Search

Sickle cell anaemia (SCA) is a monogenic disease with a highly variable clinical course. We aimed to investigate associations between microvascular function, haemolysis markers, blood viscosity and various types of SCA-related organ damage in a multicentric sub-Saharan African cohort of patients with SCA. In a cross-sectional study, we selected seven groups of adult patients with SS phenotype in Dakar and Bamako based on the following complications: leg ulcer, priapism, osteonecrosis, retinopathy, high tricuspid regurgitant jet velocity (TRV), macro-albuminuria or none. Clinical assessment, echocardiography, peripheral arterial tonometry, laboratory tests and blood viscosity measurement were performed. We explored statistical associations between the biological parameters and the six studied complications. Among 235 patients, 58 had high TRV, 46 osteonecrosis, 43 priapism, 33 leg ulcers, 31 retinopathy and 22 macroalbuminuria, whereas 36 had none of these complications. Multiple correspondence analysis revealed no cluster of complications. Lactate dehydrogenase levels were associated with high TRV, and blood viscosity was associated with retinopathy and the absence of macroalbuminuria. Despite extensive phenotyping of patients, no specific pattern of SCA-related complications was identified. New biomarkers are needed to predict SCA clinical expression to adapt patient management, especially in Africa, where healthcare resources are scarce., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the natural history of the disease on this continent remains largely unknown. Intravascular haemolysis results in activation of circulating blood cells and release of microparticles (MPs) that exert pro-inflammatory effects and contribute to vascular damage. We designed a case-control study nested in the CADRE cohort (Coeur-Artère-DRÉpanocytose, clinical trials.gov identifier NCTO3114137) and based on extreme phenotypes, to analyse blood cell-derived MPs in 232 adult SS patients at steady state in Bamako and Dakar. Thirty-six healthy adult controls matched by age and sex were recruited in Bamako. The MPs concentrations were higher in SS patients compared to AA controls with a predominance of erythrocyte- and reticulocyte-derived MPs. These erythroid-derived MPs were significantly lower in patients with retinopathy (P = 0·022). Reticulocyte-derived MPs were significantly negatively and positively associated with a history of priapism (P = 0·020) and leg ulcers (P = 0·041) respectively. We describe for the first time the comparative patterns of plasma MPs in healthy subjects and patients with SCD living in sub-Saharan Africa and exhibiting various complications. Because our present results show no clear pattern of correlation between erythroid MPs and the classical hyper-haemolytic complications, we hypothesise a weak relevance of the hyper-haemolysis versus hyper-viscous paradigm in Africa., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy., (© 2016 John Wiley & Sons Ltd.)

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

Introduction: Coinheritance of α-thalassemia influences the clinical and hematological phenotypes of β-hemoglobinopathies (β-thalassemia and sickle cell disease) and when present together in significant frequency within a population, a spectrum of clinical forms is observed. Precise molecular characterization of α-thalassemia is important in understanding their disease-modifying role in β-hemoglobinopathies and for diagnostic purposes., Patients and Methods: Because currently used approaches are labor/cost-intensive, time-consuming, error-prone in certain genotype combinations and not applicable for large epidemiological screening, we developed a systematic stepwise strategy to overcome these difficulties. We successfully applied this to characterize the α-globin gene status in 150 Omani cord blood samples with Hb Barts and 32 patients with HbH disease., Results: We observed a good correlation between α-globin genotypes and level of Hb Bart's with the Hb Bart's levels significantly higher in both deletional and non-deletional α-globin genotypes. The most common α-globin genotype in HbH cases was α(TSaudi) α/α(TSaudi) α (n = 16; 50%) followed by -α(3.7) /-(MED) (n = 10; 31%). This approach detects also the α-globin gene triplication as exemplified by the study of a family where the β-globin gene defect failed to explain the β-thalassemia intermedia phenotype., Conclusion: Molecular characterization of α-thalassemia is complex due to high sequence homology between the duplicated α-globin genes and to the existence of a variety of gene rearrangements (small and large deletions of various sizes) and punctual substitutions (non-deletional alleles). The novelty of our strategy resides, not in the individual technical steps per se but in the reasoned sequential order of their use taking into consideration the hematological phenotype as well., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The objective of our present study was to develop a warfarin dosing algorithm for the Omani patients, as performances of warfarin dosing algorithms vary across populations with impact on the daily maintenance dose. We studied the functional polymorphisms of CYP2C9, CYP4F2 and VKORC1 genes to evaluate their impact on the warfarin maintenance dose in an admixed Omani patient cohort with Caucasian, African and Asian ancestries. We observed a 64-fold inter-patient variability for warfarin to achieve stable international normalized ratio in these patients. Univariate analysis revealed that age, gender, weight, atrial fibrillation, deep vein thrombosis/pulmonary embolism and variant genotypes of CYP2C9 and VKORC1 loci were significantly associated with warfarin dose in the studied patient population. However, multiple regression model showed that only the atrial fibrillation, and homozygous CYP2C9 variant genotypes (*2/*3 and *3/*3) and VKORC1 GA and AA genotypes remained significant. A multivariate model, which included demographic, clinical and pharmacogenetic variables together explained 63% of the overall inter-patient variability in warfarin dose requirement in this microgeographically defined, ethnically admixed Omani patient cohort on warfarin. This locally developed model performed much better than the International Warfarin Pharmacogenetics Consortium (IWPC) model as the latter could only explain 34% of the inter-patient variability in Omani patients. VKORC1 3673G>A polymorphism emerged as the single most important predictor of warfarin dose variability, even in this admixed population (partial R(2)=0.45).

