gene. The maximal compliance of our five-category and seven-category classification systems with the ACMG guidelines ought to facilitate their practical application., (© 2022. The Author(s).)

Background: Venesection is the key therapy in haemochromatosis, but it remains controversial in hyperferritinaemia with moderate iron accumulation. There is substantial evidence that the results of HFE genotyping are routinely misinterpreted, while elevated serum ferritin has become more frequent in recent years in white adult populations following the increase of obesity and metabolic traits., Aims: To examine the reasons for prescribing venesection in 1,059 French patients during the period 2012-2015, determine the true prevalence of HFE-related haemochromatosis, and compare iron overload profiles between haemochromatosis and non-haemochromatosis patients., Results: Only 258 of the 488 patients referred for haemochromatosis had the p.[Cys282Tyr];[Cys282Tyr] disease causative genotype (adjusted prevalence: 24.4%). Of the 801 remaining patients, 112 (14.0%) had the debated p.[Cys282Tyr];[His63Asp] compound heterozygote genotype, 643 (80.3%) had central obesity, 475 (59.3%) had metabolic syndrome (MetS) and 93 (11.6%) were heavy drinkers. The non-haemochromatosis patients started therapeutic venesection 9 years later than haemochromatosis patients (P < 0.001). Despite similar serum ferritin values, they had lower transferrin saturation (41.1% vs 74.3%; P < 0.001), lower amounts of iron removed by venesection (1.7 vs 3.2 g; P < 0.001) and lower hepatic iron concentrations (107 vs 237 µmol/g; P < 0.001)., Conclusions: Haemochromatosis is over-diagnosed and is no longer the main reason for therapeutic venesection in France. Obesity and other metabolic abnormalities are frequently associated with mild elevation of serum ferritin, the MetS is confirmed in ~50% of treated patients. There is a minimal relationship between serum ferritin and iron overload in non-p.Cys282Tyr homozygotes. Our observations raise questions about venesection indications in non-haemochromatosis patients., (© 2022 John Wiley & Sons Ltd.)

Combining data derived from a meta-analysis of human disease-associated 5' splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15-18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Wu, Zou, Masson, Fichou, Le Gac, Cooper, Férec, Liao and Chen.)

Background: Although D variant phenotype is known to be due to genetic defects, including rare missense single nucleotide variants (SNVs), within the RHD gene, few studies have addressed the molecular and cellular mechanisms driving this altered expression. We and others showed previously that splicing is commonly disrupted by SNVs in constitutive splice sites and their vicinity. We thus sought to investigate whether rare missense SNVs located in "deep" exonic regions could also impair this mechanism., Study Design and Methods: Forty-six missense SNVs reported within exons 6 and 7 were first selected from the Human RhesusBase. Their respective effect on splicing was assessed by using an in vitro assay. An RhD-negative cell model was further generated by using the CRISPR-Cas9 approach. RhD-mutated proteins were overexpressed in the newly created model, and cell membrane expression of the D antigen was measured by flow cytometry., Results: Minigene splicing assay showed that 14 of 46 (30.4%) missense SNVs alter splicing. Very interestingly, further investigation of two missense SNVs, which both affect codon 338 and confer a weak D phenotype, showed various mechanisms: c.1012C>G (p.Leu338Val) disrupts splicing only, while c.1013T>C (p.Leu338Pro) alters only the protein structure, in agreement with in silico prediction tools and 3D protein structure visualization., Conclusion: Our functional data set suggests that missense SNVs damage quantitatively D antigen expression by, at least, two different mechanisms (splicing alteration and protein destabilization) that may act independently. These data thereby contribute to extend the current knowledge of the molecular mechanisms governing weakened D expression., (© 2021 AABB.)

The negatively charged Asp325 residue has proved to be essential for iron export by human (HsFPN1) and primate Philippine tarsier (TsFpn) ferroportin, but its exact role during the iron transport cycle is still to be elucidated. It has been posited as being functionally equivalent to the metal ion-coordinating residue His261 in the C-lobe of the bacterial homolog BbFpn, but the two residues arise in different sequence motifs of the discontinuous TM7 transmembrane helix. Furthermore, BbFpn is not subject to extracellular regulation, contrary to its mammalian orthologues which are downregulated by hepcidin. To get further insight into the molecular mechanisms related to iron export in mammals in which Asp325 is involved, we investigated the behavior of the Asp325Ala, Asp325His, and Asp325Asn mutants in transiently transfected HEK293T cells, and performed a comparative structural analysis. Our biochemical studies clearly distinguished between the Asp325Ala and Asp325His mutants, which result in a dramatic decrease in plasma membrane expression of FPN1, and the Asp325Asn mutant, which alters iron egress without affecting protein localization. Analysis of the 3D structures of HsFPN1 and TsFpn in the outward-facing (OF) state indicated that Asp325 does not interact directly with metal ions but is involved in the modulation of Cys326 metal-binding capacity. Moreover, models of the architecture of mammalian proteins in the inward-facing (IF) state suggested that Asp325 may form an inter-lobe salt-bridge with Arg40 (TM1) when not interacting with Cys326. These findings allow to suggest that Asp325 may be important for fine-tuning iron recognition in the C-lobe, as well as for local structural changes during the IF-to-OF transition at the extracellular gate level. Inability to form a salt-bridge between TM1 and TM7b during iron translocation could lead to protein instability, as shown by the Asp325Ala and Asp325His mutants.

