Jamaican Sbetañthalassaemia: mutations and haematology

The sickling disorders are a common cause of morbidity and mortality in Jamaica. Sickle cell betañthalassaemia is the fourth commonest form, occuring in one in every 3000 births. This is a heterogeneous condition, producing HbS, HbF and HbA2 with variable amounts of HbA, depending on the mutation and, within a defined population, only a few beta-thalassaemia mutations occur at high frequency. This study establishes the frequency of beta-thalassaemia mutations in Sbetañthalassaemia patients in Jamaica. In addition, comparison of the haematological phenotypes is possible by looking at the "average steady-state haematology" of the different mutational groups. Blood samples from 132 unrelated Sbetañthalassaemia patients attending the MRC Sickle Cell Unit at the University of the West Indies were analysed by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) or sequencing to determine the nature and frequencies of the underlying beta-thalassaemia mutations. Ten mutations were identified, four of which accounted for 93 percent of the patients studied. These were 29 (A --> G) in 71 (54 percent), -88 (C --> T) in 27 (20 percent), polyA (T --> C) in 17 (13 percent) and IVS1-5 (G --> C) in nine (7 percent). The six remaining mutations found at lower frequency were C24 (T --> A) in two patients and one each of IVS2-848 (C --> A), -90 (C --> T), IVS1-5 (G --> T),IVS1-6 (T --> C). In one individual, no mutation was found. The three commonest mutations were all associated with levels of greater than 10 g/dl, whereas IVS1-5 (G --> C) had a more severe haematological phenotype. The predominance of -29 (A --> G) and -88 (C --> T) is in keeping with other studies on populations of African origin. IVS1-5 (G --> C) is found chiefly in Indian populations, and all affected families acknowledged Indian ancestry, reflecting the prominent Indian community in Jamaica. (AU)

The molecular basis for genetic variation in fetal haemoglobin level in sickle cell anaemia

Raised fetal haemoglobin levels ameliorate the clinical severity of sickle cell anaemia. This provides a rationale for therapy and signals the need to elucidate the molecular basis for the variability of HbF level in sickle cell anaemia. Polymorphism within regulatory sites of the globin locus alter the affinity with which transcription factors bind their cogante recognition sites thereby modulating gene expression. A novel chromosomal haplotype utilising polymorphic variation within two enhancers hypersensitive site 2 (HS2) of the locus control region and the pre g y framework and the silencer protein BP1 binding site that spans a 53 kb interval of the globin locus was determined in 205 patients with sickle cell anaemia from the UK and Jamaica. Multiplexed polymerase chain reactions developed to facilitate rapid analysis of polymorphisms within each site allowed individual haplotype construction in a single lane of a single strand conformation polymorphism (SSCP) electrophoresis gel. SSCP banding patterns for the combined polymorphic sites were confirmed as unique chromosomal haplotypes by DNA sequence anaylsis. Three hundred and ten chromosomes with sequence TA7 N 12 TA8, GA and AT(AT)8T4 were designated class 1. Twenty-five class II with sequence TA8 N10 TA11, GG, AC(AT)6T9; 17 class III with TA9 N10 TA10, AG, AC(AT)8T4; 7 class IV with TA10 N10 TA12, AG, AC(AT)9T5 and 13 class Ia haplotype with sequence TA9 N10 TA10, GA, AT(AT)8T4 were identified. The proportion of class I chromosomes in both groups (159/210 UK; 151/200 Jamaican) is identical, however, significantly more chromosomes with sub-classes I and II are present among the Jamaican sample. There is incomplete association between the functional haplotype classes defined and conventional haplotypes based on restriction fragment length polymorphisms. The level of Hbf is significantly higher in patients with functional haplotype classes III and IV compared to those with classes I and II. Both high HbF haplotypes share a high affinity binding motif for the transcription factor GATA-1. This novel approach allows the combined effets of genetic variation in regulatory sequences within the globin locus on HbF level to be defined. (AU)

Benign clinical course in homozygous sickle cell disease: a search for predictors

