Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease.

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.

Combining full-length gene assay and SpliceAI to interpret the splicing impact of all possible SPINK1 coding variants.

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.

Identification of protease-sensitive but not misfolding PNLIP variants in familial and hereditary pancreatitis.

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.

SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing.

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/.

Expanding ACMG variant classification guidelines into a general framework.

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.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

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.

Prevalence of HFE-related haemochromatosis and secondary causes of hyperferritinaemia and their association with iron overload in 1059 French patients treated by venesection.

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.

Splicing Outcomes of 5' Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays.

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.

Insights into the Role of the Discontinuous TM7 Helix of Human Ferroportin through the Prism of the Asp325 Residue.

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.

Missense RHD single nucleotide variants induce weakened D antigen expression by altering splicing and/or protein expression.

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.

Splicing analysis of SLC40A1 missense variations and contribution to hemochromatosis type 4 phenotypes.

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.

A novel hypomorphic splice variant in EIF2B5 gene is associated with mild ovarioleukodystrophy.

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.

Biallelic variants in MAATS1 encoding CFAP91, a calmodulin-associated and spoke-associated complex protein, cause severe astheno-teratozoospermia and male infertility.

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.

Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants.

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.

Blood transcriptomic biomarker as a surrogate of ischemic brain gene expression.

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.

First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts.

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.

Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype.

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.