Beta-globin Haplotypes Research Articles

Beta-Globin Gene Haplotypes in Manipur, North-east India

A sample of 204 individuals belonging to five different population units of Manipur, viz. the Meitei, Gangte, Thadou, other Kuki, and Kabui has been analyzed for beta globin gene cluster, considering seven loci to bring out a first hand information on haplotype frequencies of beta globin in the state. A common trend of allelic frequencies observed in the present data are the high frequencies of HindIIe, Avallβ and Hinflβ and relatively low frequencies of HindlllGγ, HindIIIAγ, HindII5'ψ β and HindII3'ψβ. The frequency of Framework 1(presence of both AvaIIβ and HinfIβ sites) ranges from nil to 17.65% in the present data of Manipur. The frequency of Framework 2 ranges from 33% to 66% and that of Framework 3 from 33% to 55%. One noteworthy observation is the association of HbβEE exclusively with Framework 2, which suggests a similar mutational origin of HbE in South-east Asia and North-east India. Only five beta globin haplotypes are found in the present data. Haplotypes +——+ and +——+-, the most common in Japanese, Cambodians, Koreans, South Chinese, Samoans and Thais, are the most frequent haplotypes in Manipur too.

High Mortality Among Children with Sickle Cell Anemia and Stroke Who Discontinue Chronic Blood Transfusion After Transition to An Adult Program

AbstractAbstract 1629 Introduction:The Comprehensive Sickle Cell Disease study (CSCD) of 4082 patients showed that the likelihood of developing stroke was 11% by the age of 20 years for patients with sickle cell anemia (SS).The stroke prevention trials (STOP I and STOP II) in SS showed that blood transfusion prevents the occurrence of primary stroke in children with abnormal TransCranial Doppler (TCD) velocities (STOP1) and discontinuation of transfusions after 30 months resulted in a high rate of reversion to abnormal TCD velocities and stroke (STOP II). To that end all children with abnormal TCD velocities are managed with chronic blood transfusion without interruption as long as they are in pediatrics. The problem arises when they are due to be transferred to adult care after the age of 18 years. The transition process from pediatrics to adult programs is marred with several issues that often result in interruption or discontinuation of the quality care the patients had as children. There is little or no information in the literature about the outcome of children with stroke after transition to adult programs. In this study we report the outcome of 22 patients, 10 males and 12 females, with SS and stroke who were referred to our adult program from several Children's hospitals between 1993 and 2009. Patients & Methods:Records were kept prospectively. The mean age of patients at transition was 22.4 ± 3.10 years. Blood bank data were performed and computerized according to FDA and AABB regulations. Blood counts and % Hb S were done before and after each transfusion. Hb S was kept below 30% or 50% after transfusion. Metabolic profiles were done every 6 months or more often if needed. Molecular diagnostics including alpha genotypes and beta globin haplotypes were done by described methods. Statistical analysis was by the 2-tailed t test. Results:All patients had ischemic strokes during childhood and one had hemorrhagic stroke superimposed on cerebral infarction. Two patients developed moyamoya after transition. No alpha gene deletion was detected in 18 patients, 4 had one alpha gene deletion and none had 2 alpha gene deletion. The Ben/CAR haplotype was found in 7 patients, Ben/Ben in 5, Ben/Sen in 4 and other combinations in 6 patients; 21 patients were heterozygous for the Ben haplotype. Hb F was 1.0–7.0% except for one patient on hydroxyurea (HU) with 20% Hb F. All patients were kept on the same regimen of transfusion they had as children; 15 were kept on exchange transfusion two of whom were non-compliant, two were kept on simple transfusion plus HU and the remaining 5 patients refused to have chronic blood transfusion and were kept on normal care. Some of the causes for refusal were due to alloimmunization and difficult venous access. One of these 5 patients agreed to be on HU. Patients on blood exchange required an average of 55 ± 13.5 (40-98) units of RBC annually. Those on simple transfusion required up to 24 units annually. Alloimmunization was present in 10 patients with the number of alloantibodies between 1 and 6 per patient. The most common alloantibodies were ant-E and anti-K. Patients who were compliant with blood transfusion were rarely hospitalized for painful crises. Patients who were noncompliant with blood transfusion or who refused to be maintained on chronic transfusion were hospitalized for painful crises 5.7 ± 5.00 times annually (p<.025). All 5 patients who refused to be maintained on chronic blood transfusion died within 3 years after transition. Cause of death was massive intraventricular hemorrhage in one patient proven by autopsy, massive hemispheric infarct proven by imaging in the second patient leading to coma and death in the hospital and sudden death during hospitalization for painful crises in the remaining 3 patients. Two patients who were not compliant with chronic blood transfusion died within 2 years after transition due to ARDS. Thus the overall rate of death after transition was 32% and the major cause was discontinuation of chronic blood transfusion. The mean age at death was 27.0 ± 3.16 years whereas the mean age of patients who are alive is 35.4 ± 5.92 years (p<.001). Conclusion:Our data indicate that about one third of patients with SS and stroke die within 3 years after transition especially if chronic blood transfusion is discontinued or if compliance was poor. It is imperative that all efforts should be made to maintain adequate chronic blood transfusion for children with SCD and stroke after transition to adult care. Disclosures:No relevant conflicts of interest to declare.

Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their betaS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C-->T in the HBG1 promoter region were associated with the Central African Republic betaS-globin haplotype. In contrast, the -369 C-->G and 309 A-->G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

The changing face of homozygous sickle cell disease: 102 patients over 60 years

Earlier reports on homozygous sickle cell (SS) disease have been biased by severely affected cases. The Jamaican clinic which seeks to avoid such bias has 102 patients surviving beyond 60 years. The objective of this study was to examine the features of elderly cases and assess factors determining survival and the behaviour of this disease with advancing age. A retrospective review of all cases and prospective assessment in survivors was conducted at The Sickle Cell Clinic at the University of the West Indies, Kingston, Jamaica previously operated by the MRC Laboratories. All patients with SS disease born prior to December 31, 1943 who would, by January 2004, have passed their 60th birthday were traced and their current status ascertained. The molecular and clinical features were assessed and observations on the clinical behaviour of the disease and of haematology and biochemistry are presented. Of the 102 patients, 58 had died, four had emigrated and 40 were alive, resident in Jamaica and aged 60-87 years. Survival was associated with female gender and higher foetal haemoglobin but not with alpha-thalassaemia or beta-globin haplotype. A tendency to familial clustering among elderly survivors did not reach statistical significance. Painful crises ameliorated with age and there was a benign course in pregnancy. Mean haemoglobin levels fell with age and were generally associated with rising creatinine levels indicating the importance of renal failure. Elderly survivors present some features of intrinsic mildness but also manifest age-related amelioration of painful crises and falling haemoglobin levels from progressive renal damage.

Analysis of murine S-glutathionyl hemoglobins and beta globin haplotype by dynamic capillary isoelectric focusing

Genetic variation in the number and reactivity of beta globin sulfhydryl groups causes variation in erythrocyte redox status in mouse populations. These experiments demonstrate the use of capillary isoelectric focusing for measuring endogenous S-glutathionyl hemoglobin and identifying mouse beta globin (Hbb) haplotype in inbred and outbred mouse strains with mono-cysteinyl or di-cysteinyl beta globins. Hbb haplotype can be readily determined in all strains based on characteristic differences in peak profiles or on peak mobility shift induced by thiol exchange with glutathione disulfide in vitro. This method could prove useful for in vivo study of factors that influence thiol protein modification.

Risk Factors for Silent Cerebral Infarcts in a Pediatric Sickle Cell Anemia (SCA) Cohort

Background Silent infarcts are associated with impaired cognitive functioning and have been shown to be predictors of stroke (Miller ST J Pediatr 2001). Until now, reported risk factors for silent infarcts were low pain event rate, history of seizures, high leukocyte count and Sen bS haplotype (Kinney TR Pediatrics 1999). Here, we seek to define the prevalence and risk factors of silent infarcts in the Créteil SCA pediatric cohort comprising patients assessed at least yearly by transcranial doppler (TCD) since 1992, and by MRI/MRA.Methods This study retrospectively analyzed data from the Créteil cohort stroke-free SS/Sb0 children (280; 134 F, 146 M), according to institutional review board. Time-averaged mean of maximum velocities higher than 200 cm/sec were considered as abnormal, resulting in initiation of a transfusion program (TP). A switch to hydroxyurea was proposed to patients with normalized velocities (< 170 cm/sec) and normal MRA on TP, although TP was re-initiated in case of abnormal velocities recurrence. Patients with “conditional” velocities (170–199 cm/sec) were assessed by TCD 4 times yearly. Alpha genes and beta-globin haplotypes were determined. Baseline biological parameters (G6PD activity; WBC, PMN, Reticulocytes, Platelets counts; Hemoglobin, Hematocrit, HbF, LDH levels; MCV; SpO2) were obtained a minimum of 3 months away from a transfusion, one month from a painful episode, after 12 months of age, before the first TCD, and always before therapy intensification.Results. Patients were followed for a total of 2139 patient-years. Alpha-Thal was present in 114/254 patients (45%) and 27/241 (11.2%) had G6PD deficiency. Beta genotype, available in 240 patients, was BaBa in 102 (42.5%), BeBe in 54 (22.5%), SeSe in 19 (7.9%) and “other” in 65 (27.1%); TCD was abnormal in 52 of 280 patients (18.6%). MRA showed stenoses in 30 of 226 evaluated patients (13.3%) while MRI demonstrated presence of silent infarcts in 81/280 patients (28.9%). Abnormal TCD (p<0.001), G6PD deficiency (p=0.008), high LDH (p=0.03), and low Hb (p=0.026) were significant risk factors for stenoses by univariate analysis while multivariate analysis retained only abnormal TCD as a significant risk factor for stenoses ([OR= 10.6, 95% CI (4.6–24.4)]; p<0.001). Univariate logistic regression analysis showed that the risk of silent infarcts was not related to alpha-Thal, beta genotype, abnormal TCD, WBC, PMN, platelets, reticulocyte counts, MCV, LDH level, HbF %, pain or ACS rates but was significantly associated with stenoses detected by MRA (p<0.001), gender (male; p=0.04), G6PD deficiency (p=0.05), low Hb (p=0.016) and Hct (p=0.012). Multivariate logistic regression analysis showed that gender ([OR= 2.1, 95% CI (1.03–4.27)]; p=0.042), low Hb ([OR= 1.4, 95% CI (1.0–1.1)]; p=0.05) and stenoses ([OR= 4.8, 95% CI (1.88–12.28)]; p=0.001) were all significant independent risk factors for silent infarcts. The presence of stenoses was the only significant risk factor for silent infarcts in patients with a history of abnormal TCD ([OR= 5.9, 95% CI (1.6–21.7)]; p=0.008).Conclusion We recently showed that G6PD deficiency, absence of alpha-Thal, and hemolysis are independent significant risk factors for abnormal TCD in stroke-free SCA patients (Bernaudin et al, Blood, 2008, in press). Here, we report that an abnormal TCD is the most significant risk factor for stenoses and, expanding previous studies, we demonstrate that stenoses, low Hb and gender are significant independent risk factors for silent infarcts.