Hemochromatosis type 4, or ferroportin disease, is considered as the second leading cause of primary iron overload after HFE-related hemochromatosis. The disease, which is predominantly associated with missense variations in the SLC40A1 gene, is characterized by wide clinical heterogeneity. We tested the possibility that some of the reported missense mutations, despite their positions within exons, cause splicing defects. Fifty-eight genetic variants were selected from the literature based on two criteria: a precise description of the nucleotide change and individual evidence of iron overload. The selected variants were investigated by different in silico prediction tools and prioritized for midigene splicing assays. Of the 15 variations tested in vitro, only two were associated with splicing changes. We confirm that the c.1402G>A transition (p.Gly468Ser) disrupts the exon 7 donor site, leading to the use of an exonic cryptic splicing site and the generation of a truncated reading frame. We observed, for the first time, that the p.Gly468Ser substitution has no effect on the ferroportin iron export function. We demonstrate alternative splicing of exon 5 in different cell lines and show that the c.430A>G (p.Asn144Asp) variant promotes exon 5 inclusion. This could be part of a gain-of-function mechanism. We conclude that splicing mutations rarely contribute to hemochromatosis type 4 phenotypes. An in-depth investigation of exon 5 auxiliary splicing sequences may help to elucidate the mechanism by which splicing regulatory proteins regulate the production of the full length SLC40A1 transcript and to clarify its physiological importance., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Multiple morphological abnormalities of the flagella (MMAF) consistently lead to male infertility due to a reduced or absent sperm motility defined as asthenozoospermia. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analysed remain unresolved, suggesting that many yet uncharacterised gene defects account for this phenotype METHODS: Exome sequencing was performed on 167 infertile men with an MMAF phenotype. Immunostaining and transmission electron microscopy (TEM) in sperm cells from affected individuals were performed to characterise the ultrastructural sperm defects. Gene inactivation using RNA interference (RNAi) was subsequently performed in Trypanosoma ., Results: We identified six unrelated affected patients carrying a homozygous deleterious variants in MAATS1, a gene encoding CFAP91, a calmodulin-associated and spoke-associated complex (CSC) protein. TEM and immunostaining experiments in sperm cells showed severe central pair complex (CPC) and radial spokes defects. Moreover, we confirmed that the WDR66 protein is a physical and functional partner of CFAP91 into the CSC. Study of Trypanosoma MAATS1's orthologue (TbCFAP91) highlighted high sequence and structural analogies with the human protein and confirmed the axonemal localisation of the protein. Knockdown of TbCFAP91 using RNAi impaired flagellar movement led to CPC defects in Trypanosoma as observed in humans., Conclusions: We showed that CFAP91 is essential for normal sperm flagellum structure and function in human and Trypanosoma and that biallelic variants in this gene lead to severe flagellum malformations resulting in astheno-teratozoospermia and primary male infertility., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To identify the genetic cause in an adult ovarioleukodystrophy patient resistant to diagnosis., Methods: We applied whole-exome sequencing (WES) to a vanishing white matter disease patient associated with premature ovarian failure at 26 years of age. We functionally tested an intronic variant by RT-PCR on patient's peripheral blood mononuclear cells (PBMC) and by minigene splicing assay., Results: WES analysis identified two novel variants in the EIF2B5 gene: c.725A > G (p.Tyr242Cys) and an intronic noncanonical mutation (c.1156 + 13G>A). This intronic mutation resulted into generation of various isoforms both in patient's PBMC and in the minigene splicing assay, showing that ~20% residual wild-type isoform is still expressed by the intronic-mutated allele alone, concordant with an hypomorphic effect of this variant., Conclusion: We report two novel variants in EIF2B5, one of them a noncanonical intronic splice variant, located at a +13 intronic position. This position is mutated only in 0.05% of ClinVar intronic mutations described so far. Furthermore, we illustrate how minigene splicing assay may be advantageous when validating splice-altering variants, in this case highlighting the coexistence of wild-type and mutated forms, probably explaining this patient's milder, late-onset phenotype., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Introduction: Subarachnoid hemorrhage (SAH) from intracranial aneurysm rupture is an unpredictable event responsible for significant morbidity and mortality. Despite inconsistencies, some studies suggest a potential role of climate conditions in SAH onset. The purpose of this study was to determine the impact of climatic and lunar factors on onset of SAH in an oceanic climate such as that of Brittany, France., Methods: All adults with SAH admitted to the neurosurgery department and intensive care unit of the University Hospital of Rennes (France) between January 1st, 2011 and December 31st, 2012 were included. Meteorological variables, their variations, lunar phases and tidal coefficients were compared between days with and without SAH., Results: We retrospectively included 295 patients with SAH. Mean minimum temperature was significantly lower during days with SAH (7.7±4.7°C versus 8.3±4.6°C; P=0.039); temperature variation between 2 successive days was significantly greater for days with SAH (8.6±4.1°C versus 7.9±3.8°C; P<0.01). Multivariate analysis showed that a 2-day temperature drop greater than or equal to 8°C was associated with 35% increased risk of SAH (odds ratio 1.35 [1.03-1.77]). There were no significant effects of other meteorological variables, lunar phase or tidal coefficient on SAH occurrence., Conclusion: Low temperature and sudden temperature drop were associated with increased occurrence of SAH in Brittany, France., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss., Results: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%., Conclusions: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