AIMS: (1) To estimate the proportion of subjects with homozygous sickle cell disease who have a benign clinical course, and (2) to assess factors that may be predictive of benign disease. MATERIAL: Subjects (n = 280) were participants in a longitudinal cohort study of sickle cell disease. They were classified as benign or control based on clinical history from birth to age 13 years old. Associations with growth, hematology, and an index of social status were investigated. RESULTS: Benign disease occurred in 43 (15 percent) patients. Neither growth nor social status were related to benign disease. There were only two statistically independent association: alpha thalassemia status and average steady state fetal hemoglobin (HbF). Patients with a normal complement of alpha globin genes were 2.2 (1.0, 4.9) times more likely to have frequent painful crises, dactylitis, and bone necrosis. The odds of having benign disease were 1.09 (1.02, 1.17) times higher for each unit increase in HbF, and 44 percent of subjects with HbF in the top decile (HbF > 13.8 percent) of the distribution had benign disease. There was no evidence for a threshold effect of high HbF on benign disease. CONCLUSION: A benign clinical course of sickle cell disease may occur in Jamaica and is associated with a normal alpha globin gene complement, and high levels of HbF. Ability to predict benign disease at birth is limited.(AU)

Factors affecting prepubertal growth in homozygous sickle cell disease

OBJECTIVE: To investigate the role of hematological indices, socioeconomic status, and morbidity in prepubertal growth in homozygous sickle cell (SS) disease from birth to 9 years at the sickle cell clinic of the University Hospital of the West Indies, Kingston, Jamaica. RESULTS: Height increment between 3 and 9 years correlated positively with total haemoglobin at age 7 years in boys but not girls. Attained height and weight at 7 years correlated positively with haemoglobin and fetal haemoglobin in boys but not girls. Only the correlation between haemoglobin and weight showed a significant gender difference. Partial correlation analysis suggested that the effect of haemoglobin was accounted for by the effect of fetal haemoglobin and further analysis indicated that height correlated with F reticulocyte count (a measure of fetal haemoglobin production) in both sexes but not with the ratio of F cells to F reticulocytes (a measure of F cell enrichment). Growth was not significantly related to mean red cell volume, proportional reticulocyte count, alpha thalassaemia, socioeconomic status, or morbidity. CONCLUSION: A high concentration of fetal haemoglobin in boys with SS disease is associated with greater linear growth. It is postulated that in boys, low concentrations of fetal haemoglobin increase haemolysis and hence metabolic requirements for erythropoiesis, putting them at greater risk of poor growth. Differences in the relationship to haemotoloy and growth between boys and girls with SS disease dictate that future analyses of growth take gender into account (AU).x

An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the x-linked factor, á-Globin haplotypes, O-globin gene number, gender, and age

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the O-globin gene number, á-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), i.e., the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a cohort group identified by newborn screening and from a sib pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < 0.05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40 percent of Hb F variation. á-Globin haplotypes, O-globin genes, and age accounted for less than 10 percent of the variation. The association between the á-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common á-globin haplotypes and other as yet unidentified factor(s).(AU)

Haematological risk factors for pregnancy outcome in Jamaican women with homozygous sickle cell disease

OBJECTIVE; To examine the association between fetal outcome and the steady state haematology of mothers with homozygous sickle cell disease. DESIGN; A retrospective observational study. The data were taken from the dockets, kept at the Sickle Cell Clinic at the University Hospital of the West Indies or two peripheral clinics operated by the staff of the MRC Laboratories. SUBJECTS; All women aged 14 years or older with homozygous sickle cell disease who had experienced at least one pregnancy in the period 1977 to 1986. MAIN OUTCOME MEASURES; Three fetal outcomes including miscarriages, perinatal deaths, and birthweight. RESULTS; There were 270 singleton pregnancies in 175 women with an overall fetal wastage of 32.2 percent. There was a significant increased risk of perinatal death with low maternal fetal haemoglobin level, but there was no haematological associations with miscarriages or birthweight. CONCLUSIONS: These data suggest that maternal steady-state haemoglobin has little influence on fetal outcome, with the exception that mothers with high HbF levels are less prone to perinatal deaths. Further study is required to investigate acute haematological changes associated with pregnancy. (AU)

