Molecular Crowding: The History and Development of a Scientific Paradigm.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

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.

First investigation of RH gene polymorphism in patients with sickle cell disease and associated blood donors in Cameroon, Central Africa.

BACKGROUND: Although genetic polymorphism of the RH blood group system is well known in sub-Saharan Africa, national/regional specificities still remain to be described precisely. For the first time in Cameroon, Central Africa, and in order to better characterize the molecular basis driving RH phenotype variability, as well as to identify the main antigens that may be potentially responsible for alloimmunization, we sought 1) to study the RH genes in a cohort of 109 patients with sickle cell disease; 2) to study the same genes in the corresponding donors whose red blood cells (RBCs) were transfused to the patients (108 donors in 98 patients); 3) to predict RH phenotype on the basis of the molecular data and compare the results with serologic testing; and 4) to identify retrospectively patients at risk for alloimmunization. MATERIALS AND METHODS: In order to generate an exhaustive dataset, the RH genes of all patient and donor samples were systematically investigated 1) by quantitative multiplex PCR of short fluorescent fragments (QMPSF) for characterization of RHD gene zygosity and potential structural variants (SVs), and 2) by Sanger sequencing for identification of single nucleotide variants (SNVs). Subsequent to molecular analysis, the genotypes and RH phenotype were deduced and predicted, respectively, from reference databases. RESULTS: In a total of 217 Cameroonian individuals, as many as 24 and up to 22 variant alleles were identified in the RHD and RHCE genes, respectively, in addition to the reference alleles. Interestingly, 65 patients with SCD (66.3%) were assumed to be exposed to one or more undesirable RH antigen(s) with varying degrees of clinical relevance. DISCUSSION: Beyond the comprehensive report of the nature and distribution of RH variant alleles in a subset of Cameroonian patients treated by transfusion therapy, this work highlights the need for an extensive review of current practice, including routine serologic typing procedures, preferably in the near future.

Mutations in Tau Protein Promote Aggregation by Favoring Extended Conformations.

Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Some tauopathies can be inherited due to mutations in the gene encoding tau, which might favor the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the disease-associated single-point mutations promote amyloid formation. We combined biochemical and biophysical characterization, notably, small-angle X-ray scattering (SAXS), to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions, and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radii of gyration. We show that mutations disfavor intramolecular protein interactions, which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate the aggregation propensity of IDPs.

Chemical Imaging of RNA-Tau Amyloid Fibrils at the Nanoscale Using Tip-Enhanced Raman Spectroscopy.

In the presence of cofactors, tau protein can form amyloid deposits in the brain which are implicated in many neurodegenerative disorders. Heparin, lipids, and RNA are used to recreate tau aggregates in vitro from recombinant protein. However, the mechanism of interaction of these cofactors and the interactions between cofactors and tau are poorly understood. Herein, we use tip-enhanced Raman spectroscopy (TERS) to visualize the spatial distribution of adenine, protein secondary structure, and amino acids (arginine, lysine and histidine) in single polyadenosine (polyA)-induced tau fibrils with nanoscale spatial resolution (<10-20 nm). Based on reference unenhanced and surface-enhanced Raman spectra, we show that the polyA anionic cofactor is incorporated in the fibril structure and seems to be superficial to the ß-sheet core, but nonetheless enveloped within the random-coiled fuzzy coat. TERS images also prove the colocalization of positively charged arginine, lysine, and histidine amino acids and negatively charged polyA, which constitutes an important step forward to better comprehend the action of RNA cofactors in the mechanism of formation of toxic tau fibrils. TERS appears as a powerful technique for the identification of cofactors in individual tau fibrils and their mode of interaction.

Anti-D alloimmunization by Asia type DEL red blood cell units in a D-negative Thai patient.

