Beta-globin Haplotypes Research Articles

Sickle Cell Anemia Screening in Newborns and Analysis of Haplotypes in Patients from Santiago Island, Cape Verde.

Sickle cell anemia (SCA) results from a mutation in the β-globin gene, leading to the production of mutant hemoglobin, known as hemoglobin S (HbS). Despite being a genetic disorder, the phenotype of SCA can be influenced by the level of fetal hemoglobin (HbF), which is associated with beta S-globin haplotypes. In this study, we conducted newborn screening (NBS) using samples collected from umbilical cord blood in two hospitals on Santiago Island, Cape Verde. In newborns, HbS was detected using high-performance liquid chromatography (HPLC) on dried blood spot, with confirmation through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). In addition, we assessed the hematological and clinical characteristics of a second population group consisting of patients diagnosed with SCA. Haplotype determination was performed on both newborns with HbS and patients with SCA. Beta S-globin haplotypes were determined using PCR-RFLP. Hematological values were analyzed using standard methods. Out of 346 newborns, 21 (6%) were carriers of the sickle cell trait (HbAS) while none were identified as homozygous for sickle cell disease (HbSS). Among both groups of individuals, four haplotypes were identified: Senegal, Arabi-Indian, Bantu, and Benin. The Senegal haplotype was the most prevalent, possibly reflecting the ethnic origin of the mutations observed. Hematological values did not differ significantly among haplotypes. However, higher levels of HbF were associated with better hematological values. These findings suggest a positive impact of elevated HbF levels on reducing the severity of SCA. Finally, we demonstrated how the combination of technics, HPLC and molecular analysis, provided a consistent and reproducible results that can be used for NBS for SCA.

Phenotypic variation in sickle cell disease: the role of beta globin haplotype, alpha thalassaemia and fetal haemoglobin in HbSS

Introduction The haematological and clinical feature vary markedly between the different genotypes of sickle cell disease. Even within the single genotype of homozygous sickle cell disease (HbSS), there is marked variability which is presumed to result from interacting genetic and environmental factors. Areas Covered The classification of the different genotypes of sickle cell disease with approximate prevalence at birth in different communities and some of the major clinical and haematological differences. This assessment includes three potential genetic factors influencing haematology and clinical outcome in HbSS, the beta globin haplotype, alpha thalassaemia and persistence of fetal haemoglobin (HbF). Expert Opinion The author is a clinician with experience of sickle cell disease primarily in Jamaica but also in Greece, Uganda, Saudi Arabia and India. It is therefore necessarily an account of clinical data and does not address current debates on molecular mechanisms. Most data derive from Jamaica where efforts have been made to reduce any symptomatic bias by long term follow-up of patients all over the island and further reduced by a cohort study based on newborn screening which has been in operation for over 48 years.

Alpha thalassemia, but not βS-globin haplotypes, influence sickle cell anemia clinical outcome in a large, single-center Brazilian cohort.

Alpha thalassemia and beta-globin haplotype are considered classical genetic disease modifiers in sickle cell anemia (SCA) causing clinical heterogeneity. Nevertheless, their functional impact on SCA disease emergence and progression remains elusive. To better understand the role of alpha thalassemia and beta-globin haplotype in SCA, we performed a retrospective study evaluating the clinical manifestations of 614 patients. The univariate analysis showed that the presence of alpha-thalassemia -3.7-kb mutation (αα/-α and -α/-α) decreased the risk of stroke development (p = 0.046), priapism (p = 0.033), and cholelithiasis (p = 0.021). Furthermore, the cumulative incidence of stroke (p = 0.023) and cholelithiasis (p = 0.006) was also significantly lower for patients carrying the alpha thalassemia -3.7-kb mutation. No clinical effects were associated with the beta-globin haplotype analysis, which could be explained by the relatively homogeneous haplotype composition in our cohort. Our results reinforce that alpha thalassemia can provide protective functions against hemolysis-related symptoms in SCA. Although, several genetic modifiers can impact the inflammatory state of SCA patients, the alpha thalassemia mutation remains one of the most recurrent genetic aberration and should therefore always be considered first.

Estimating the age of Hb G-Coushatta [β22(B4)Glu→Ala] mutation by haplotypes of β-globin gene cluster in Denizli, Turkey.