This is the first study to evaluate the spectrum and prevalence of dose-predictive genetic polymorphisms of the CYP2C9, CYP4F2 and VKORC1 loci together, in a geographically defined, ethnically admixed healthy adult Omani population sharing common lifestyle/environmental factors. Since the present-day Omani population is the result of an admixture of Caucasian, African and Asian ancestries, we compared the pharmacogenetic profile of these three loci in this population. Interestingly, the Omani pharmacogenetic profile, in terms of allele and genotype distribution, has values that are intermediate between Caucasians and African Americans, the African admixture further substantiated by the presence of the CYP2C9*8 allele. However, limitations and usefulness of such comparisons warrant caution, as the data from pharmacogenetic literature do not always represent bona fide population categories. Furthermore, definition of study population based on microgeographical scale would be more appropriate in pharmacogenetic research rather than the flawed racial, ethnic, or social categorizations since pharmacogenetic variation is clinal, and genetic influences will be further altered by lifestyle and environmental factors.

Background and Objective: The clinical hallmarks of sickle cell disease (SCD) are vaso-occlusive crises (VOC) triggered by red blood cells (RBC) stiffening and abnormal adhesion to vascular endothelial cells (VEC) in the context of chronic inflammation, cell activation, and vascular tone abnormalities. Hydroxycarbamide (HC) is the only drug with a proven efficacy in decreasing VOC frequency. HC decreases RBC stiffening, modulates adhesion protein expression by RBC and VEC, and reduces endothelin-1 production by VEC. Our objective was to test whether HC could also affect inflammation through its action on VEC., Methods: We used microarrays to study the effect of HC on the transcriptome of transformed human bone marrow endothelial cell, a cell line derived from bone marrow microcirculation (the predilection site of VOC), in basal and proinflammatory conditions. Microarray results were confirmed by real-time quantitative PCR and protein analysis on transformed human bone marrow endothelial cell (TrHBMEC) and on two other VEC types in the primary culture: human pulmonary microcirculation endothelial cell (HPMEC) and human umbilical vein endothelial cell (HUVEC a classical model for the macrocirculation)., Results: HC had a significant effect on the expression of genes of the 'inflammation pathway'. Strikingly, it stimulates the expression of proinflammatory genes such as IL1A, IL1B, IL6, IL8, CCL2, CCL5, CCL20, and CCL8 in all the tested VEC types., Conclusion: Our study confirms that VECs are significant targets of HC in the context of SCD and identifies its earlier unsuspected action on another major component of SCD pathophysiology, that is, the 'inflammation pathway'.

Human mannose- binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p=0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%,respectively; p=0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV- positive patients.

Human mannose-binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p = 0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%, respectively; p = 0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV-positive patients.

An earlier study of human globin gene polymorphism in two Adriatic islands of Olib and Silba showed an abnormal arrangement of alpha-globin genes in two different individuals. The next step was to determine the degree of the kinship relationship between the two probands, one with a deleted and another with triplicated alpha-globin gene on the island Silba, and to determine the stability of this disorder through generations. We reviewed the parish registers (Status Animarum) of the island of Silba, dating from the year 1527, and constructed family trees for the two probands. Restriction endonuclease mapping was performed to study the arrangement of the alpha-globin genes in the offspring of our probands. A total of 183 ancestors completed the two family trees. The kinship relationship between them was established in the 5th, 6th, and 7th generation. The analysis of alpha-globin genes in the offspring of our probands showed the triplicated alpha-globin genes in two persons. We also found alpha-globin gene triplication in other three relatives. We did not find any deleted alpha-globin genes. We determined the kinship relationship between the two probands, one with deleted and the other with triplicated alpha-globin genes. This finding enabled us to determine the stability of this gene disarrangement through generations. It also showed new possibilities in anthropogenetic research, by combining the analyses of parish registers with those of modern genetic methods, such as restriction endonuclease mapping.

