Hemoglobin Phenotype Research Articles

Three-dimensional visualization of human hemoglobin phenotypes with HPLC

Hemoglobinopathies were included in the Brazilian Neonatal Screening Program on June 6, 2001. Automated high-performance liquid chromatography (HPLC) was indicated as one of the diagnostic methods. The amount of information generated by these systems is immense, and the behavior of groups cannot always be observed in individual analyses. Three-dimensional (3-D) visualization techniques can be applied to extract this information, for extracting patterns, trends or relations from the results stored in databases. We applied the 3-D visualization tool to analyze patterns in the results of hemoglobinopathy based on neonatal diagnosis by HPLC. The laboratory results of 2520 newborn analyses carried out in 2001 and 2002 were used. The "Fast", "F1", "F" and "A" peaks, which were detected by the analytical system, were chosen as attributes for mapping. To establish a behavior pattern, the results were classified into groups according to hemoglobin phenotype: normal (N = 2169), variant (N = 73) and thalassemia (N = 279). 3-D visualization was made with the FastMap DB tool; there were two distribution patterns in the normal group, due to variation in the amplitude of the values obtained by HPLC for the F1 window. It allowed separation of the samples with normal Hb from those with alpha thalassemia, based on a significant difference (p < 0.05) between the mean values of the "Fast" and "A" peaks, demonstrating the need for better evaluation of chromatograms; this method could be used to help diagnose alpha thalassemia in newborns.

Newborn screening for sickle cell disease in a Nigerian hospital

Sickle cell disease (SCD) is the most common genetic disorder to affect Blacks. The mortality rate associated with SCD has remained high despite the use of appropriate interventions to manage the various forms of crisis. In developed countries, newborn screening programmes are conducted routinely, which has resulted in a reduction in the SCD mortality rate from 16% to <1%. In developing countries where the disease is prevalent, newborn screening programmes are yet to be established, and the acceptability of such programmes by the parents of newly delivered infants is unknown. This study was carried out to determine the acceptability of newborn screening for SCD on mothers of newly delivered infants, and to establish disease prevalence amongst a newborn population in Nigeria. This prospective cross-sectional study was conducted at St. Philomena's Hospital, Benin City with mothers of newly delivered infants and their newborn babies. Newly delivered mothers were recruited consecutively into the study. Knowledge of their own haemoglobin phenotype status was assessed, and their wishes regarding SCD screening of their babies were determined. Babies were screened using isofocusing electrophoresis. Six hundred and thirty mothers, delivered of 649 babies, were recruited into this study. Nineteen sets of babies were twins. Two mothers refused screening for their babies and 628 mothers or caregivers accepted screening; hence the acceptance rate was 99.7%. Four hundred and fifty-seven (71%) mothers did not know their own haemoglobin phenotype. Six hundred and forty-seven babies were screened for SCD. Of these, two samples were lost to testing and one baby had an indeterminate result; these three cases were not included in the analysis. Of the 644 babies whose results were analysed, 332 (51.6%) were male, 312 (48.4%) were female, 485 (75.3%) were AA, 133 (20.6%) were AS, seven (1.1%) were AC, 18 (2.8%) were SS, and one (0.2%) was SC. The majority of mothers in this study did not know their haemoglobin phenotype. Newborn screening for SCD was acceptable to 99.7% of the mothers. The prevalence of SCD in the newborn population was 3% (2.8% SS and 0.2% SC).

A Novel Frameshift Mutation (+A) at Codon 18 of the β-Globin Gene Associated with High Persistence of Fetal Hemoglobin Phenotype and δβ-Thalassemia

We report in this paper a novel thalassemia mutation (insertion of a single A nucleotide within the exon 1, at codon 18, of the β-globin gene) associated with a deletion of the δβ-globin gene region, in a patient exhibiting high persistence of fetal hemoglobin. The novel mutation causes a frameshift with the generation of a UGA stop codon. Analysis of the parent’s DNA demonstrates that the A insertion and frameshift mutation are inherited from the father, while the δβ-globin gene deletion is inherited from the mother. Gene dosage analysis and deletion-specific PCR demonstrate that the deletion is the (δβ)⁰ Sicilian deletion, involving a 13.4-kb δβ-globin gene region.