Cerebral Vasculopathy Outcome in a Sickle Cell Anemia (SCA) Newborn Cohort Screened Early with Transcranial Doppler

Background: Stroke is the most severe complication in children with SCA and has been reported to concern 11% of patients by the age of 18 years. Transcranial Doppler detects patients at risk for stroke and transfusion program significantly reduces the risk of stroke in patients with abnormal TCD (Adams, NEJM 1998, 2005). However, the estimate of overt and silent strokes occurrence in a newborn SCA cohort explored early with TCD has not yet been reported.Methods: In our Créteil pediatric center, TCD was systematically performed annually since 1992 as soon as 12–18 months of age and patients were assessed by MRI/MRA every 2 years after the age of 5 years or earlier in case of abnormal TCD. Time-averaged mean of maximum velocities higher than 200 cm/sec were considered as abnormal, resulting in initiation of a transfusion program (TP). A switch to hydroxyurea was proposed to patients with normalized velocities on TP (< 170 cm/sec) and normal MRA, but TP was reinitiated if abnormal velocities recurred. Patients with “conditional velocities” (170–199 cm/sec) were assessed with TCD every three months. Alpha genes and beta-globin haplotypes were determined. Baseline biological parameters (G6PD activity; WBC, PMN, Reticulocytes, Platelets counts; Hemoglobin, Hematocrit, HbF%; LDH levels; MCV; SpO2) were obtained after 12 months of age, before the first TCD, a minimum of 3 months away from a transfusion, one month from a painful episode, and always before intensive therapy. One patient refused initiation of TP for abnormal TCD and was excluded from the analysis.Results: This study included 217 SS/Sb0 children (114 M, 103 F), annually explored by TCD before age 5 (mean age at first TCD±SD: 2.2±1.1yr). They were followed for a total of 1627 patient-years. Eighty-two of 187 patients had alpha-Thal (44%); beta genotype, available in 157, was BaBa in 67 (43%), BeBe in 36 (23%), SeSe in 11 (7%) and “other” in 43 (27%); 21 of 182 (11.5%) had G6PD deficiency. TCD became “conditional” in 57 of 217 (26.3%) at the median age of 3.3 yr (range 1.2–8), and abnormal in 45 of 217 patients (20.7%) at the median age of 3.2 yr (range 1.3–8.3). Three patients had a stroke at the ages of 1.6, 4.2 and 4.4 yr: the first one had a first abnormal TCD at the age of 1.5 but had a stroke just before the TCD control at 1.6 yr; the second one had normal TCD on one side but unaivalable window on the other side and had a stroke before the first MRI/MRA; the third one had normal TCD just before the occurrence of an ACS and had a massive thrombotic stroke in intensive unit. The Kaplan-Meier (KM) estimate of stroke occurrence was 1.9% by the age of 18 yr. MRA, available in 126, was normal in 104 and showed stenoses in 22 (17.5%) at the median age of 4.8 yr (range 2.6–11.5). In stroke-free patients, MRI showed silent infarcts in 31 of 122 patients (25.4%) at the median age of 5.9 yr (range 3.5–15.3); 10 of the 31 had a history of abnormal TCD. KM estimate for abnormal TCD was 29.7% by the ages of 10 and 18 yr. KM was higher in case of G6PD deficiency: 52% vs. 26% (Log Rank, p=0.059), significantly lower in case of alpha-Thal: 16.3% vs. 36.9% (Log Rank p=0.005) and significantly dependent on the degree of hemolysis: LDH IU/l <750:11.6%; 750–1209:27%; >1209: 40.3% (Log-rank < 0.026). KM estimate of stenoses on MRA was 23.6% by the age of 18 yr, was highly significantly higher in case of G6PD deficiency: 53.3% vs 15.8% (Log Rank p<0.001), lower in case of alpha-Thal: 8.7% vs 29.9% (p=0.022), and significantly dependent on hemolysis: LDH IU/l <750: 0%; 750–1209:13.9%; >1209: 37.1% (Log Rank p=0.013). KM estimate of silent infarcts was significantly dependent on the presence of stenoses on MRA (Log Rank p=0.002), and on gender (male older than 8) (Log Rank p=0.038).Conclusion:This monocenter prospective study reports a significant decrease in the risk of stroke at 18 yr (KM 1.9% vs. 11–11.5%, previously published by Ohene-Frempong [Blood, 1998] and Quinn [Blood, 2004]) demonstrating the strong efficacy of systematic and early screening by TCD in association with TP to prevent stroke. It confirms our recent study that G6PD deficiency, absence of alpha-Thal and hemolysis significantly and independently increase the risk for abnormal velocities (Blood 2008 in press), which are themselves highly significantly predictive of stenosis occurrence. Morever, this study shows that the risk of silent infarcts remains high, even in patients with normal TCD, and is higher in males than in girls after the age of 8 years.