Screening for opthalmic manifestations of sickle cell disease in the United Kingdom

There are marked variations in the manifestations of sickle cell disease in different populations. The occular complications of this condition amongst the Afro-Caribbeans living in the United Kingdom have not previously been reported. We present the preliminary results of an opthalmic screening programme at King's College Hospital, London. One hundred eyes of 50 patients with sickle cell disease were assessed. Full ocular examination was performed including fundus fluorescein angiography. We have looked at the haemotological and clinical profile of the patients involved as well as the number of days spent in the hospital during the year preceding the examination. The incidence of Grade 11 retinopathy was found to be significantly higher than grade 1 in SC disease. This concurs with the results of the Jamaican screening and confirms that the patients are at higher risk of visual impairment than those wuth the SS disease. Our results also agrees with the Jamaican experience which suggests that visual morbidity is mostly due to complications of proliferative sickle cell retinopathy (PSR). However, the findings in patients without proliferative changes are different; in particular, angloid streaks leading to disciforms are an important cause of visual loss in Jamaica, but were not seen in any of the 98 eyes examined in this study. No correlation was found between the grade of retinopathy and age, sex, systemic complications and various haematological parameters except for the percentage of haemoglobin F, which was significantly higher in patients with grade I (7.6) compared with grade II (4.2) retinopathy (p=0.0127)(AU)

Resting metabolic rate in homozygous sickle cell disease

The resting metabolic rate in 20 patients with homozygous sickle cell (SS) disease was 19 percent higher than in 20 age-and sex-matched control subjects with a normal hemoglobin genotype (AA). The difference was not accounted for by differences in lean body mass. It is postulated that this increased energy expenditure reflects the energy expenditure of erythropoietic hyperplasia and leads to a marginal nutritional state that may contribute to the abnormal growth in SS disease. (AU)

Resting metabolic rate in homozygous sickle-cell disease - abstract

Erythropoietic hyperplasia and a hyperdynamic circulation might be expected to increase the resting metabolic rate (RMR) of patients with homozygous sickle-cell (SS) disease. Therefore we used the ventilated hood technique to determine the RMRs of the representative sample of adolescents with SS disease and controls with a normal haemoglobin (AA) genotype matched for age, sex, and pubertal stage. The RMR in 19 pairs was corrected for the lean body mass which was derived from the measurement of total body water. The RMR of SS subjects expressed as kcal/kg LBM was greater than that of AA controls (median, range: 38.6, 30.7 - 45.4 and 33.2, 25.4 - 35.7, respectively, z = 3.1, p = 0.002). We postulate that the increased RMR in SS disease is not compensated for by a reduction in physical activity or increased energy intake and that this leads to a marginal nutritional state which may contribute to the abnormal growth of children with SS disease(AU)

Serum transferrin receptor levels in homozygous sickle-cell disease - abstract

Transferrin receptors are used by rapidly dividing tissues such as bone marrow for the extraction of iron necessary for their metabolic requirements. As cells in the erythropoietic series mature, transferrin receptors are cleaved from the cell surface and may be measured free in the plasma. Serum transferrin receptors (STR) levels are therfore believed to reflect the extent of erythropoiesis and are elevated in anaemias associated with iron deficiency, haemolysis and thalassaemia and decreased in aplastic anaemia. Levels have been measured in stored sera from 182 children with homozygous sickle-cell (SS) disease and 42 controls with a normal haemoglobin (AA) genotype age 8 years (ñ 3 months) in a cohort study from birth. Levels is SS children (mean, SD: 38.3, 12.7) greatly exceeded those in AA controls (6.7, 1.9) (test = 32.1, p0.001) and were negatively correlated with total haemoglobin and foetal haemoglobin levels in both sexes but not with reticulocyte counts. The serum transferrin receptor level may be a useful indicator of the degree of eryreticulocyte counts. The serum transferrin receptor level may be a useful indicator of the degree of erythropoietic expansion in SS disease, and requires further study to determine its clinical value (AU)