BACKGROUND: DEL phenotype is a rare Rh variant that cannot be detected by routine serological typing, and DEL individuals are thus typed D-negative (D-). Anti-D alloimmunization has been reported in "true" D- patients receiving DEL red blood cells (RBCs). CASE PRESENTATION: A 17-year-old, D- Thai male patient suffering from immunodeficiency syndrome with negative antibody screening received RBC units from 17 serological D- donors over a period of seven months due to acute respiratory failure with anemia. Before the 12th transfusion, anti-D production was detected. He was later transfused with RBCs from six other apparent D- donors. In order to elucidate anti-D production, all 17 blood donors were investigated by replicative serological testing and molecular analysis to identify potential RHD gene variants. All donors were confirmed D- by routine method, but as many as 12/17 were positive by adsorption-elution testing. Molecular analysis showed that five donors, including four whose blood was transfused before anti-D production occurred, carry the Asia type DEL allele, and are thus predicted to express a DEL phenotype. These data clearly suggest that 1/ the alloimmunized D- patient was exposed to D antigen, 2/ our adsorption-elution test is currently defective to identify DEL RBCs, and 3/ molecular analysis is highly valuable for Asia type DEL allele screening. CONCLUSION: For the first time in Thailand, we report anti-D alloimmunization in a serological D- patient transfused by Asia type DEL RBC units. This work definitely supports the implementation of a dedicated policy for DEL blood management including molecular testing.

Patients with Asian-type DEL can safely be transfused with RhD-positive blood.

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

Mapping Phase Diagram of Tau-RNA LLPS Under Live Cell Coculturing Conditions.

Protein liquid-liquid phase separation (LLPS) has been associated with biological functions and pathological aggregation. Mapping the phase separation conditions is the first step to identify and quantify the driving forces of LLPS. Here, we describe the protocols to draw the phase diagram of tau-RNA LLPS and use the mapped diagram to guide experimental conditions for LLPS-cell coculturing, electron resonance spectroscopy in particular double electron-electron resonance spectroscopy, crosslinking immunoprecipitation, and isothermal titration calorimetry.

Molecular and serological analysis of the D variant in the Chinese population and identification of seven novel RHD alleles.

BACKGROUND: The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary. STUDY DESIGN AND METHODS: Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three-step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon-intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti-D investigation was performed. RESULTS: DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty-two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti-D was detected in two DVI type 3 pregnant women. DISCUSSION: The three-step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.

Serologically D-negative blood donors in Thailand: molecular variants and diagnostic strategy.

BACKGROUND: Discriminating individuals with "Asian type DEL" from those who are "true D-negative" (D-) among serologically D- donors/patients in Asia would be very valuable, as clinical outcomes are different in these groups. Here we investigated the molecular basis of D-negativity in Thai blood donors, designing a specific strategy for this purpose. MATERIALS AND METHODS: After routine testing, a total of 1,270 serologically D- blood donors originating from Central, Northeastern and South Thailand underwent analysis of the RHD gene by (i) quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF); (ii) direct sequencing of exon 9 to identify the c.1227G>A variant defining the Asian type DEL allele; and (iii) direct sequencing of the other exons. RESULTS: The most common observation was whole deletion of the gene (i.e. RHD*01N.01; allele frequency: 86.81%), followed by the Asian type DEL allele (RHD*01EL.01; 7.60%) and a D-negative hybrid allele (RHD*01N.03; 3.46%). Four novel alleles, including one with a 13.1 kilobase-deletion, were identified and characterized. All but one RHD*01EL.01 allele carriers (183/184) were C-positive (C+), suggesting that this latter subset may be screened specifically when investigating the c.1227G>A variant, which can be identified with 100% accuracy by a specific Tm-shift genotyping assay. DISCUSSION: On the basis of our extensive molecular findings, we have designed a dedicated diagnostic strategy based on Rh C antigen typing followed by a genotyping test. Implementation of this method in all or selected groups of serologically D- donors/patients will contribute to improve the management of transfusion and pregnancy in Thailand.

