P-selectin mediates the adhesion of sickle erythrocytes to the endothelium.

The adherence of sickle red blood cells (RBCs) to the vascular endothelium may contribute to painful vaso-occlusion in sickle cell disease. Sickle cell adherence involves several receptor-mediated processes and may be potentiated by the up-regulated expression of adhesion molecules on activated endothelial cells. Recent results showed that thrombin rapidly increases the adhesivity of endothelial cells for sickle erythrocytes. The current report presents the first evidence for the novel adhesion of normal and, to a greater extent, sickle RBCs to endothelial P-selectin. Studies of the possible interaction of erythrocytes with P-selectin revealed that either P-selectin blocking monoclonal antibodies or sialyl Lewis tetrasaccharide inhibits the enhanced adherence of normal and sickle cells to thrombin-treated endothelial cells. Both RBC types also adhere to immobilized recombinant P-selectin. Pretreating erythrocytes with sialidase reduces their adherence to activated endothelial cells and to immobilized recombinant P-selectin. Herein the first evidence is presented for the binding of normal or sickle erythrocytes to P-selectin. This novel finding suggests that P-selectin inhibition be considered as a potential approach to therapy for the treatment of painful vaso-occlusion in sickle cell disease.

In vivo blood flow abnormalities in the transgenic knockout sickle cell mouse.

The accepted importance of circulatory impairment to sickle cell anemia remains to be verified by in vivo experimentation. Intravital microscopy studies of blood flow in patients are limited to circulations that can be viewed noninvasively and are restricted from deliberate perturbations of the circulation. Further knowledge of sickle blood flow abnormalities has awaited an animal model of human sickle cell disease. We compared blood flow in the mucosal-intestinal microvessels of normal mice with that in transgenic knockout sickle cell mice that have erythrocytes containing only human hemoglobin S and that exhibit a degree of hemolytic anemia and pathological complications similar to the human disease. In sickle cell mice, in addition to seeing blood flow abnormalities such as sludging in all microvessels, we detected decreased blood flow velocity in venules of all diameters. Flow responses to hyperoxia in both normal and sickle cell mice were dramatic, but opposite: Hyperoxia promptly slowed or halted flow in normal mice but markedly enhanced flow in sickle cell mice. Intravital microscopic studies of this murine model provide important insights into sickle cell blood flow abnormalities and suggest that this model can be used to evaluate the causes of abnormal flow and new approaches to therapy of sickle cell disease.

Enhanced adherence of sickle erythrocytes to thrombin-treated endothelial cells involves interendothelial cell gap formation.

The adherence of sickle erythrocytes to vascular endothelium has the capacity to initiate vasoocclusion. The known effects of thrombin on endothelial cell function and the increased activity of thrombin in sickle cell disease led us to examine the effect of thrombin on the adhesivity of cultured endothelial cells for sickle erythrocytes. In particular, we studied whether the effect of thrombin on interendothelial cell gap formation (ICGF) was involved in endothelial cell adhesivity for sickle erythrocytes. Those endothelial cell monolayers stimulated by thrombin to maximal levels of static sickle erythrocyte adherence also underwent striking cell contraction and enlargement of interendothelial cell gaps. Adhesivity also increased when gaps were induced with antilaminin antibodies or EDTA. Maximally adhesogenic thrombin conditions failed to increase adhesivity when gap formation was prevented by pretreatment of the monolayers with 8-bromo-cyclic adenosine monophosphate (bromo-cAMP) or glutaraldehyde, agents that respectively inhibit actin-myosin-dependent cell contraction or cross-link adjacent cells in the monolayer. The influence of these two agents on EDTA-enhanced adhesivity was linked to their ability to prevent gap formation. Glutaraldehyde prevented both increased adherence and gap formation; bromo-cAMP prevented neither. Interendothelial cell gap formation may contribute to vasoocclusion by facilitating sickle erythrocyte adherence.

Revised transthyretin Ile 122 allele frequency in African-Americans.

The transthyretin (TTR) Ile 122 variant is associated with cardiac amyloidosis in individuals of African descent. To determine the prevalence of the allele encoding TTR Ile 122 in African-Americans, we have used PCR and restriction analysis to test DNA from African-Americans from various geographic areas, and found an allele frequency of 66/3376 (0.020), which is higher than the value we previously reported in a much smaller pilot study. Our data indicate that this TTR variant is present at equal frequency in African-Americans throughout the U.S., and suggest that this mutation may be a common, often unrecognized cause of cardiac disease in African-Americans.