The majority of the chromosomes with the beta(S) gene have one of the five common haplotypes, designated as Benin, Bantu, Senegal, Cameroon, and Arab-Indian haplotypes. However, in every large series of sickle cell patients, 5-10% of the chromosomes have less common haplotypes, usually referred to as "atypical" haplotypes. In order to explore the genetic mechanisms that could generate these atypical haplotypes, we extended our analysis to other rarely studied polymorphic markers of the beta(S)-gene cluster, in a total of 40 chromosomes with uncommon haplotypes from Brazil and Cameroon. The following polymorphisms were examined: seven restriction site polymorphisms of the epsilongammadeltabeta-cluster, the pre-(G)gamma framework sequence including the 6-bp deletion/insertion pattern, HS-2 LCR (AT)xR(AT)y and pre-beta (AT)xTy repeat motifs, the GC/TT polymorphism at -1105-1106 of (G)gamma-globin gene, the C/T polymorphism at -551 of the beta-globin gene, and the intragenic beta-globin gene framework. Among the Brazilian subjects, the most common atypical structure (7/16) was a Bantu 3'-subhaplotype associated with different 5'-sequences, while in two chromosomes a Benin 3'-subhaplotype was associated with two different 5'-subhaplotypes. A hybrid Benin/Bantu configuration was also observed. In three chromosomes, the atypical haplotype differed from the typical one by the change of a single restriction site. In 2/134 chromosomes identified as having a typical Bantu RFLP-haplotype, a discrepant LCR repeat sequence was observed, probably owing to a crossover 5' to the epsilon-gene. Among 80 beta(S) chromosomes from Cameroon, 22 were associated with an atypical haplotype. The most common structure was represented by a Benin haplotype (from the LCR to the beta-gene) with a non-Benin segment 3' to the beta-globin gene. In two cases a Bantu LCR was associated with a Benin haplotype and a non-Benin segment 3' to the beta-globin gene. In three other cases, a more complex structure was observed that can be considered as a hybrid of Benin, Bantu, Senegal, or other chromosomes was observed. These data suggest that the atypical beta(S) haplotypes are not uncommon in America and in Africa. These haplotypes are probably generated by a variety of genetic mechanisms including (a) isolated nucleotide changes in one of the polymorphic restriction sites, (b) simple and double crossovers between two typical beta(S) haplotypes or much more frequently between a typical beta(S) haplotype and a different beta(A)-associated haplotype that was present in the population, and (c) gene conversions., (Copyright 2000 Wiley-Liss, Inc.)

HIV-1 and related viruses require co-receptors, in addition to CD4, to infect target cells. The chemokine receptor CCR-5 (ref.1) was recently demonstrated to be a co-receptor for macrophage-tropic (M-tropic) HIV-1 strains, and the orphan receptor LESTR (also called fusin) allows infection by strains adapted for growth in transformed T-cell lines (T-tropic strains). Here we show that a mutant allele of CCR-5 is present at a high frequency in caucasian populations (allele frequency, 0.092), but is absent in black populations from Western and Central Africa and Japanese populations. A 32-base-pair deletion within the coding region results in a frame shift, and generates a non-functional receptor that does not support membrane fusion or infection by macrophage- and dual-tropic HIV-1 strains. In a cohort of HIV-1 infected caucasian subjects, no individual homozygous for the mutation was found, and the frequency of heterozygotes was 35% lower than in the general population. White blood cells from an individual homozygous for the null allele were found to be highly resistant to infection by M-tropic HIV-1 viruses, confirming that CCR-5 is the major co-receptor for primary HIV-1 strains. The lower frequency of heterozygotes in seropositive patients may indicate partial resistance.

Two blaTEM-like genes were characterized that encoded IRT beta-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with beta-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 beta-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic pI (pI = 5.2) of these IRT enzymes compared to that of TEM-1 (pI = 5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this beta-lactamase to inhibition by p-chloromercuribenzoate.

During normal human development, a switch is classically observed in the relative expression of the two gamma globin genes, the G gamma/A gamma ratio varying from 70/30 at birth to 40/60 by the end of the first year. An exception to this developmental pattern is linked to the presence of an XmnI restriction site at a position -158 to the Cap site of the G gamma gene. Another exception is observed in individuals homozygous for two easily detectable variations of the A gamma gene: the presence of a threonine residue at codon 75 and a HindIII site within the second intron. A 4-bp deletion has been described around position -225 in some thalassemic patients presenting with these variations. In this study, we find this deletion to be haplotype-linked in a series of 156 individuals of various ethnic origins and presenting with various normal and pathological phenotypes. In sickle cell patients heterozygous for this 4-bp deletion, the relative expression of the A gamma genes on the two chromosomes can be measured by estimating the A gamma T and A gamma I chains, the former always being synthesized at a lower rate. These results suggest a functional role for the deleted sequence.