From the investigation of RHD-CE hybrid genes to the recognition of RHCE variants and RHD zygosity. Expanding the analysis by QMPSF in Brazilian donors and in patients with sickle cell disease.

BACKGROUND: Hybrid genes are responsible for the formation of Rh variants and are common in patients with sickle cell disease (SCD). However, it is not usually possible to detect them by conventional molecular protocols. In the present study, hybrid genes were investigated using the Quantitative Multiplex Polymerase chain reaction of Short Fluorescent Fragments (QMPSF), a molecular protocol that quantifies the copy number of RHD and RHCE exons. In addition, we explored additional relevant information obtained with QMPSF, such as recognition of variant RHCE and RHD zygosity. MATERIALS AND METHODS: Three groups of subjects were selected for the study: patients with SCD, self-declared African descent donors (SDA), and D-negative donors. RHD and RHCE hybrids genes were investigated by the QMPSF method. Real-time multiplex polymerase chain reaction (PCR) assay was used to confirm the copy number of the RHD in two samples. Cloning was performed to investigate the allele. Relative RhD antigen density was investigated by flow cytometry, and RhCE phenotyping was performed with both tube and gel methods. RESULTS: In the 507 samples analysed, hybrid allele frequencies were found in 20.08% of patients with SCD, in 18.22% of individuals in the SDA group, and 3.67% of D-negative donors. The SCD and SDA groups had a higher frequency of hybrid alleles, most commonly involving exon 8, with which we found an association with c.733C>G, a common polymorphism observed in individuals of African descent. Of note, two patients with SCD were shown to carry three gene copies, as confirmed by quantitative PCR; no increase in D expression was observed in these patients. In addition, the QMPSF guided the investigation of 144 RHCE variants and RHD zygosity, and two novel alleles were identified. DISCUSSION: The QMPSF was shown to identify hybrid alleles involved in altered Rh phenotypes in Brazilian donors and patients with SCD. The association of the hybrid RHCE-D(8)-CE allele with c.733C>G suggests this hybrid allele may be used as a marker to detect the most frequent variants found in patients with SCD.

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.

Illuminating the Structural Basis of Tau Aggregation by Intramolecular Distance Tracking: A Perspective on Methods.

The aggregation of the tau protein is central to several neurodegenerative diseases, collectively known as tauopathies. High-resolution views of tau tangles accumulated under pathological conditions in post-mortem brains have been revealed recently by cryogenic electron microscopy. One of the striking discoveries was that fibril folds are unique to and homogeneous within one disease family, but typically different between different tauopathies. It is widely believed that seeded aggregation can achieve structural propagation of tau fibrils and generate pathological fibril structures. However, direct molecular level measurement of structural evolution during aggregation is missing. Here, we discuss our perspective on the biophysical approaches that can contribute to the ongoing debate regarding the prion-like propagation of tau and the role of cofactors. We discuss the unique potential of double electron-electron resonance (DEER)-based intramolecular distance measurement, sensitive to two to several nanometers distances. DEER can track the structural evolution of tau along the course of aggregation from the completely disordered state, to partially ordered and highly ordered fibril states, and has the potential to be a key tool to elucidate the disease-specific tau aggregation pathways.

Total Internal Reflection Tip-Enhanced Raman Spectroscopy of Tau Fibrils.

Total internal reflection tip-enhanced Raman spectroscopy (TIR-TERS) has recently emerged as a promising technique for noninvasive nanoscale chemical characterization of biomolecules. We demonstrate that the TERS enhancement achieved in this experimental configuration is nearly 30 times higher than that in linear polarization and 8 times higher than that in radial polarization using traditional bottom-illumination geometry. TIR-TERS is applied to the study of Tau amyloid fibrils formed with the human full-length Tau protein mixed with heparin. This technique reveals the possibility to perform TERS imaging with 1-4 nm axial and 5-10 nm lateral spatial optical resolution. In these Tau/heparin fibrils, spectral signatures assigned to aromatic amino acid residues (phenylalanine, histidine, and tyrosine) and nonaromatic ones (e.g., cysteine, lysine, arginine, asparagine, and glutamine) are distinctly observed. Amide I and amide III bands can also be detected. In a fibril portion, it is shown that antiparallel ß-sheets and fibril core ß-sheets are abundant and are often localized in amino acid-rich regions where parallel ß-sheets and random coils are present in lower proportions. This first TIR-TERS study on a nonresonant biological sample paves the way for future nanoscale chemical and structural characterization of biomolecules using this performant and original technique.

