Orah S. Platt

Mortality In Sickle Cell Disease -- Life Expectancy and Risk Factors for Early Death

BACKGROUND Information on life expectancy and risk factors for early death among patients with sickle cell disease (sickle cell anemia, sickle cell-hemoglobin C disease, and the sickle cell-beta-thalassemias) is needed to counsel patients, target therapy, and design clinical trials. METHODS We followed 3764 patients who ranged from birth to 66 years of age at enrollment to determine the life expectancy and calculate the median age at death. In addition, we investigated the circumstances of death for all 209 adult patients who died during the study, and used proportional-hazards regression analysis to identify risk factors for early death among 964 adults with sickle cell anemia who were followed for at least two years. RESULTS Among children and adults with sickle cell anemia (homozygous for sickle hemoglobin), the median age at death was 42 years for males and 48 years for females. Among those with sickle cell-hemoglobin C disease, the median age at death was 60 years for males and 68 years for females. Among adults with sickle cell disease, 18 percent of the deaths occurred in patients with overt organ failure, predominantly renal. Thirty-three percent were clinically free of organ failure but died during an acute sickle crisis (78 percent had pain, the chest syndrome, or both; 22 percent had stroke). Modeling revealed that in patients with sickle cell anemia, the acute chest syndrome, renal failure, seizures, a base-line white-cell count above 15,000 cells per cubic millimeter, and a low level of fetal hemoglobin were associated with an increased risk of early death. CONCLUSIONS Fifty percent of patients with sickle cell anemia survived beyond the fifth decade. A large proportion of those who died had no overt chronic organ failure but died during an acute episode of pain, chest syndrome, or stroke. Early mortality was highest among patients whose disease was symptomatic. A high level of fetal hemoglobin predicted improved survival and is probably a reliable childhood forecaster of adult life expectancy.

Association between Microdeletion and Microduplication at 16p11.2 and Autism

BACKGROUND Autism spectrum disorder is a heritable developmental disorder in which chromosomal abnormalities are thought to play a role. METHODS As a first component of a genomewide association study of families from the Autism Genetic Resource Exchange (AGRE), we used two novel algorithms to search for recurrent copy-number variations in genotype data from 751 multiplex families with autism. Specific recurrent de novo events were further evaluated in clinical-testing data from Childrens Hospital Boston and in a large population study in Iceland. RESULTS Among the AGRE families, we observed five instances of a de novo deletion of 593 kb on chromosome 16p11.2. Using comparative genomic hybridization, we observed the identical deletion in 5 of 512 children referred to Childrens Hospital Boston for developmental delay, mental retardation, or suspected autism spectrum disorder, as well as in 3 of 299 persons with autism in an Icelandic population; the deletion was also carried by 2 of 18,834 unscreened Icelandic control subjects. The reciprocal duplication of this region occurred in 7 affected persons in AGRE families and 4 of the 512 children from Childrens Hospital Boston. The duplication also appeared to be a high-penetrance risk factor. CONCLUSIONS We have identified a novel, recurrent microdeletion and a reciprocal microduplication that carry substantial susceptibility to autism and appear to account for approximately 1% of cases. We did not identify other regions with similar aggregations of large de novo mutations.

Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia

Hydroxyurea, a widely used cytotoxic/cytostatic agent that does not influence methylation of DNA bases, increases fetal hemoglobin production in anemic monkeys. To determine its effect in sickle cell anemia, we treated two patients with a total of four, 5-d courses (50 mg/kg per d, divided into three oral doses). With each course, fetal reticulocytes increased within 48-72 h, peaked in 7-11 d, and fell by 18-21 d. In patient I, fetal reticulocytes increased from 16.0 +/- 2.0% to peaks of 37.7 +/- 1.2, 40.0 +/- 2.0, and 32.0 +/- 1.4% in three successive courses. In patient II the increase was from 8.7 +/- 1.2 to 50.0 +/- 2.0%. Fetal hemoglobin increased from 7.9 to 12.3% in patient I and from 5.3 to 7.4% in patient II. Hemoglobin of patient I increased from 9.0 to 10.5 g/dl and in patient II from 6.7 to 9.9 g/dl. Additional single-day courses of hydroxyurea every 7-20 d maintained the fetal hemoglobin of patient I t 10.8-14.4%, and the total hemoglobin at 8.7-10.8 g/dl for an additional 60 d. The lowest absolute granulocyte count was 1,600/mm3; the lowest platelet count was 390,000/mm3. The amount of fetal hemoglobin per erythroid burst colony-forming unit (BFU-E)-derived colony cell was unchanged, but the number of cells per BFU-E-derived colony increased. Although examination of DNA synthesis in erythroid marrow cells in vitro revealed no decreased methylcytidine incorporation, Eco RI + Hpa II digestion of DNA revealed that hypomethylation of gamma-genes had taken place in vivo after treatment. This observation suggests that hydroxyurea is a potentially useful agent for the treatment of sickle cell anemia and that demethylation of the gamma-globin genes accompanies increased gamma-globin gene activity.