Haemoglobin Hokusetsu [β52 (D3)] Asp→Gly in German families associated with inclusion body

Inclusion bodies associated with Hb Hokusetsu have never been published. We investigated the autoxidation of this variant as a cause for the inclusion bodies in three unrelated families. Moreover, haplotype analysis was carried out to unravel the origin of this variant also found in the Japanese population. The presence of inclusion bodies was revealed by incubating the fresh peripheral blood with brilliant cresyl blue. We further characterised this variant using mass spectrometry and DNA analysis. The generation of superoxide radical (ROS) during the autoxidation was assayed by electron spin resonance spectrometry. Inclusion bodies were seen in about 25% of red cells. Hb Hokusetsu turned out to be less thermostable than the control. It showed a tenfold-enhanced ROS formation versus control. The analysis of the beta-globin haplotypes for the three unrelated families showed that Hb Hokosetsu was linked with haplotype I (5' + - - - - + + 3'). This is the first case published in the German population. The inclusion bodies could be due to the instability of the variant. This is supported by the increased autoxidation. The absence of anaemia evokes an elimination of the inclusion bodies by the proteolytic mechanism of the red cells. The association of the variant in three unrelated families with the five polymorphisms of haplotype I indicates a single common mutation event. In the presence of Hb Hokusetsu, HbA 1C standard methods used to assess glycaemic control are mistaken.

High LDH Level, G6PD Deficiency and Absence of alpha-Thalassemia Are Significant Independent Risk Factors of Abnormally High Cerebral Velocities in Patients with Sickle Cell Anemia.

Background Predicting the severity of sickle cell anemia (SCA) is important for providing better informed genetic counseling and for better targeting of intensive therapies. Stroke is the most severe complication in children with SCA and is predicted by abnormally high cerebral velocities by transcranial doppler (TCD). We attempted to define the risk factors associated with high velocities.Methods This study concerned the Créteil pediatric SCA cohort, composed of newborn patients, screened and followed at our Center since birth, and of patients secondarily referred to the Center because of the severity of their disease. Time-averaged mean of maximum velocities (TAMMX) higher than 200 cm/sec were considered as abnormal, resulting in initiation of a transfusion program initiated</DEL>. Cerebral MRI/MRA was done after the age of 5 years or earlier in case of abnormal TCD. Alpha </DEL>genes and beta-globin haplotypes were determined. Baseline biological parameters (G6PD activity; WBC, PMN, Reticulocytes, Platelets counts; Hemoglobin, Hematocrit, HbF, LDH levels; MCV; SpO2) were obtained a minimum of 3 months away from a transfusion, one month from a painful episode, after 18 months of age and, before intensive therapy.Results SS children (390; 189 F, 201 M) were annually explored by TCD (n=2286) since 1992, and followed for a total of 1962 patient-years. The follow-up before initiation of intensive therapy was 1032 patient-years. Nineteen patients experienced an overt stroke. TCD was abnormal in 65 of 390 patients (17%). MRI (n=850) was performed in 268 patients, was abnormal in 86 cases and showed silent infarcts in 67 of 249 patients (27%). Silent infarcts were seen in 33% of patients with abnormal TCD. Alpha genes study, available in 336 patients, demonstrated alpha-thalassemia in 158 patients (47%): 31 had a deletion of 2 genes (7.9%) and 127 of 1 gene (32.6%). G6PD deficiency was present in 26 of 228 evaluated patients (11%). Beta-globin haplotypes studied in 316 patients were Car/Car in 125 (40%), Ben/Ben in 76 (24%), Sen/Sen in 30 (9%) and, “other” in 85 (27%). Univariate analysis showed that the risk of abnormally high velocities was not related to sex, beta-globin haplotypes, pain and acute chest syndrom rates, WBC, PMN, platelets counts, HbF level and SpO2 but was significantly associated with the absence of alpha-thalassemia (p< 0.001), G6PD deficiency (p=0.012), low Hb and Ht levels (p< 0.001), high reticulocyte count (p=0.008), high MCV (p=0.004) and high LDH level (p<0.001). Multivariate logistic regression analysis showed that absence of alpha-thalassemia [OR= 14.5, 95% CI (2.6–79.7)], (p=0.002); G6PD deficiency [OR=6.7, 95% CI (1.7–26.1)], (p=0.006) and, LDH > 1200 UI/L [OR=4.5, 95% CI (1.5–13.5)], (p=0.007) were independent risk factors of abnormally high velocities.Conclusion This study confirms that the risk of high velocities in patients with SCA is significantly decreased by the presence of alpha-thalassemia. It shows for the first time that hemolysis is a more significant risk factor than the degree of anemia and that absence of alpha-thalassemia, G6PD deficiency and hemolysis are significant independent risk factors of cerebral vasculopathy in patients with SCA.