Advances in molecular diagnosis of inherited hemoglobin disorders.

Molecular diagnosis for inherited disorders of hemoglobin production has been driven largely by the need for improved prenatal diagnosis. In turn, DNA-based testing has engendered better methods of fetal sampling. The sensitivity of DNA-based testing was increased tremendously by the polymerase chain reaction. Currently there are numerous polymerase chain reaction-based methods for diagnosing specific mutant globin alleles and detecting unknown mutations. These rely on restriction analysis, allele-specific hybridization or amplification, alterations in electrophoretic mobility, and DNA sequence analysis. The advantages and disadvantages of each are important to their specific application. The reverse dot blot method, with its capability for screening multiple alleles with a single hybridization reaction, is currently the most advantageous method for prenatal and routine clinical diagnosis.

Advances in the prenatal and molecular diagnosis of the hemoglobinopathies and thalassemias.

Prenatal diagnosis is available for pregnancies at risk for virtually all inherited disorders of hemoglobin production. The field of reproductive genetics must confront many ethical, legal, and social concerns regarding its use, many of which derive from a woman's desire to bear children but legal right to abortion. The goal of more widespread utilization of prenatal diagnosis is sought in the context of questioning the ethical control to be exerted over the biological makeup of future generations. Its appropriate application would be facilitated greatly by the availability of reliable DNA markers of disease severity. Advances in fetal sampling and in detecting mutant globin genes have provided the safe, accurate methodology required for prenatal diagnosis. Chorionic villus sampling in the first trimester has become standard practice, but second trimester amniocentesis also is used for sampling fetal DNA. The use of preimplantation diagnosis and testing fetal cells from the maternal circulation will soon be practical. DNA-based detection of globin gene mutations has been facilitated greatly by the polymerase chain reaction revolution, and several reliable diagnostic methods are available. Polymerase chain reaction-based methods rely on restriction analysis, allele-specific hybridization or amplification, DNA sequence analysis, and new non-polymerase chain reaction methods for DNA amplification in vitro. These methods are available for detecting hemoglobinopathy, thalassemia, and thalassemic-hemoglobinopathy genes that affect alpha- or beta-globin loci.

Reverse dot-blot detection of the African-American beta-thalassemia mutations.

DNA-based diagnosis of the beta thalassemias provides accuracy to newborn screening genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different-beta-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the beta-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two beta-globin target DNA fragments that encompass all known African-American beta-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele-specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 beta-thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common beta-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common beta-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of beta thalassemia among African-Americans and other ethnic groups in which beta thalassemia occurs.

Reverse dot-blot detection of Thai beta-thalassaemia mutations.

Pending curative therapy, newborn screening and prenatal diagnosis are essential to the management of beta thalassaemia. Diagnosis using electrophoretic methods is difficult in the presence of composite phenotypes and high Hb F levels. Direct DNA detection of mutant alleles circumvents both problems, but the enormous diversity of beta-thalassaemia mutations poses challenges for this approach. Among PCR-based tests, the reverse dot-blot method enables screening several mutations with a single hybridization reaction. Unfortunately it has often been targeted to only the common mutations of a particular ethnic population, necessitating the use of more arduous detection methods for the less common mutations. We developed a reverse dot-blot strip for the 10 beta-thalassaemia mutations, including the beta-thalassaemic haemoglobinopathies Hb E and Hb Malay, that account for 96% of beta thalassaemia in Thailand, and another strip for six less common Thai mutations. The second strip precludes the need for more technically challenging methods. To avoid problems associated with secondary structure of amplified full-length target DNA, we amplified and labelled beta-globin DNA as two shorter fragments that encompassed all known Thai mutations. Reverse dot-blotting is a rapid, accurate method for detecting beta-thalassaemia mutations.

Chronic pulmonary disorders in sickle cell disease: findings at thin-section CT.