Muscle fibre number varies with haemoglobin phenotype in Atlantic cod as predicted by the optimal fibre number hypothesis

Atlantic cod (Gadus morhua L.) with the HbI-(2/2) haemoglobin phenotype have a higher blood oxygen affinity at low temperatures and a lower routine metabolic rate than individuals with the HbI-(1/1) phenotype. In the present study, muscle structure was found to be related to haemoglobin phenotype in a coastal population of Atlantic cod from the Saltenfjord region of Northern Norway. The maximum number of fast muscle fibres (FNmax) was reached at approximately 39 cm fork length and was 15% greater in the HbI-(1/1) than in the HbI-(2/2) phenotypes whereas the average fibre diameter for fish of the same fork length was significantly lower. Theoretically, the higher oxygen affinity of the HbI-(2/2) phenotype in the cold water of northern latitudes could have resulted in a relaxation of diffusional constraints at the level of individual muscle fibres, permitting the observed increase in fibre diameter. The results support the optimal fibre number hypothesis which envisages a trade-off between diffusional constraints and the energy cost of maintaining ionic homeostasis with fewer larger diameter muscle fibres in the HbI-(2/2) phenotype contributing to a lower routine metabolic rate.

Does temperature preference relate to the anaerobic capacity of Atlantic cod (Gadus morhua L.) with different haemoglobin phenotype?

The effect of Hb-I* phenotype on white muscle lactate dehydrogenease (LDH, E. C. 1.1.1.27) activity and buffering capacity was studied in Atlantic cod (Gadus morhua), acclimated and measured at temperatures near their behavioral temperature preference. It was hypothesized that these conditions would optimize biochemical processes but no difference was found in LDH activity between the Hb-I* phenotype after 56 d of acclimation to 6 and 14°C. However, LDH activity was both mass- and temperature-dependent; mean activity was 162.2±5.0 and 275.9±6.4 IU g−1 wet mass (mean±SEM) at 6 and 14°C respectively and larger fish had the highest rate of enzyme activity. White muscle buffer capacity was unaffected by Hb-I* phenotype but higher in cod held at 14°C.

γ-Globin Gene Expression in Chemical Inducer of Dimerization (CID)-dependent Multipotential Cells Established from Human β-Globin Locus Yeast Artificial Chromosome (β-YAC) Transgenic Mice

Identification of trans-acting factors or drugs capable of reactivating gamma-globin gene expression is complicated by the lack of suitable cell lines. Human K562 cells co-express epsilon- and gamma-globin but not beta-globin; transgenic mouse erythroleukemia 585 cells express predominantly human beta-globin but also gamma-globin; and transgenic murine GM979 cells co-express human gamma-and beta-globin. Human beta-globin locus yeast artificial chromosome transgenic mice display correct developmental regulation of beta-like globin gene expression. We rationalized that cells established from the adult bone marrow of these mice might express exclusively beta-globin and therefore could be employed to select or screen inducers of gamma-globin expression. A thrombopoietin receptor derivative that brings the proliferative status of primary mouse bone marrow cells under control of a chemical inducer of dimerization was employed to institute and maintain these cell populations. Human beta-globin was expressed, but gamma-globin was not; a similar expression pattern was observed in cells derived from fetal liver. gamma-Globin expression was induced upon exposure to 5-azacytidine, in cells derived from -117 Greek hereditary persistence of fetal hemoglobin human beta-globin locus yeast artificial chromosome (beta-YAC) mice, showing that the hereditary persistence of fetal hemoglobin (HPFH) phenotype was maintained in these cells or was reactivated by an artificial zinc finger-gamma-globin transcription factor and the previously identified fetal globin transactivators fetal Krüppel-like factor (FKLF) and fetal globin-increasing factor (FGIF). These cells may be useful for identifying transcription factors that reactivate gamma-globin synthesis or screening gamma-globin inducers for the treatment of sickle cell disease or beta-thalassemia.