Classical sickle beta-globin haplotypes exhibit a high degree of long-range haplotype similarity in African and Afro-Caribbean populations

BackgroundThe sickle (βs) mutation in the beta-globin gene (HBB) occurs on five "classical" βs haplotype backgrounds in ethnic groups of African ancestry. Strong selection in favour of the βs allele – a consequence of protection from severe malarial infection afforded by heterozygotes – has been associated with a high degree of extended haplotype similarity. The relationship between classical βs haplotypes and long-range haplotype similarity may have both anthropological and clinical implications, but to date has not been explored. Here we evaluate the haplotype similarity of classical βs haplotypes over 400 kb in population samples from Jamaica, The Gambia, and among the Yoruba of Nigeria (Hapmap YRI).ResultsThe most common βs sub-haplotype among Jamaicans and the Yoruba was the Benin haplotype, while in The Gambia the Senegal haplotype was observed most commonly. Both subtypes exhibited a high degree of long-range haplotype similarity extending across approximately 400 kb in all three populations. This long-range similarity was significantly greater than that seen for other haplotypes sampled in these populations (P < 0.001), and was independent of marker choice and marker density. Among the Yoruba, Benin haplotypes were highly conserved, with very strong linkage disequilibrium (LD) extending a megabase across the βs mutation.ConclusionTwo different classical βs haplotypes, sampled from different populations, exhibit comparable and extensive long-range haplotype similarity and strong LD. This LD extends across the adjacent recombination hotspot, and is discernable at distances in excess of 400 kb. Although the multi-centric geographic distribution of βs haplotypes indicates strong subdivision among early Holocene sub-Saharan populations, we find no evidence that selective pressures imposed by falciparum malaria varied in intensity or timing between these subpopulations. Our observations also suggest that cis-acting loci, which may influence outcomes in sickle cell disease, could lie considerable distances away from β-globin.

Determinants of Sickle Cell Density Distribution: A Study of Benin Twins.

Red cell density distribution affects both hemolysis and vaso-occlusion; however, currently recognized factors cannot account for all of the variation seen. We hypothesized that a range of genetically controlled factors contributes to red cell density distribution and hemolysis, which has recently received a great deal of attention from Gladwin et al for its role in sickle cell anemia (SCA) and its impact on NO metabolism. Our previous studies have demonstrated that although RBC density distribution varies significantly from patient to patient, the pattern for individual patients is stable in the absence of disease. Some genetically determined factors that affect red cell density distribution have been defined, such as alpha-thalassemia and % HbF (Fabry et al, Blood, 1982); however, neither of these factors completely predicts density distribution. The study of identical twins offers the unique opportunity to minimize some of the genetic variability between individuals that may not be relevant to RBC density while allowing the remaining differences to be detected. Because SCA patients from the US have a complex mixture of Caucasian, other ethnicities, and genes from all parts of Africa including all of the sickle haplotypes, we have chosen to recruit our population from Benin that has a single beta-globin haplotype, thus further minimizing differences arising from admixture from inside and outside of Africa. We have collected samples from six sets of monozygous twins from Benin and validated their monozygosity by DNA analysis. Of the 6 twin sets analyzed to date, 4 have alpha-thalassemia. We compared density gradients on two separate occasions, approximately one year apart, for these twins and found that density gradients for both members of all twin sets without medical complications (malaria, painful crisis) were indistinguishable. This is not true for pairs of randomly chosen individuals even after their % HbF and alpha-thalassemia status has been determined and matched. After elimination of WBCs, we were able to isolate sufficient RNA to obtain microarray data without amplification. We compared subjects with a low % dense cells vs a high % dense cells. In the combinations that were analyzed, we found a consistent pattern of up- and down-regulated genes. Down-regulated genes included the Gardos channel (KCNN4), K-Cl cotransporter (KCC1), NOS3, CAIV, and PKC. Up-regulated genes included ferritin heavy chain (probably in mitochondria that are present in reticulocytes) and 2,3-bisphosphomutase that was elevated in twins with higher MCHC (density). The latter is consistent with our previous observations and those of Poillon et al that DPG can affect polymer formation and RBC density. We conclude that the study of twins demonstrates that there is a strong genetic component in the control of sickle cell density distribution and that a better understanding of factors controlling density distribution may lead to new forms of treatment.