It has long been known that canonical 5' splice site (5'SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5'SSs capable of generating wild-type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta-analysis of 45 human disease-causing 5'SS GT>GC variants and a cell culture-based full-length gene splicing assay of 103 5'SS GT>GC substitutions, we estimate that ~15-18% of canonical GT 5'SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5'SSs in which substitution of GT by GC-generated normal transcripts exhibit stronger complementarity to the 5' end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5'SS GT>GC variants that generated wild-type transcripts from those that did not. Our findings imply that 5'SS GT>GC variants in human disease genes may not invariably be pathogenic., (© 2019 Wiley Periodicals, Inc.)

Objectives: Blood biomarkers for cerebral tissue ischemia are lacking. The goal was to identify a blood transcriptomic signature jointly identified in the ischemic brain., Methods: A nonhuman primate model with middle cerebral artery (MCA) territory infarction was used to study gene expression by microarray during acute ischemic cerebral stroke in the brain and the blood. Brain samples were collected in the infarcted and contralateral non-infarcted cortex as well as blood samples before and after occlusion. Gene expression was compared between the two brain locations to find differentially expressed genes. The expressions of these genes were then compared in the blood pre- and post-occlusion., Results: Hierarchical clustering of brain expression data revealed strong independent clustering of ischemic and nonischemic brain samples. The top five enriched, up-regulated gene sets in the brain were TNF α signaling, apoptosis, P53 pathway, hypoxia, and UV response up. A comparison of differentially expressed genes in the brain and blood revealed a significant overlap of gene expression patterns. Stringent analysis of blood expression data from pre- and post-occlusion samples in each monkey identified nine genes highly differentially expressed in both the brain and the blood. Many of these up-regulated genes belong to pathways involved in cell death and DNA damage repair., Interpretation: Common gene expression profile can be identified in the brain and blood and clearly differentiates ischemic from nonischemic conditions. Therefore, specific blood transcriptomic signature may represent a surrogate for brain ischemic gene expression., (© 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.)

Background: We previously showed that several variations in the RHD gene, including synonymous changes, can be classified as splice site variants and may play a direct role in D variant phenotype expression. We sought to extend our study to additional candidates, notably in the first and last exons of the gene, by engineering a novel universal splice reporting vector, i.e., minigene., Study Design and Methods: Our previous plasmid construct was modified to allow subcloning of any exon(s) of interest for assessing effect of variations on splicing. Seventeen novel and/or uncharacterized variations of the RHD gene were selected for the study and tested in our novel model., Results: We engineered and validated a novel universal minigene for assessing virtually any variations of interest for splicing defect. Of the 17 variants tested in the novel model, 11 were shown to alter splicing either totally or partially, including the silent c.1065C>T variation, which induces major skipping of exon 7, and may therefore be responsible for reducing D antigen expression. We also showed that while all three missense variations c.1154G>C, c.1154G>T, and c.1154G>A in exon 9 are splice site variants, splicing is differentially altered and D-negative phenotype observed in the presence of the latter substitution is likely due to a defect in RhD protein folding., Conclusion: Overall, we hypothesize that splicing alteration is likely to be a common mechanism of D phenotype variation that has been underestimated so far. Further large-scale studies are necessary to demonstrate this statement definitely., (© 2019 AABB.)