An adult Algerian patient with homozygous beta thalassemia was found to have a unique beta thalassemia gene. Cloning and sequencing revealed that the only abnormality present in this beta gene is a transversion in the polypyrimidine stretch at the 3' end of the large intervening sequence (IVS 2) six bases 5' to the consensus AG dinucleotide sequence (CCGCCCACAG instead of CCTCCCACAG). In addition, digestion of the cloned fragment by the enzyme Mnl I demonstrates the disappearance of a restriction site as expected. This is the first example of a defect in the consensus sequence at the 3' end of an IVS leading to a thalassemia phenotype presumably due to decreased splicing.

Homozygous beta-thalassemia intermediate (TI) differs from thalassemia major (TM) in being less severe clinically. Associated alpha-thalassemia could account for the TI phenotype by reducing the alpha/non-alpha chain imbalance. We have analyzed the alpha loci of 9 TI and 11 TM patients by restriction endonuclease mapping. All the TM and 7 of the TI patients have the normal complement of four alpha-globin genes (alpha alpha/alpha alpha). One TI patient has three alpha-globin genes (alpha alpha/-alpha), and another TI patient has five alpha genes (alpha alpha/alpha alpha alpha).

The clinical and laboratory criteria which distinguish thalassaemia intermedia (T.I.) from thalassaemia major were analyzed in a series of 30 patients with homozygous beta- thalassaemia, 8 of whom had T.I. The appearance of the first symptoms after the age of 2 years, the moderate spleen enlargement, the haemoglobin levels approaching 8 g/100 ml and the response to moderate transfusions over 1-year observation period were in favour of T.I. Since patients who had transfusions were clinically better than those who had none, it is suggested that T.I. patients should be treated with regular transfusions and iron chelating agents.

The association between the polymorphism of the A gamma chain of human fetal haemoglobin and the DNA polymorphism at the beta gene cluster has been investigated. The A gamma 75 threonine mutation was found in association with haplotypes II and VI described by Orkin et al. (1982), which share the HindIII cleavage site in the A gamma IVS 2 sequence. The distance between the two polymorphic sites is 868 base-pairs. In contrast, haplotypes I, III, V and IX which do not possess this HindIII cleavage site were associated with the normal A gamma 75 isoleucine allele. The simple detection of the gamma gene polymorphism at the protein level can be useful for identifying DNA haplotypes, and therefore beta thalassaemic mutations.

A new abnormal hemoglobin, Hb J Amiens beta 17 (A 14) Lys replaced by Asn, has been discovered during the exploration of a recent polycythemia in a 65-year-old patient of Spanish extraction. Oxygen affinity of washed red blood cells was found to be normal at pH 7.13 (P 50 = 30.0 mmHg, N = 29.5 +/- 1). Cooperativity is unchanged, and no instability was detected. From this study, it is concluded that there is no relation between this functionally silent hemoglobin and the polycythemia. In fact, the recent appearance of the polycythemia, the involvement of the other blood cell lines, particularly the thrombocytosis, the high score of leukocyte alkaline phosphatases, and the results of the bone marrow biopsy led to the diagnosis of polycythemia vera.

To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.

Titration curves of reduced and alkylated polypeptide chains can be successfully performed in 8 M urea-polyacrylamide gel plates by electrophoresis perpendicular to a stationary stack of focused carrier ampholytes. All buffers and thiol reagents with pK values in the range pH 3--10 should be removed, since their pH-dependent ionization affects the migration and apparent pI values of the protein chains. No blurring of the patterns below pH 4.5 is observed, as usually found in titration curves in the absence of urea, thus allowing the direct titration of Glu and Asp residues. It is not possible by the present technique to titrate any group below pH ca. 3 and above pH ca. 10, due to the lack of suitable carrier ampholytes and to a "flooding" phenomenon, with concomitant identical electrophoretic mobility for all protein species, irrespective of their relative pI values and amino acid composition. The "electrophoretic titration curves" thus obtained were well correlated with the overall amino acid composition of the polypeptide chains analyzed.

Expression of a cloned human beta thalassemia gene with a single base change at position 5 of IVS 1 has been analyzed 48 hours after transfer of the gene into HeLa cells (transient expression). Little or no normal beta globin mRNA accumulates in the presence of the abnormal beta gene in contrast to significantly more normal beta mRNA produced with other mutations at this same position. By contrast, large amounts of an abnormal beta globin mRNA are present; this is due to the use of a cryptic 5' splice site in exon 1 rather than the normal 5' splice site of IVS 1. The results indicate the variability of the effect on RNA splicing of different single base defects within IVS.