High-resolution Neutron Spectroscopy to Study Picosecond-nanosecond Dynamics of Proteins and Hydration Water.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state. Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer. The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into µm sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

The novel c.634+4A>G splicing variant in RHCE results in weak C and e antigen expression in a pregnant woman originated from Japan.

BACKGROUND: In the RH blood group genes, molecular variants that alter antigen expression with potential clinical relevance are frequently identified and reported in the literature. STUDY DESIGN AND METHODS: A pregnant woman in her first pregnancy, who originates from Japan, was typed by routine serological testing. The RHCE gene was investigated to identify single nucleotide variants (SNVs) and/or structural variants by a commercial platform, Sanger sequencing, and quantitative multiplex PCR of short fluorescent fragments. The haplotypes were determined by sequencing PCR fragments generated from genomic DNA and subcloned into a plasmid vector. Effect on splicing was predicted by bioinformatics tools, including SpliceAI and the splicing module of Alamut. In parallel, functional analysis was carried out by a minigene splicing assay. RESULTS: A patient with no transfusion history was typed RH:1,2w,3,4,5w. An unreported single variant was identified in RHCE intron 4 at the heterozygous state: c.634+4A>G. Minigene splicing assay showed that this SNV decreases significantly the relative abundance of the full-length transcript, in accordance with the predictions made by the Alamut tools, but not SpliceAI, suggesting expression of a normal RhCE protein. CONCLUSION: Overall, the novel RHCE*02(c.634+4A>G) allele alters quantitatively, but not qualitatively, the expression of C and e in the RH blood group system, indicating that the patient is not at risk for alloimmunization and may safely receive C+e+ red blood cell units. This report illustrates the relevance of functional assays for the interpretation of rare variants and, specifically, how it may help guide transfusion management in patients.

Molecular characterization of rare D--/D-- variants in individuals of Indian origin.

BACKGROUND: Rh antigens are critical in haemolytic disease of the foetus and newborn (HDFN). The D-- phenotype is a rare blood group characterised by the lack of expression of C, c, E and e antigens at the surface of red blood cells because of mutations in both RHCE alleles inactivating the expression of a "normal" protein. The aim of the study was to determine the molecular basis of D-- individuals of Indian origin. MATERIALS AND METHODS: Ten Rh D-positive postnatal women who had produced antibodies against all Rh antigens, except D, leading to HDFN and foetal loss, were investigated. Extensive serological and molecular (polymerase chain reaction [PCR] using sequence-specific primers), quantitative multiplex PCR of short fluorescent fragments (QMPSF), and Sanger sequencing analyses were carried out. RESULTS: Serological testing with anti-C, anti-c, anti-E, and anti-e reagents showed absence of the four antigens in all ten index cases, as well as in three siblings. Flow cytometry indicated absence of these antigens with a typical exalted expression of the D antigen, thus confirming the rare D-- phenotype. Molecular analysis by QMPSF suggested homozygous CE-D hybrid alleles causing the D-- phenotype: RHCE-D(3-9)-CE (n = 11), RHCE-D(3-8)-CE (n=1), and RHCE-D(2-6)-CE (n=1). DISCUSSION: For the first time, we report the molecular basis of the D-- phenotype in the Indian population. Identification and characterisation of RHCE-null variants by molecular methods can help resolve transfusion-related problems in these individuals. Family studies of index cases helped to identify rare blood donors and offer counselling to females of child-bearing age on the complications involved in such pregnancies.

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.