BackgroundHb G‐Coushatta variant was reported from various populations’ parts of the world such as Thai, Korea, Algeria, Thailand, China, Japan and Turkey. In our study, we aimed to discuss the possible historical relationships of the Hb G‐Coushatta mutation with the possible migration routes of the world. For this purpose, associated haplotypes were determined using polymorphic loci in the beta globin gene cluster of hemoglobin G‐Coushatta and normal populations in Denizli, Turkey.MethodsWe performed statistical analysis such as haplotype analysis, Hardy–Weinberg equilibrium, measurement of genetic diversity and population differentiation parameters, analysis of molecular variance using F‐statistics, historical‐demographic analyses, mismatch distribution analysis of both populations and applied the test statistics in Arlequin ver. 3.5 software program.ResultsThe diversity of haplotypes has been shown to indicate different genetic origins for two populations. However, AMOVA results, molecular diversity parameters and population demographic expansion times showed that the Hb G‐Coushatta mutation develops on the normal population gene pool. Our estimated τ values showed the average time since the demographic expansion for normal and Hb G‐Coushatta populations ranged from approximately 42,000 to 38,000 ybp, respectively.ConclusionOur data suggest that Hb G‐Coushatta population originate in normal population in Denizli, Turkey. These results support the hypothesis that the multiple origin of Hb G‐Coushatta and indicate that mutation may have been triggered the formation of new variants on beta globin haplotypes.

Association between Oxidative Stress, Genetic Factors, and Clinical Severity in Children with Sickle Cell Anemia

To investigate the associations between several sickle cell disease genetic modifiers (beta-globin haplotypes, alpha-thalassemia, and glucose-6-phosphate dehydrogenase deficiency) and the level of oxidative stress and to evaluate the association between oxidative stress and the rates of vaso-occlusive events. Steady-state oxidative and nitrosative stress markers, biological variables, genetic modulators, and vaso-occlusive crisis events requiring emergency admissions were measured during a 2-year period in 62 children with sickle cell anemia (58 SS and 4 Sβ0). Twelve ethnic-matched children without sickle cell anemia also participated as healthy controls (AA) for oxidative and nitrosative stress level measurement. Oxidative and nitrosative stress were greater in patients with sickle cell anemia compared with control patients, but the rate of vaso-occlusive crisis events in sickle cell anemia was not associated with the level of oxidative stress. The presence of alpha-thalassemia, but not glucose-6-phosphate dehydrogenase deficiency or beta-globin haplotype, modulated the level of oxidative stress in children with sickle cell anemia. Mild hemolysis in children with alpha-thalassemia may limit oxidative stress and could explain the protective role of alpha-thalassemia in hemolysis-related sickle cell complications.

The First Two Years of Life in Sickle Cell Anemia Infants: Results of a Comprehensive Longitudinal Study

Background and hypothesis: Infancy is a critical time during which the first complications of sickle cell anemia (SCA) emerge. Very early prognostic factors of severity are needed to select high-risk children to offer precisely tailored therapy. Our hypothesis was that clinical, biological or genetic parameters measurable soon after birth (3 to 6 months of age) could predict severe outcomes in the first two years of life in SCA infants.Methods: Infants with SCA were offered to participate in a prospective study (ClinicalTrials.gov: NCT01207037) that included clinical and laboratory assessment at enrollment (3-6 months) and at 12, 18 and 24 months of age. The prognostic performance of conventional and SCA-specific biomarkers (notably erythroid adhesion markers, dense cells, ektacytometry indices) on disease severity was assessed using Cox's proportional hazard regression models. The disease severity was defined as time to first occurrence of either acute splenic sequestration (ASS), vaso occlusive (VOC) event requiring hospitalization, transfusion, conditional or abnormal Trans Cranial Doppler, acute chest syndrome and death. Hazard ratios (HRs) were calculated with 95% Cis.Results: Fifty-seven infants (55 SS; 2 Sβ°; 54.4% males), diagnosed through neonatal screening, were included in the study at a mean age of 4.4 ± 1 months and longitudinally assessed with a median follow-up of 19.4 months (range 3.1-23.2). Only 1 of 57 infants had experienced a SCA-related event prior to enrolment (dactilytis). At inclusion, clinical examinations were unremarkable except for pallor found in 15 (26,3%) and none of the infants had an enlarged or palpable spleen. Growth parameters were normal. Main biological characteristics were a mild hemolytic anemia (Hb 9.3 ±1.3 g/dL; reticulocytes count: 151.2 ±76 x 109/L), increased HbF level (3.8 ±1.3 g/dL. or 41.4 ±11.7%) and presence of dense red cells (23.1 % ±10.8). Genetic analysis showed one alpha globin deletion in 18 (32.1%) and a balanced distribution of beta globin haplotypes: 16 (30.8%), 18 (34.6%) and 18 (34.6%) in the favorable (SEN/SEN, BEN/SEN, CAM/SEN or SEN/ATYP), unfavorable (CAR/CAR, CAM/CAR, CAR/ATYP or BEN/CAR) and intermediate category (other) respectively.During the 2 years duration of the study, 44 of 57 (77%) infants required 157 hospital admissions, median (range): 2 (1-12) per patient. Infection was a leading cause of hospitalization although no serious adverse event related to pneumococcal infection was noted. Eight (14%) children experienced an episode of ASS at a median (range) age of 13.4 months (7.8- 15.9) while 13 infants (22.8%) experienced at least one VOC event at a median age of 12.7 months (7-22.5), with 6 experiencing ≥ 2 episodes. Nineteen infants (33.3%) required at least one transfusion with 10 (17.6%) requiring more than 2. Altogether, 22 (38.6%) infants of this cohort experienced a SCA-related severe clinical event by 24 months of age. The Kaplan Meier estimate of the 24-month event-free rate was 54.4% (95% CI, 39.7 to 74.5%) (Figure).Univariate analysis of potential prognostic markers at inclusion showed that higher HbF % and concentration were the strongest protective parameters for ASS in particular and for all severe outcomes, except for VOC (p < 0.001). Unfavorable haplotypes were also associated with severe outcome (HR (95% CI) 4.73 (1.31-17.01), p=0.017). Protective factors for VOC were higher Hb level (threshold > 8 g/dL) and low % of circulating dense cells (p=0.04 and 0.02, respectively). In a multivariate analysis, Hb level ≥ 8 g/dL and HbF ≥ 2.8 g/dL proved to be two independent prognostic factors of a SCA-related severe event (adjusted HRs (95% CI) 0.27 (0.11-0.73) and 0.16 (0.06-0.43) respectively). Interestingly, absolute neutrophil or reticulocyte counts, level of expression of known potentially pathogenic erythroid adhesion markers (CD36, Lu/B-CAM, ICAM-4/LW), % of red dense cell, or deformability parameters failed to be prognostic factors of specific complications or overall severity in this very young cohort.Conclusion: HbF and Hb levels measured between 3 and 6 months of age in SCA infants predict the risk of subsequent severe clinical outcome in the next 2 years. These events are frequent even in a high-income setting where neonatal screening is implemented. These findings further support the early use of HbF inducers such as hydroxyurea in high-risk infants to sustain a protective HbF level. [Display omitted] DisclosuresBrousse:Add Medica: Membership on an entity's Board of Directors or advisory committees. De Montalembert:Novartis: Consultancy, Honoraria, Research Funding; Addmedica: Consultancy, Honoraria, Research Funding.