Tracing the origin of Brazilian HTLV-1 as determined by analysis of host and viral genes

We compared the genetic diversity of the Brazilian human T-cell lymphotropic virus type 1 isolates with those found in KwaZulu-Natal (KZN), South Africa, and with the genetic background of the hosts. The seroprevalence rate in KZN was 1.7%. All sequences belonged to the A subgroup. The presence of South African sequences in two different clusters from Brazil, and the finding of the beta-globin haplotype in infected hosts are consistent with the transmission of this virus from southern Africa to Brazil.

Influence of Globin Gene Modifiers on Baseline and Hydroxyurea-Induced Fetal Hemoglobin Levels in Children with Sickle Cell Anemia.

Fetal hemoglobin (HbF) is recognized as a major determinant of clinical disease severity in children and adults with sickle cell anemia (SCA). Patients with elevated HbF levels have a milder disease course, and many current therapeutic protocols for SCA include pharmacological induction of HbF. However, baseline and treatment HbF levels vary widely due to presumed genetic and environmental factors. Recognized globin gene modifiers of HbF include the beta globin haplotype and a potential contribution from concomitant alpha thalassemia. To characterize more fully the influence of globin gene modifiers on both baseline and treatment HbF levels, we retrospectively determined the beta globin haplotype (Benin, CAR, Senegal, Cameroon, or Arab-Indian) by selective gamma globin gene nucleotide sequencing and the alpha globin gene number (2, 3, or 4) by PCR for 67 African-American children with SCA receiving hydroxyurea therapy at stable maximal tolerated dose (MTD). The four beta globin haplotypes and frequencies identified in our cohort of children include Benin (0.61), CAR (0.17), Senegal (0.12), and Cameroon (0.10). The number of alpha globin genes and frequencies identified were 4 genes (0.72), 3 genes (0.25) and 2 genes (0.03). Baseline and MTD HbF levels were analyzed according to each variable. The average baseline HbF value for the entire cohort of children was 7.7 ± 4.4% (median 7.6%, range 1.3 – 19.3%), while the average treatment HbF value was 23.9 ± 7.2 % (median 22.9%, range 10.2 – 40.7%). All 67 children increased their HbF in response to hydroxyurea therapy (median 16.7%, range 5.0 – 28.8%). There was a modest but statistically significant correlation between the baseline and treatment HbF (r=0.66, p<.0001). The estimated effect of one unit change in baseline HbF on treatment HbF was 1.11 (95% CI of 0.78, 1.43). When baseline %HbF was analyzed according to the beta globin haplotype, the overall ANOVA had a p-value of 0.02, indicating a statistically significant influence. Further analysis confirmed associations previously identified in adults with SCA, i.e. children with at least one copy of the CAR haplotype had a lower baseline HbF (5.9% vs 8.4%, p=.05), while those with at least one copy of the Senegal haplotype had a higher baseline HbF (11.1% vs 6.7%, p<.001). When hydroxyurea MTD (treatment) HbF values were analyzed according to beta globin haplotype while adjusting for baseline HbF, however, the effect of beta globin haplotype was not statistically significant (p=.13). Analyses of HbF according to alpha globin gene number revealed no statistically significant effects on either baseline or treatment HbF values. Taken together, these data support the hypothesis that beta globin haplotype significant influences baseline HbF values for children with SCA, but has no significant effects on hydroxyurea MTD HbF values. Accordingly, children with SCA should be offered hydroxyurea based solely on clinical indications, without consideration of baseline HbF or beta globin haplotype. Even children with low baseline HbF values or the CAR beta globin haplotype can respond to hydroxyurea therapy with an elevated %HbF. Future studies designed to identify genetic modifiers of treatment HbF values should focus on sequence polymorphisms in non-globin genes that have trans-acting effects on gamma globin gene expression.

The influence of uridine diphosphate glucuronosyl transferase 1A promoter polymorphisms, βS‐globin gene haplotype, co‐inherited α‐thalassemia trait and Hb F on steady‐state serum bilirubin levels in sickle cell anemia

Homozygosity for the (AT)7 allele of uridine diphosphate glucuronosyl transferase 1A (UGT1A1) gene polymorphism is associated with increased bilirubin levels in sickle cell anemia (SCA). In the present study, in addition to UGT1A1 promoter genotype, serum bilirubin level was related to other genetic modifiers -beta(S)-globin gene haplotype, Hb F, co-inherited alpha-thal trait, age and gender. The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A1 promoter polymorphisms were determined using automated sequencing. Other investigations were with standard techniques. There were 67 SCA patients (41 males and 26 females), aged 2-44 yr (mean of 20.6 +/- 10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A1 allele, 35 (52.2%) were heterozygous for (AT)6 and (AT)7 alleles while 22 (32.8%) were homozygous for (AT)7. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7 +/- 1.5, 3.8 +/- 2.3 and 5.6 +/- 2.4 mg/dL, respectively). It was also significantly higher in males than females and in patients aged >10 yr. There was a significant negative linear correlation (r = -0.304, P = 0.016) of serum bilirubin with Hb F. The beta-globin haplotype and co-existing alpha-thal trait did not have any significant influence on serum bilirubin levels. Patients on hydroxyurea were older, had lower Hb F, but higher mean serum bilirubin. The latter also was signifcantly higher among those with UGT1A1 (AT)7 homozygosity. Apart from UGT1A1 (AT)7 homozygosity, Hb F, age and gender are the other factors that significantly influence serum bilirubin level in SCA.