PURPOSE: To characterize the non-acute abnormalities seen at computed tomography (CT) in patients with sickle cell (SC) disease and a prior history of acute chest syndrome (ACS)-pneumonia. MATERIALS AND METHODS: Twenty-nine patients with SC disease who had experienced one to more than 10 (median, six) previous episodes of ACS-pneumonia were prospectively studied with thin-section CT of the thorax. Scans were graded for interstitial disease and assigned a disease index ranging from 0 to 3. Twenty-four patients underwent pulmonary function tests (PFTs) and measurement of their blood gasses. RESULTS: Twelve of the 29 patients (41%) had significant interstitial disease that was multifocal. A correlation was found between the disease index and number of episodes of ACS-pneumonia (P = .02) but not between the disease index and PFT results. CONCLUSION: Thin-section CT demonstrates significant multifocal interstitial lung abnormalities in 41% of selected patients with SC disease. The pattern is most consistent with scarring from episodes of infarction or infection.

Osteonecrosis of the humeral head in sickle cell disease.

The prevalence and incidence of osteonecrosis (ON) of the humeral head in sickle cell disease was determined by a study of 2524 patients who were entered into a prospective study and followed for an average of 5.6 years. At entry, 5.6% had roentgenographic evidence of ON in one or both shoulders. There was little difference in age-adjusted prevalence among genotypes, but there were striking differences in age-specific rates. Observed at ages ranging from five to 24 years, 3.25% of sickle cell anemia (S/S) patients, but only 1.1% of sickle cell disease (S/C) patients, had ON. No S/beta+ thalassemia patients younger than 25 years of age had ON on entry. The highest age-adjusted incidence rate was found in S/S patients with concomitant alpha-thalassemia (4.85 per hundred patient-years), followed by S/beta zero-thalassemia (4.84 per hundred patient-years), S/beta+ thalassemia (2.61 per hundred patient-years), S/S without alpha-thalassemia (2.54 per hundred patient-years), and S/C (1.66 per hundred patient-years). Only 20.9% of patients reported pain or had limited range of movement at the time of diagnosis. Sickle cell disease is a frequent cause of ON of the humeral head, especially in children and young adults.

Hemoglobin constant spring in Bangkok: molecular screening by selective enzymatic amplification of the alpha 2-globin gene.

Hemoglobin Constant Spring (Hb CS) is a hemoglobin variant with an elongated alpha-globin chain secondary to a chain termination mutation. The diagnosis of HbCS by electrophoresis is difficult because it is present in very low amounts in the red cells of heterozygotes. Selective enzymatic amplification of the alpha 2-globin gene and allele-specific hybridization for Hb CS gene provided accurate diagnosis of Hb Constant Spring. We have used this approach to detect the alpha cs mutation in the cord blood that contained all four alpha-globin genes but had Hb Bart on electrophoresis. The alpha cs mutation was found in six subjects whose Hb Bart levels were 3.0, 3.2, 3.7, 4.0, 4.9, and 9.8%. The latter also had -alpha mutation on the other chromosome, giving rise to the genotype alpha cs alpha/-alpha, which produced high Hb Bart. The gene frequency for alpha cs in the Thai calculated from a total of 406 cord blood studied in Bangkok was found to be approximately 0.008.

Sickle cell disease as a cause of osteonecrosis of the femoral head.

BACKGROUND AND METHODS: Osteonecrosis of the femoral head is an important complication of sickle cell disease. We studied 2590 patients who were over 5 years of age at entry and followed them for an average of 5.6 years. Patients were examined twice a year, and radiographs of the hips were taken at least twice: at study entry and approximately three years later. RESULTS: At study entry, 9.8 percent of patients were found to have osteonecrosis of one or both femoral heads. On follow-up, patients with the hemoglobin SS genotype and alpha-thalassemia were at the greatest risk for osteonecrosis (age-adjusted incidence rate, 4.5 cases per 100 patient-years, as compared with 2.4 in patients with the hemoglobin SS genotype without alpha-thalassemia and 1.9 in those with the hemoglobin SC genotype). Although the rate of osteonecrosis in patients with the hemoglobin SC genotype did not differ significantly from that in patients with the hemoglobin SS genotype without alpha-thalassemia, osteonecrosis tended to develop in these patients later in life. Intermediate rates of osteonecrosis were observed among patients with the hemoglobin S-beta zero-thalassemia and the hemoglobin S-beta(+)-thalassemia genotypes (3.6 and 3.3 cases per 100 patient-years, respectively). Osteonecrosis was found in patients as young as five years old (1.8 cases per 100 patient-years for all genotypes). The frequency of painful crises and the hematocrit were positively associated with osteonecrosis. The mean corpuscular volume and serum aspartate aminotransferase level were negatively associated. Twenty-seven patients had hip arthroplasty during the study; 10 were under 25 years of age. Five of the 27 required reoperation 11 to 53 months after the initial operation. CONCLUSIONS: Osteonecrosis of the femoral head is common in patients with sickle cell disease, with an incidence ranging from about 2 to 4.5 cases per 100 patient-years. Patients with the hemoglobin SS genotype and alpha-thalassemia and those with frequent painful crises are at highest risk. The overall prevalence is about 10 percent. The results of hip arthroplasty are poor.