Comparison of homozygous sickle cell disease in Northern Greece

The clinical and hematological features of homozygous sickle cell (SS) disease were compared in 30 Greek and 310 Jamaican patients. Deletional O-thallassaemia, which modifies SS disease, is rare among Greek patients, so only Jamaican patients with four O-globin genes were included in the control group. Greek patients had higher total haemoglobin concentration and red cell counts, and lower mean cell haemoglobin concentration (MCHC) and reticulocyte counts. They also had a more normal body build and more adults had persistent splenomegaly. Fewer had a history of leg ulceration or priapism but more reported acute chest syndrome. The comparitively mild disease in Greek patients is consistent with less haemolysis and sickling and therefore less bone marrow expansion. In the absence of amelioriating factors such as high HbF concentration or O-thalassaemia, these findings may be explained by the low MCHC. (AU)

Sickle cell retinopathy in Jamaican children: further observations from a cohort study

Serial retinal examinations were performed in children aged 5 years and older and fluorescein angiography/angioscopy in children 6 years and older participating in a cohort study of sickle cell disease. There were 1229 patient years of observation among 389 children aged 5 - 13 years. Peripheral retinal vessel closure was present in approximately 50 percent of children with SS and SC genotypes at age 6 years and increased to affect 90 percent of children by age 12 years. A matched pair analysis, comparing groups with minimal and complete closure, indicated that complete closure was associated with significantly lower total haemoglobin and fetal haemoglobin levels and significantly lower weight in SS disease, whereas in SC disease the risk factors appeared to be high mean cell volume and low platelet count. Proliferative retinopathy was rare, occurring only once in an 8-year-old boy with SC disease, despite 592 patient years of observation in children over this age. (AU)

Fetal hemoglobin levels and beta s globin haplotypes in an Indian population with sickle cell disease

To further explore the cause for variation in hemoglobin F (HbF) levels in sickle cell disease, the á globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126SS, 141AS, 17Sá§, 7Aá§, and 12AA) Indians from the state of Orissa. The ás globin gene was found to be linked almost exclusively to a ás haplotype (+++-++-), which is also common in Saudi Arabian patients from the Eastern province (referred to as the Asian ás haplotype). By contrast, the majority of áA and ᧠thalassemia globin genes are linked to hoplotypes common in all European and Asian populations (+-----[+/-];--++-++). Family studies showed that there is a genetic factor elevating HbF levels dominantly in homozygotes (SS). This factor appears to be related to the Asian ás globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary presistance of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.(AU)

Sickle cell disease in Orissa State, India

A study of 131 patients with homozygous sickle cell (SS) disease in Orissa State, India, indicated that, compared with Jamaican patients, Indian patients have higher frequencies of alpha thalassaemia, higher fetal haemoglobin, total haemoglobin, and red cell counts, and lower mean cell volume, mean cell haemoglobin concentration, and reticulocyte counts. Indian patients have a greater frequency and later peak incidence of splenomegaly, and hypersplenism is common. Painful crises and dactylitis are not uncommon in Indian patients but chronic leg ulceration is rare. Homozygous sickle cell disease in Orissa is similar to that in the Eastern Province of Saudi Arabia and is very different from that in populations of West Africa origin.(Summary)

Alpha thalassemia and the hematology of homozygous sickle cell disease in childhood

Alpha thalassemia modifies the gematolic expression of homozygous sickle cell (SS) disease, resulting in increased total hemoglobin and HbA2 and decreased HbF, mean cell volume, reticulocytes, irreversibly sickled cells, and biliru-bin levels. The age at which these changes develop in children with SS disease is unknown. Ascertainment of globin gene status in a large representative sample of study the gematologic indices in nine children homozygous for Alpha thalassemia 2 (two-gene group), 90 children heterozygous for Alpha thalassemia 2 (three-gene group), and 167 children with a normal Alpha globin gene complement (four-gene group). The two-gene group had significantly lower mean cell volumes from birth, higher red cell counts from one month, lower reticulocytes from three months, and higher HbA2 levels from one year, as compared with the four-gene group. Children with three genes had intermediate indices but resembled more closely the four-gene group. Differences in total hemoglobin or in fetal hemoglobin between the groups were not apparent by eight years of age. The most characteristic differences of the two-gene group were the raised proportional HbA2 level and low mean cell volume, the latter having some predictive value for Alpha thalassemia status at birth.(AU)