Are there clinical phenotypes of homozygous sickle cell disease?

The distribution of clinical features was examined in subjects with homozygous sickle cell (SS) disease in the Jamaican Cohort Study to determine whether there is evidence of distinct clustering of symptoms or clinical phenotypes. A twofold model yielded groups that could be interpreted as painful crisis or leg ulcer phenotypes and 78% of patients were classified with 95% confidence into one of these. The painful crisis phenotype also manifested higher frequencies of dactylitis, meningitis/septicaemia, acute chest syndrome and stroke. Attempts to define a three-group model were less convincing although 43% of patients could be allocated with 95% confidence. The three-group model essentially divided subjects with the leg ulcer phenotype into subgroups with higher and lower frequencies of painful crisis, dactylitis, meningitis/septicaemia and acute chest syndrome. In the three-group model, the painful crisis phenotype had lower total haemoglobin, fetal haemoglobin, mean cell volume and higher reticulocytes but there was no apparent influence of alpha thalassaemia or beta globin haplotype. Both environmental and genetic factors are likely to contribute to most manifestations of SS disease and the evidence for different clinical phenotypes suggests that a search for associated genetic polymorphisms may provide insights into the mechanisms of clinical variability in SS disease.

ßS-Haplotypes in sickle cell anemia patients from Salvador, Bahia, Northeastern Brazil

BetaS-Globin haplotypes were studied in 80 (160 betaS chromosomes) sickle cell disease patients from Salvador, Brazil, a city with a large population of African origin resulting from the slave trade from Western Africa, mainly from the Bay of Benin. Hematological and hemoglobin analyses were carried out by standard methods. The betaS-haplotypes were determined by PCR and dot-blot techniques. A total of 77 (48.1%) chromosomes were characterized as Central African Republic (CAR) haplotype, 73 (45.6%) as Benin (BEN), 1 (0.63%) as Senegal (SEN), and 9 (5.63%) as atypical (Atp). Genotype was CAR/CAR in 17 (21.3%) patients, BEN/BEN in 17 (21.3%), CAR/BEN in 37 (46.3%), BEN/SEN in 1 (1.25%), BEN/Atp in 1 (1.25%), CAR/Atp in 6 (7.5%), and Atp/Atp in 1 (1.25%). Hemoglobin concentrations and hematocrit values did not differ among genotype groups but were significantly higher in 25 patients presenting percent fetal hemoglobin (%HbF) > or = 10% (P = 0.002 and 0.003, respectively). The median HbF concentration was 7.54+/-4.342% for the CAR/CAR genotype, 9.88 3.558% for the BEN/BEN genotype, 8.146 4.631% for the CAR/BEN genotype, and 4.180+/-2.250% for the CAR/Atp genotype (P = 0.02), although 1 CAR/CAR individual presented an HbF concentration as high as 15%. In view of the ethnic and geographical origin of this population, we did not expect a Hardy-Weinberg equilibrium for CAR/CAR and BEN/BEN homozygous haplotypes and a high proportion of heterozygous CAR/BEN haplotypes since the State of Bahia historically received more slaves from Western Africa than from Central Africa.

Beta-globin gene cluster haplotypes as evidence of African gene flow to the northeastern coast of Venezuela.

In order to study the origin of mutation HBB*S in Sucre and Anzoátegui states and the genetic affinities of these Venezuelan populations with other human groups, the beta-globin gene cluster haplotypes were determined for 28 sickle cell and/or S-beta thalassemia patients and for 37 individuals with normal hematological parameters. Bantu, Benin, Senegal, and atypical haplotypes were identified in 50%, 36%, 2%, and 12% of the HBB*S chromosomes, respectively. Similar results have been published for Venezuelan patients from the central states, but a different trend is shown in a publication based on a group of patients from different regions of the country. For HBB*A, haplotype 2 (+ - - - -), characteristic of non-African groups, was the most common (39%), followed by haplotype 3 (- - - - +) of African origin, and haplotype 6 (- + + - +), also typical of non-Africans. The results reveal a high level of admixture of the Sucre-Anzoátegui population. The importance of specific conditions which have acted differently in the Venezuelan populations, such as founder effect, genetic drift, isolation, and endogamy are discussed. Genetic distances between the Sucre-Anzoátegui sample and several other human populations calculated on the basis of the HBB*S and HBB*A haplotypes revealed similar results, the closest genetic relationships being observed in relation to Bantu-speaking groups. These results confirm the utility of the beta-globin haplotypes for population studies and contribute to knowledge of the Venezuelan gene pool.