T to C Substitution at –175 or –173 of the γ-Globin Promoter Affects GATA-1 and Oct-1 Binding in Vitro Differently but Can Independently Reproduce the Hereditary Persistence of Fetal Hemoglobin Phenotype in Transgenic Mice

The T to C substitution at position -175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma-to beta-globin gene with the natural chromosome arrangement but with the -175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the mu'LCRAgamma(-173)psibetadeltabeta construct, in which the -175 T to C Agamma gene was substituted with the -173 T to C Agamma gene. In vitro experiments proved that the -175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the -173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the -175 mutation or the -173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences gamma-globin gene expression in normal adult erythrocytes. Both the -173 and -175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the gamma-globin gene in adult erythrocytes.

Effects of beta thalassemia minor on results of six glycated hemoglobin methods

Background Beta-thalassemia minor (BTM) is a common benign condition that can be present in patients with diabetes mellitus. There are conflicting reports about the effect of BTM on glycated hemoglobin (gHb) measurements. We evaluated 6 gHb methods using samples from non-diabetic subjects with BTM. Methods Samples submitted for hemoglobin phenotype analysis were evaluated. A total of 57 samples (30 controls and 27 with BTM) from non-diabetic subjects were selected. GHb analysis was performed by Tosoh A1c 2.2+, Primus CLC 330, Bayer DCA 2000, Beckman Coulter, Synchron CX7 and LX20, and Roche Tina-quant II assays. Results The A1c 2.2+, CLC 330, DCA 2000 and Tina-quant II assays showed no statistically significant difference between the control and BTM groups. In contrast, BTM results were significantly higher than controls on the Synchron CX7 analyzer and borderline significant on the Synchron LX20 ( p=0.051). Further investigation demonstrated an increase in Synchron %HbA 1c results with decreasing hemoglobin concentrations. Conclusions In this study using samples from subjects with normal or near-normal gHb, BTM does not affect gHb measurements per se. However, the Synchron methods yielded higher results for samples with lower hemoglobin concentrations, like those that can be seen in BTM. The Synchron method was improved at the end of 2003, which minimized this problem.

Asthma and acute chest in sickle-cell disease.

Our objective was to determine if physician-diagnosed asthma increases the risk of acute chest syndrome (ACS) in children with sickle-cell disease (SCD) hospitalized for pain. Our study design was a retrospective case-control study of all SCD patients, aged 2-21 years, hospitalized for pain during the interval 1999-2000. Medical records of first admissions during the interval were reviewed to determine the presence of ACS during the admission. Cases were defined as patients with ACS, and controls were patients without ACS. Independently, medical records of admissions prior to the study interval were reviewed for evidence of physician-diagnosed asthma. Sixty-three cases with ACS and 76 controls without ACS were identified. No significant differences in gender, age, and hemoglobin phenotype were found. Patients with physician-diagnosed asthma were 4.0 times (95% CI, 1.7, 9.5) more likely to develop ACS during the admission than patients without asthma. Individuals with physician-diagnosed asthma had a longer hospitalization for ACS, i.e., 5.6 days vs. 2.6 days, respectively (P = 0.01). In conclusion, our preliminary data suggest that asthma in children with SCD admitted to the hospital for pain may be a risk factor for ACS and may increase the duration of hospitalization when compared to children with SCD and without asthma.

Relation between the response to iron supplementation and sickle cell hemoglobin phenotype in preschool children in western Kenya

Iron supplementation has been associated with greater susceptibility to malaria and lower hematologic responses in pregnant Gambian women with sickle cell trait (HbAS) than in similar women with the normal (HbAA) phenotype. It is not known whether a similar interaction exists in children. Our aim was to determine the influence of the HbAS phenotype on hematologic responses and malaria after iron supplementation in anemic (hemoglobin: 70-109 g/L) children aged 2-35 mo. We conducted a double-blind, randomized, placebo-controlled trial (HbAS, n = 115; HbAA, n = 408) of intermittent preventive treatment with sulfadoxine pyrimethamine (IPT-SP) at 4 and 8 wk and daily supervised iron for 12 wk. The mean difference in hemoglobin concentrations at 12 wk between children assigned iron and placebo iron, after adjustment for the effect of IPT-SP, was 9.1 g/L (95% CI: 6.4, 11.8) and 8.2 g/L (4.0, 12.4) in HbAA and HbAS children, respectively (P for interaction = 0.68). Although malaria parasitemia and clinical malaria occurred more often in HbAS children in the iron group than in those in the placebo iron group, this difference was not significant; incidence rate ratios were 1.23 (95% CI: 0.64, 2.34) and 1.41 (0.39, 5.00), respectively. The corresponding incidence rate ratios in HbAA children in the same groups were 1.07 (95% CI: 0.77, 1.48) and 0.59 (0.35, 1.01), respectively. The corresponding interactions between the effects of iron and hemoglobin phenotype were not significant. There was no evidence for a clinically relevant modification by the hemoglobin S phenotype of the effects of iron supplementation in the treatment of mild anemia. The benefits of iron supplementation are likely to outweigh possible risks associated with malaria in children with the HbAA or HbAS phenotype.