Association between beta globin haplotypes, HBBP1 and HMOX1 polymorphisms in relation to HbF among sickle cell anemia patients: a study in Southwest Iran

The haplotypes of β-globin gene cluster in patients with sickle cell anemia are associated with different clinical manifestations and fetal hemoglobin (HbF) levels. In addition, SNP in some genes within the β-globin gene cluster (HBBP1) and outside it (HMOX1) has been recently reported to be related to HbF levels. In this study, rs2071348 and rs2071746 polymorphisms in HBBP1 and heme oxygenase 1 (HMOX1) genes associated with different haplotypes and HbF levels in patients with sickle cell anemia were evaluated in comparison with healthy subjects. In this case-control study, 150 patients with sickle cell anemia (90 homozygous and 60 sickle-cell trait) were evaluated and compared with 50 healthy individuals referred to Shafa tertiary hospital in Ahvaz city of Khuzestan Province, Iran. HbF level was measured by alkali and acid electrophoresis. PCR-RFLP and ARMS-PCR methods were used to determine β-globin gene haplotypes and HBBP1 gene polymorphism, respectively. Arab-Indian was the most common genotype found in both homozygous and sickle-cell trait patients with a respective prevalence of 55.5 and 50% followed by homozygous Benin with 11.1 and 18.3% prevalence, respectively. The highest and lowest level of HbF were observed in Arab-Indian (24.1 ± 6.35) and Benin/Benin haplotypes (7.63 ± 2.29), respectively. A significant relationship was found between HbF level with rs2071746 A>T polymorphism of HMOX1 gene (P < 0.019) as well as with rs2071348 polymorphism of HBBP1 gene (P < 0.034). However, there was no significant relationship between haplotypes with gene polymorphisms. Arab-Indian was the most common haplotype in this region. The HbF level was significantly higher in patients with Arab-Indian haplotype, patients with C genotype in rs2071348 polymorphism locus of HBBP1 gene, and patients with T genotype in rs2071746 polymorphism locus of HMOX gene.

Clinical Genotypic Correlation of Beta S-Globin Haplotypes in Sickle Cell Anemia

Sickle cell disease played a pioneering role in the establishment of the field of molecular medicine. Even though this disease was identified many years ago its clinical course is still not clear. Clinical severity ranges across individuals, even within the same ethnic group. Molecular studies have identified different haplotypes across the globin gene cluster. Correlating individual haplotypes with clinical severity has become the primary focus in the endeavour to establish a successful treatment. Even though numerous studies have been performed, most have shown a negative correlation between beta S-globin haplotypes and clinical phenotype. After a review of recent medical literature, the authors conclude that further research translating genetic analysis to positive therapeutic response for sickle cell disease patients is needed.