Outcome in hemoglobin SC disease: a four-decade observational study of clinical, hematologic, and genetic factors.

Over the past 40 years, we observed 284 subjects with hemoglobin SC disease (Hb SC) for 2,837 person-years. We examined the association of the course of clinical events with hematologic and genetic factors. The mean entry age was 21 years, although 15% entered before one year of age. The mean Hb concentration was 11.3 g/dL, the mean fetal hemoglobin was 2.5%, and the mean MCV was 84.4 fL. Twenty-five subjects died at a median age of 37 years. Chronic organ-specific complications occurred in 112 subjects (39.4%), with advanced retinopathy in 65 subjects (22.9%) and osteonecrosis (avascular necrosis) in 42 subjects (14.8%). We identified the beta-globin haplotypes in 82 subjects and the alpha-gene status in 79. Twenty-nine percent had alpha-thalassemia-2. The beta(CI) haplotype was present in 85.4%. We found a decreased incidence of retinopathy in the beta(CI) subjects compared to the non-beta(CI) subjects (33% vs. 67%; P = 0.049) with a later mean onset age (29 years vs. 21 years; log-rank test, P= 0.026). We also found a consistent pattern of decreased morbidity in subjects who had alpha-thalassemia-2 in comparison to those who did not. We found a reduced risk of chronic organ-specific complications (log-rank test, P= 0.003), lower incidence of sickle crisis (48% vs. 80%, P= 0.001), later onset of gallbladder disease (age of onset: 55 years vs. 34 years; P= 0.055), and lower risk of osteonecrosis (log-rank test, P= 0.024). Our findings suggest that Hb SC subjects who have not inherited alpha-thalassemia-2 might benefit from erythrocyte rehydration therapy.

Thalassemia and abnormal hemoglobin.

Thalassemia and abnormal hemoglobins are common genetic disorders in Asia. Thalassemia is not only an important public health problem but also a socio-economic problem of many countries in the region. The approach to deal with the thalassemic problem is to prevent and control birth of new cases. This requires an accurate identification of the couple at high risk for thalassemia. However, the diagnosis of thalassemia carrier states need several tests which are not practical for screening the population at large. Recently we have used two simple laboratory tests to screen for potential thalassemia carriers and hemoglobin E individuals. There is also a new development in using the automatic HPLC to diagnose thalassemic diseases and the carriers. This system gives both qualitative and quantitative analysis of hemoglobin components in the same run with good precision and reproducibility. The system has been applied to study thalassemia and abnormal Hb in adult and cord blood. This system has enabled us to do both prenatal and postnatal diagnosis of thalassemia within the few minutes. However, none of these screening tests can accurately give specific diagnosis of the thalassemia genotype. Specific thalassemia mutation can be carried out by DNA analysis. Many DNA techniques have been used for point mutation detection and small deletion. For the last few years there is a development of DNA chip technology that has been applied for thalassemia mutation as well. Clinically, thalassemia is very heterogeneous in the manifestation. In spite of seemingly identical genotypes, severity of beta thalassemic patients can vary greatly. Heterogeneity in the clinical manifestation of beta thalassemic diseases may occur from the nature of beta globin gene mutation, alpha thalassemia gene interaction and difference in the amount of Hb F production which is partly associated with a specific beta globin haplotype. However, there is still some beta thalassemia cases that have a mild clinical symptom without those known genetic fators interaction suggesting that there are other additional factors responsible for the mildness of the disease.

The peopling of São Tomé (Gulf of Guinea): origins of slave settlers and admixture with the Portuguese.

The geographic origins of African slave settlers and the Portuguese genetic contribution to the population of São Tomé (Gulf of Guinea) were assessed through the analysis of beta-globin haplotypes in 44 chromosomes bearing the betaS allele and through the study of the genetic variation in eight autosomal markers (APOA1, AT3, FY, LPL, OCA2, RB1, Sb19.3, and GC) informative for admixture in a sample of 224 individuals. The observed betaS haplotype distribution (36.4% Bantu, 52.3% Benin, 4.5% Cameroon, 4.5% Senegal, and 2.3% atypical) is in accordance with the historical information on the major geographic sources of slave settlers of São Tomé, although it captures a more important contribution of Central-West Africa regions than previously anticipated. European admixture, estimated to be 10.7 +/- 0.9%, has created a considerable level of genetic structure, as indicated by the finding of significant linkage disequilibrium between 33% of unlinked marker loci pairs. Recent admixture was found to have an important contribution to these values, since removal of individuals with Portuguese or Cape Verdian parents or grandparents from the sample dropped the miscegenation level to 6.5 +/- 0.8% and reduced significant linkage disequilibrium to 11% of unlinked marker pairs. Taken together, these results indicate that the peopling of São Tomé might have provided one of the first examples of the combination of diverse African contributions and European admixture that emerged from the overseas population relocations promoted by the Atlantic slave trade.