Hydroxyurea induces fetal hemoglobin by the nitric oxide–dependent activation of soluble guanylyl cyclase

Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of γ-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced γ-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.

Sickle cell trait carriage: imbalanced distribution of IgG subclass antibodies reactive to Plasmodium falciparum family-specific MSP2 peptides in serum samples from Gabonese children

Several mechanisms have been proposed for explaining the protection of young children with hemoglobin AS from severe Plasmodium falciparum malaria. In a previous study carried out in Gabon, we have shown an association between hemoglobin AS carriage and a greater P. falciparum infection complexity. In the present study, we have investigated the presence and fine specificity of merozoite surface protein 2 (MSP2) reactive antibodies using different peptides covering conserved and polymorphic regions (Blocks 1–3) of P. falciparum MSP2 molecules. A cross-sectional study was conducted in the city of Bakoumba (Gabon), where malaria is hyperendemic with perennial P. falciparum transmission. Among the 641 children included, 135 were heterozygous for the sickle cell trait (HbAS). There was no significant difference in age distribution (mean age: 5 years, 0.5–11 years) and sex ratio in both hemoglobin groups (HbAA vs. HbAS). Blood group O was, however, associated with the sickle cell trait ( P=0.02). P. falciparum isolates obtained from children with HbAS had a trend to higher infection complexity before the age of 5 years. Plasma samples were tested for the presence of antibodies to the different MSP2 peptides. Total IgG antibodies with a predominant reactivity against the FC27 type (the predominant P. falciparum MSP2 genotype) were found in serum samples from both groups. The profile of the IgG subclasses varied according to the hemoglobin phenotype. IgG3 and IgG2 were predominantly detected in plasma samples from HbAS children, whereas mainly IgG3 was found in children with HbAA. The role of the high multiclonal carriage associated with high family-specific antibodies reactive to MSP2 in HbAS children with asymptomatic P. falciparum parasitism is discussed.

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.

Hb Nancy and Hb Osler: two distinct genetic variants with identical clinical and hemoglobin phenotype

Three hemoglobin variants (Hb Nancy, Osler and Fort Gordon), carrying the same Tyr→Asp substitution at position β145(HC2), have been independently described in 1975 in patients with marked polycythemia. The first one was found in a French Caucasian family from Lorraine, and the two others in African Americans. Two unrelated individuals with Hb Osler have been recently reinvestigated at the DNA level and surprisingly, in their β gene, codon 145 was found to be AAT which encodes for asparagine and not for aspartic acid, the aspartate at the protein level resulting, thus, from a very efficient posttranslational event. We reinvestigated a patient from the family of Hb Nancy and found that codon 145 was GAT, encoding for aspartate. This demonstrates that Hb Nancy is geneticaly distinct from Hb Osler despite an almost identical phenotype.

Pharmacologic Hemoglobin Reversal: The Importance of Lipid Intermediaries and the Proposed Involvement of the Camp and Phosphatidylinositol Second Messenger Systems

Humoral and microenvironmental influences have played a major role in recent research into reversing the Hb F to Hb A switch. Early research in this area focused on hormonal influences and showed both thyroid hormone and prolactin could induce small but statistically significant reversals in hemoglobin phenotype. Recent research has focused on the effect of certain lipids in this process. The current study shows a synergy between thyroid hormone and prolactin in inducing a significant switch in adult rat hemoglobin patterns toward the neonatal pattern. Further, it is hypothesized that this synergy is due to the horjmones' effect on lipid intermediaries whose effect in turn are proposed to be mediated by the cAMP and phosphatidylinositol second messenger systems.