Hydroxyurea for the Treatment of Sickle Cell Anemia

An 18-year-old woman with sickle cell anemia presents with recurrent painful crises, and treatment with hydroxyurea is recommended. Hydroxyurea causes a shift toward the production of red cells containing fetal hemoglobin. A possible increase in the risk of acute leukemia due to hydroxyurea therapy remains the subject of debate.

Sickle cell anemia as an inflammatory disease

The classical view of sickle cell anemia has always focused on the primary genetic defect — the abnormal sickle hemoglobin that polymerizes when deoxygenated. Polymerization within the red cell causes it to deform, to become rigid, to obstruct blood flow, and to produce acute and chronic tissue damage because of poor perfusion. A more holistic view sees the sickle red cell with its abnormal contents and membrane in a larger context — as it interacts with, damages, and stimulates the vascular endothelium and surrounding tissues and cells. This view allows examination of the hypothesis that the sticky, stiff, oxidizing sickle red cell is an irritant that provokes an inflammatory response as it obstructs flow. The tissues are not only under-perfused, but also exposed to inflammatory cytokines, growth factors, and the actions of the activated cells that respond to and produce them. The article by Kaul and Hebbel in this issue of the JCI (1) suggests that reperfusion injury plays a major role in sickle pathophysiology and provides new insights that contribute to this broader view of the disease process. Using a transgenic sickle mouse model and directly visualizing the microcirculation in a living open cremaster muscle preparation, the authors make three key observations that implicate reperfusion injury (1). First, after 3 hours of mild hypoxia followed by reoxygenation, the vascular bed of the sickle mouse (but not control) showed a distinct inflammatory response with increased leukocyte rolling, adhesion, and emigration. Second, the reoxygenation resulted in conversion in endothelial cells of the oxidant-sensitive probe dihydrorhodamine to the fluorescent compound rhodamine, suggesting local production of H2O2. Third, the abnormal rolling, adhesion, and migration of leukocytes were prevented by infusion with a monoclonal murine P-selectin antibody before reoxygenation. This theme of reactive oxygen species generation and P-selectin-sensitive leukocyte adhesion and migration is consonant with a variety of reperfusion injury models. The implication is that once oxygen is restored to ischemic tissues, oxygen radicals are formed, and inflammatory endothelial and tissue injury occurs. This is a particularly attractive model for sickle cell anemia — especially for the chronic subliminal organ damage that typically occurs in the spleen, lung, and kidney. In the vascular beds of these organs, young reticulocytes periodically attach to endothelial cells and, if conditions are right, cause a transient logjam of relatively rigid deoxygenating mature red cells in their wake (2). As the obstruction eventually clears, the reperfusion pathophysiology plays out, escalating the likelihood of additional rounds of reticulocyte adhesion to the inflamed endothelium that is becoming increasingly decorated with large unyielding leukocytes. These repetitive episodes of localized ischemia and reperfusion can set up a low-grade chronic inflammatory tissue-injuring state. The authors support the concept of chronic inflammation by pointing out the abnormally high base-line leukocyte count in the sickle mouse, an abnormality that is also found in humans with sickle cell disease (3). Even more impressive is the increasing number of epidemiological studies that implicate base-line leukocyte count as a major risk factor for severity in sickle cell anemia. Despite the fact that all individuals with sickle cell anemia have the identical globin gene mutation, there is a wide range of clinical severity. For example, the most common clinical manifestation of the disease, the pain crisis, varies tremendously among individuals, with rates ranging from 0 per year to more than 10 per year (4). It was no surprise to discover that some of the typical clinical complications of sickle cell anemia were less common among those individuals with higher levels of fetal hemoglobin (4–6). What has been surprising is the emerging evidence that the base-line leukocyte count is also a very important independent risk factor for disease severity. Patients with high base-line leukocyte counts are more likely to die at a younger age (5). Such patients are also more susceptible to the acute chest syndrome, an effect that is of similar magnitude to that of fetal hemoglobin over the range of fetal hemoglobin and leukocyte counts in the population (6). Base-line leukocyte counts are also risk factors for the development of silent brain infarcts (7). Most recently, the base-line leukocyte count was shown to be a strong predictor of which infants with sickle cell anemia will develop clinically severe disease (8). In the context of these epidemiological studies, it is impossible to determine whether the base-line leukocyte count simply reflects ongoing base-line chronic inflammation, or whether it indicates the propensity to develop an inflammatory response to a given stimulus. The observation that base-line leukocyte count has a heritability estimate of 0.62 in the general population (9) suggests that a genetic modulator of inflammatory response may be related to the clinical variability of sickle cell anemia. Exploring the role of inflammation in the pathophysiology of sickle cell anemia is of critical importance as we search for new therapeutic approaches. The paper by Kaul and Hebbel (1) sets the stage for therapies designed to interfere with P-selectin in order to decrease leukocyte-endothelial interactions. This is just the beginning.

Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.

Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy.

Pulmonary hypertension and nitric oxide depletion in sickle cell disease.

During the past decade a large body of experimental and clinical studies has focused on the hypothesis that nitric oxide (NO) depletion by plasma hemoglobin in the microcirculation plays a central role in the pathogenesis of many manifestations of sickle cell disease (SCD), particularly pulmonary hypertension. We have carefully examined those studies and believe that the conclusions drawn from them are not adequately supported by the data. We agree that NO depletion may well play a role in the pathophysiology of other hemolytic states such as paroxysmal nocturnal hemoglobinuria, in which plasma hemoglobin concentrations are often at least an order of magnitude greater than in SCD. Accordingly, we conclude that clinical trials in SCD designed to increase the bioavailability of NO or association studies in which SCD clinical manifestations are related to plasma hemoglobin via its surrogates should be viewed with caution.

Influence of sickle hemoglobinopathies on growth and development.

To determine the influence of hemoglobinopathy on growth and development, we examined the height, weight, and sexual maturation of 2115 patients 2 to 25 years old who had homozygous sickle-cell disease (SS), SC disease (SC), sickle beta+ thalassemia (S beta+), or sickle beta O thalassemia (S beta O). Using regression analysis of these cross-sectional data to generate growth and maturation curves for each hemoglobinopathy, we found that the curves for all hemoglobinopathy groups were significantly different from published norms for black subjects (P less than 0.001), and that subjects with SS and S beta O were consistently smaller and less sexually developed than those with SC and S beta+ (P less than 0.001). For both sexes and all hemoglobinopathies, low weight was more pronounced than short height and was most apparent in subjects over the age of seven. The median age of the female subjects who had attained at least Tanner Stage V was 17.3 years for those with SS, 17.2 years for S beta O, 16.0 years for SC, and 16.5 years for S beta+; among male subjects the corresponding values were 17.6, 18.8, 16.6, and 16.6 years. Discriminant analysis of menarche status, weight, age, and hemoglobinopathy revealed that the influences of age and weight on menarche were similar regardless of hemoglobinopathy. This relationship suggests a constitutional rather than a primary endocrinologic cause of sexual immaturity in patients with hemoglobinopathies.

Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease.

Intracellular polymerization and sickling depend markedly on the cellular concentration of sickle hemoglobin (Hb S). A possible therapeutic strategy for sickle cell disease is based on reducing the cellular concentration of Hb S through prevention of erythrocyte dehydration. The K-Cl cotransporter is a major determinant of sickle cell dehydration and is inhibited by increasing erythrocyte Mg content. We studied 10 patients with sickle cell disease before treatment and after 2 and 4 wk of treatment with oral Mg supplements (0.6 meq/kg/d Mg pidolate). Hematological parameters, erythrocyte Na, K, and Mg content, erythrocyte density, membrane transport of Na and K, and osmotic gradient ektacytometry were measured. We found significant increases in sickle erythrocyte Mg and K content and reduction in the number of dense sickle erythrocytes. Erythrocyte K-Cl cotransport was reduced significantly. We also observed a significant reduction in the absolute reticulocyte count and in the number of immature reticulocytes. Ektacytometric analysis showed changes indicative of improved hydration of the erythrocytes. There were no laboratory or clinical signs of hypermagnesemia. Mild, transient diarrhea was the only reported side effect. We conclude that oral Mg supplementation reduces the number of dense erythrocytes and improves the erythrocyte membrane transport abnormalities of patients with sickle cell disease.

The Acute Chest Syndrome of Sickle Cell Disease

It has become a cliche to say that sickle cell anemia is the first “molecular disease.”1 The substitution of valine for glycine in β-globin has been known as the cause of sickle cell anemia since 1...