Asymmetrically primed selective amplification/temperature shift fluorescence polymerase chain reaction to detect the hemoglobin Constant Spring mutation.

Hemoglobin (Hb) Constant Spring is an alpha-thalassemic hemoglobinopathy that is a major cause of severe alpha-thalassemia in Southeast Asians. The difficulty of diagnosing Hb Constant Spring using standard electrophoretic methods has led to interest in DNA-dependent diagnostic methods. The methods developed have had to contend with the high degree of homology of the alpha 2-globin gene (the site of the Hb Constant Spring mutation) and the alpha 1-globin gene. We have developed a single reaction polymerase chain reaction-based method that uses asymmetric priming and a temperature shift to accomplish dual ends, selective amplification of alpha 2 but not alpha 1 DNA and discrimination of normal and Hb Constant Spring alpha 2 genes by allele-specific fluorescence polymerase chain reaction. Advantages of this method over previous approaches include avoiding radioisotopes, precluding the need for electrophoresis, and serving as its own control for successful amplification. It is readily applicable to routine diagnosis, population screening, and prenatal diagnosis.

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping.

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine-labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.

Human embryonic zeta-globin chains in fetal and newborn blood.

A sensitive and specific radioimmunoassay (RIA) for human embryonic zeta-globin chains was used to study normal fetal blood and newborn cord blood as well as cord blood from newborns with alpha-thalassemias. From 17 weeks until 37 weeks of gestation, zeta-globin chains were present in almost all fetal and cord blood samples (0.27% +/- 0.15% in samples of weeks 17 through 30; 0.14% +/- 0.11% in samples of weeks 31 through 37). zeta-Globin chains were present in greater than 80% of cord blood hemolysates from normal, full-term newborns (0.15% +/- 0.11%) as well as from 16 near-term newborns of diabetic mothers (0.13% +/- 0.13%). zeta-Globin chains were not detected in normal infants aged 3 months to 2 years. In cord blood hemolysates from alpha-thalassemic newborns, the levels of zeta-globin chain content varied from very high to undetectable levels. Gene mapping of the zeta-alpha-globin gene cluster was performed in 12 newborns in whom cord blood zeta-globin chains had been determined. Newborns who were carriers of alpha-thalassemia-1 due to the (--SEA/) deletion had very high levels of zeta-globin chains (greater than 1.5%).

Selective enzymatic amplification of alpha 2-globin DNA for detection of the hemoglobin Constant Spring mutation.

Hemoglobin Constant Spring is an elongation mutation of the alpha 2-globin locus that results in a thalassemic phenotype. It has a high prevalence in Asian populations. When inherited with other alpha-thalassemia determinants, the Constant Spring gene has the potential to cause severe forms of alpha-thalassemia. Accurate diagnosis of the condition with standard hemoglobin electrophoresis is unreliable due to the small to undetectable amounts of the mutant hemoglobin present. Because of the extensive sequence homology of the alpha 1 and alpha 2 loci, allele-specific hybridization to total genomic DNA containing the Constant Spring gene would not distinguish between heterozygous and homozygous hemoglobin Constant Spring. Selective enzymatic amplification of alpha 2-globin DNA sequences, however, allows unambiguous diagnoses to be made using allele-specific hybridization. This method is useful for providing accurate genetic counseling and prenatal diagnosis in populations and specific families in which precise diagnosis is important.