Hemochromatosis type 4 is one of the most common causes of primary iron overload, after HFE -related hemochromatosis. It is an autosomal dominant disorder, primarily due to missense mutations in SLC40A1 This gene encodes ferroportin 1 (FPN1), which is the sole iron export protein reported in mammals. Not all heterozygous missense mutations in SLC40A1 are disease-causing. Due to phenocopies and an increased demand for genetic testing, rare SLC40A1 variations are fortuitously observed in patients with a secondary cause of hyperferritinemia. Structure/function analysis is the most effective way of establishing causality when clinical and segregation data are lacking. It can also provide important insights into the mechanism of iron egress and FPN1 regulation by hepcidin. The present study aimed to determine the pathogenicity of the previously reported p.Arg178Gln variant. We present the biological, clinical, histological and radiological findings of 22 patients from six independent families of French, Belgian or Iraqi decent. Despite phenotypic variability, all patients with p.Arg178Gln had elevated serum ferritin concentrations and normal to low transferrin saturation levels. In vitro experiments demonstrated that the p.Arg178Gln mutant reduces the ability of FPN1 to export iron without causing protein mislocalization. Based on a comparative model of the 3D structure of human FPN1 in an outward facing conformation, we argue that p.Arg178 is part of an interaction network modulating the conformational changes required for iron transport. We conclude that p.Arg178Gln represents a new category of loss-of-function mutations and that the study of "gating residues" is necessary in order to fully understand the action mechanism of FPN1., (Copyright© 2018 Ferrata Storti Foundation.)

Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5' and 3' consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

Background: HFE hemochromatosis is an inborn error of iron metabolism linked to a defect in the regulation of hepcidin synthesis. This autosomal recessive disease typically manifests later in women than men. Although it is commonly stated that pregnancy is, with menses, one of the factors that offsets iron accumulation in women, no epidemiological study has yet supported this hypothesis. The aim of our study was to evaluate the influence of pregnancy on expression of the predominant HFE p.[Cys282Tyr];[Cys282Tyr] genotype., Methods: One hundred and forty p.Cys282Tyr homozygous women enrolled in a phlebotomy program between 2004 and 2011 at a blood centre in western Brittany (France) were included in the study. After checking whether the disease expression was delayed in women than in men in our study, the association between pregnancy and iron overload was assessed using multivariable regression analysis., Results: Our study confirms that women with HFE hemochromatosis were diagnosed later than men cared for during the same period (52.6 vs. 47.4 y., P < 0.001). Compared to no pregnancy, having at least one pregnancy was not associated with lower iron markers. In contrast, the amount of iron removed by phlebotomies appeared significantly higher in women who had at least one pregnancy (e β  = 1.50, P = 0.047). This relationship disappeared after adjustment for confounding factors (e β  = 1.35, P = 0.088)., Conclusions: Our study shows that pregnancy status has no impact on iron markers level, and is not in favour of pregnancy being a protective factor in progressive iron accumulation. Our results are consistent with recent experimental data suggesting that the difference in disease expression observed between men and women may be explained by other factors such as hormones.

Background: Hepatocellular carcinoma (HCC) is an aggressive cancer with limited therapeutic options. Retrospective studies have shown that the administration of local anesthetics (LAs) during cancer surgery could reduce cancer recurrence. Besides, experimental studies reported that LAs could inhibit the growth of cancer cells. Thus, the purpose of this study was to investigate the effects of LAs on human HCC cells., Methods: The effects of 2 LAs (lidocaine and ropivacaine) (10 to 10 M) were studied after an incubation of 48 hours on 2 HCC cell lines, namely HuH7 and HepaRG. Cell viability, cell cycle analysis, and apoptosis and senescence tests were performed together with unsupervised genome-wide expression profiling and quantitative real-time polymerase chain reaction for relevant genes., Results: We showed that LAs decreased viability and proliferation of HuH7 cells (from 92% [P < .001] at 5 × 10 M to 40% [P = .02] at 10 M with ropivacaine and from 87% [P < .001] to 37% [P = .02] with lidocaine) and HepaRG progenitor cells (from 58% at 5 × 10 M [P < .001] to 29% at 10 M [P = .04] with lidocaine and 59% [P < .001] with ropivacaine 5 × 10 M) in concentration-dependent manner. LAs have no effect on well-differentiated HepaRG. Ropivacaine decreased the mRNA level of key cell cycle regulators, namely cyclin A2, cyclin B1, cyclin B2, and cyclin-dependent kinase 1, and the expression of the nuclear marker of cell proliferation MKI67. Lidocaine had no specific effect on cell cycle but increased by 10× the mRNA level of adenomatous polyposis coli (P < .01), which acts as an antagonist of the Wnt/β-catenin pathway. Both LAs increased apoptosis in Huh7 and HepaRG progenitor cells (P < .01)., Conclusions: The data demonstrate that LAs induced profound modifications in gene expression profiles of tumor cells, including modulations in the expression of cell cycle-related genes that result in a cytostatic effect and induction of apoptosis.