Plasmodium falciparum:Sickle-Cell Trait Is Associated with Higher Prevalence of Multiple Infections in Gabonese Children with Asymptomatic Infections

Through PCR amplifications of the gene encoding the merozoite surface antigen 2, utilizing allele-specific 3D7 and FC27 probes, we have examined the prevalence ofPlasmodium falciparumin children aged from 7 to 14 years living in a village located in the equatorial forest region of Central Africa (Gabon). Using this technique, 61% (100/163) of the blood samples were shown to be infected withP. falciparumwith 24 alleles distinguished by size polymorphism and sequence type. The two main families (3D7 and FC27) and hybrid alleles were detected regardless of sex and hemoglobin phenotype. No age-related changes in prevalence ofP. falciparumstrains were observed; however, the prevalence of infection (42%) was significantly lower in individuals with the sickle-cell trait compared with their normal-hemoglobin counterparts (68%). Mixtures of genetically distinct parasite clones were present in 82% of children carrying the sickle-cell trait but in only 58% of normal-hemoglobin carriers. The significance of these observations regarding the design and interpretation of epidemiological investigations is discussed in the context of malaria transmission in the region studied.

PAIN MANAGEMENT IN SICKLE CELL ANEMIA

Sickle cell anemia was first identified in the United States in 1910 by Herrick:' who observed sickle-shaped red blood cells (RBCs) in an anemic African-American medical student in Chicago. In 1923, the disease was shown to be inherited as an autosomal dominant and in 1927, Hahn and GillespieZO reported that the sickled morphology of RBCs occurs when the cells are placed in an environment of decreased oxygen tension. Pauling noted that the sickled hemoglobin had a slower rate of migration on electrophoretic testing; in 1959, Ingram,2z using a technique of electrophoretic fingerprinting, demonstrated that a digested sickled hemoglobin fragment was the cause of the slowed migration. More recently, amino acid sequencing of this peptide fragment demonstrated that glutamic acid was replaced by valine and that this single amino acid substitution in the globin chain was responsible for the sickled phenotype.I0 The hemoglobin molecule is a protein that consists of two polypeptide chains. Normal adult hemoglobin contains two a and two /3 globin chains (Hb A, a2, p2). Sickle hemoglobin (Hb S, a2p26@u-va1) consists of two normal a chains and the substitution of glutamic acid for valine in the sixth amino acid position of the /3 chain. This single amino acid substitution affects the physicochemical composition of the altered hemoglobin in the presence of deoxygenated blood which polymerizes into rigid tubules; the membrane of these RBCs also becomes less distensible, and the cells become more dense. These changes characterize the classic sickled hemoglobin phenotype; these cells become trapped in small capillaries and occlude the microcirculation, causing pain, infarction, and endorgan failure.'* Fetal hemoglobin (hemoglobin F) contains two a and two y globin chains (Hb F, aZ yz) and persists into the first few months of life. Fetal

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Diagnosing and preventing inherited disease: Detection of sickle gene by coelocentesis in early pregnancy: a new approach to prenatal diagnosis of single gene disorders

Coelomic fluid, placental tissue and maternal blood were collected at 7-10 weeks gestation from each of 58 women undergoing elective termination of pregnancy for psychological indications. In all samples, a 364 bp fragment of the human beta-globin gene spanning positions -23 to 341 was amplified. The restriction endonuclease Ddel was used to detect the sickle mutation which abolishes its restriction site. beta-Globin DNA was successfully amplified from all samples. In 53 cases a normal maternal beta-globin genotype was detected. In three out of five cases, where the maternal haemoglobin phenotype was HbAS, heterozygosity for the sickle mutation was demonstrated on analysis of coelomic fluid. In the remaining two cases a normal beta-globin genotype was observed. Three further coelomic fluid samples were found to be heterozygous for the sickle mutation. In these instances the maternal haemoglobin phenotype was normal, indicating paternal transmission of the sickle gene. The results of the present study have established that the diagnosis of sickle cell anaemia, and potentially other human single gene disorders, is feasible by coelocentesis.