A genetic marker for elevated levels of haemoglobin F in homozygous sickle cell disease

Ten patients with sickle cell (SS) disease from a Jamaican family were found to have unusually high levels of haemoglobin F for this population. Each of them has inherited one sickle cell gene on a chromosome characterized by an arrangement of restriction fragment length polymorphisms (haplotype) which is very rare in the Jamaican population. Genetic analysis of the family suggests that there is a determinant linked to the á-globin gene cluster, charaterized by this haplotype, which is responsible for increased haemoglobin F production in response to anaemia. Interestingly this particular haplotype appears to be common in patients with SS disease in eastern Saudi Arabia in whom a high level of haemoglobin F is the rule rather than the exception. Hence it is possible that this haplotype (++-++) acts as a genetic marker for elevated levels of haemoglobin F in sickle cell disease (AU)

Clinical presentation of homozygous sickle cell disease

The pattern of initial clinical symptoms and signs developing in a representative sample of 305 children with homozygous sickle cell (SS) disease diagnosed at birth was analyzed. Specific symptoms were present by age 6 months in 6 percent of the group, and had developed by the first to eighth birthdays in 32 percent, 61 percent, 78 percent, 86 percent, 90 percent, 92 percent, 94 percent and 96 percent, respectively. Inclusion of nonspeccific symptoms in the analyst led to earlier recognition by a mean of 3 months in the first year and by a mean of approximately 1 year between the ages of 2 and 4 years. Dactylitis was the most common initial symptom, noted in 40 percent of the group overall and in 50 percent in the first 2 years. Painful crisis was the first symptom in more than one fourth of the patients and was the most frequent symptom after the age of 2 years. Acute splenic sequestration led to presentation in one-fifth of the group overall and in one third of the patients younger than 2 years. The most common nonspecific symptom was pneumonia. There was a significant trend of earlier presentation in children with low fetal hemoglobin levels. The age of presentation did not appear to be affected by O-thalassemia status. (AU)

Alpha-Thalassemia changes erythrocyte heterogeniety in sickle cell disease

Homozygous alpha thalassemia has the beneficial effect in sickle cell anaemia of reducing the hemolytic severity while changing several other hematological parameters. We examined in detail some of these hematological alterations. We find that the broad distribution in erythrocyte density and the large proportion of dense cells associated with sickle cell anaemia are both reduced with co-existing alpha-thalassemia. Measurements of glycosylated hemoglobin levels as a function of cell density indicate that the accelerated increase in cell density, beyond normal cell ageing, in sickle cell anaemia is also reduced with alpha thalassemia. The patients with homozygous alpha-thalassemia and sickle cell disease have slightly lower levels of hemoglobin F than non-thalassemic patients. Examination of hemoglobin F production revealed that the proportion of hemoglobin F containing reticulocytes remained unchanged, as did the proportion of hemoglobin F in cells containing hemoglobin F (F cells). Preferential survival of F cells occurs in sickle cell anaemia, with or without alpha-thalassemia, and the slight difference in hemoglobin F levels appear to reflect differences in numbers of circulating F cells. Thus in sickle cell disease with co-existing alpha-thalassemia, the change in the erythrocyte density profile, possibly due to inhibition of polymerisation-related increases in cell density, explains the hematological improvement.(Summary)

The haematology of steady state homozygous sickle cell disease: frequency distibutions, variation with age and sex, longitudinal observations

The steady state haematological characteristics observed in 1071 patients with homozygous sickle cell (SS) disease aged 5-66 years are presented (Summary)

Production of F cells in sickle cell anemia: regulation by a genetic locus or loci separate from the beta-globin gene cluster

Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2 percent to 50 percent in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the á- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from ás. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9 percent confidence limits or were alike by t test (Po .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always ó .005 and usually ó 10 to the 4th power. We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of á-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the á-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to á to the s power, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one (AU)