Sickle cell disease in India: A perspective.

Sickle cell disease is an inherited blood condition which is most common among people of African, Arabian and Indian origin. In disease of African origin, research has led to models of care which prevent serious complications, improve the quality of life, and increase survival1. In India, the disease is largely undocumented. Thus, there is an urgent need to document the features of Indian disease so that locally appropriate models of care may be evolved. The sickle cell mutation affects the beta chain of adult haemoglobin which changes the behaviour of sickle cell haemoglobin. Possession of a single HbS gene results in the generally harmless sickle cell trait (AS genotype) but inheritance of the HbS gene from both parents results in homozygous sickle cell (SS) disease which is often a severe condition destroying red blood cells rapidly and blocking flow in blood vessels with painful and often serious complications2. The HbS mutation has occurred on at least three occasions in Africa, named after the areas where these were first described3, Benin, Senegal and Bantu (Central African Republic) and referred to as the beta globin haplotypes. A separate and fourth occurrence of the mutation was seen around the Arabian Gulf and India and designated the Arab-Indian or Asian haplotype4.

Beta-Globin Haplotypes and Alpha-Thalassemia 3.7 kb Deletion in Sickle Cell Disease Patients From the Occidental Brazilian Amazon

Background: Sickle cell disease (SCD) includes a group of inherited red blood cell disorders. SCD patients vary widely from person to person. We describe β S and β C haplotypes and α-thalassemia 3.7 kb genotypes from SCD patients Fundacao Hospitalar de Hematologia e Hemoterapia do Amazonas, Manaus, AM. Methods: Our survey included 139 HbSS and 11 HbSC patients. Molecular genotypes have been identified by PCR-RFLP and α-thalassemia 3.7 kb deletion by ASO-PCR. Male/female distribution was 42.3%/57.7%. Results: The average age at enrolment was 19.1 years for HbSS and 25.85 years for HbSC. Average fetal hemoglobin was 11.27% for HbSS and 7.86% for HbSC. Anemia in HbSS patients was more severe and hemolysis twice as stronger as HbSC individuals. The frequency distribution of the most common β-globin haplotypes among HbSS patients was 52.5% CAR/CAR, 23.7% CAR/Ben and 18% Ben/Ben. For the HbSC group, the haplotype distribution was 36.3% CAR/CI, 27.3% Benin/CI, 18.2% CAR/CII, 9.1% CAR/CIII and 9.1% Benin/CII. Of the HbSS patients, 13.7% and 2.8% were heterozygous and homozygous for the α-thalassemia 3.7 kb deletion, respectively. No HbSC patients presented the deletion. Conclusions: Here we present the distribution of haplotypes β S and β C and α-thalassemia deletion 3.7 kb in a population sample with SCD from the Occidental Brazilian Amazon. Results have been analyzed in the context of hematological and biochemical profiles. J Hematol. 2016;5(4):123-128 doi: https://doi.org/10.14740/jh310w

Beta-globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B.

Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta-globin haplotypes. Haplotype A is very similar to the goat beta-globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta-C globin, which encodes a globin with high oxygen affinity. We surveyed the beta-globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2-3 million years old. Approximately 40 kb of the sequence flanking the ~37-kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta-globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40-kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40-kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non-Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries.

FOXO3 Variants Are Associated With Lower Fetal Hemoglobin Levels In Children With Sickle Cell Disease

Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia (SCA) exhibit wide variability in their degree of clinical severity. One of the most powerful and reproducible predictors of disease severity is the level of endogenous fetal hemoglobin (HbF). Several genetic modifiers of HbF levels have previously been identified, by association with beta globin haplotypes, or by genome wide association studies. These methods are only able to detect common variants, with a minor allele frequency (MAF) greater than 5%. We have applied whole exome sequencing (WES) to find new genetic variants associated with baseline HbF in SCA, using 171 pediatric SCA genomes from participants in two clinical trials, HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980). WES allows identification of both common and rare exonic variants, and use of burden analysis testing. In order to capture the association between the phenotype variants with a MAF below 1%, burden tests maximize power by grouping low frequency variants together by gene. This allows us to transition from a single variant theory, in which a variant is associated with a phenotype, to a gene-based theory, in which a collection of rare variants within a single gene are associated with the phenotype. Burden analysis (T1), found seven unique non-synonymous variations in a Forkhead box O transcription factor, FOXO3, to be significantly associated with lower HbF (p=5.6x10-4, b-value ln HbF -0.66). All variants produced the same effect, a lowering of HbF. HbF values were normalized using natural log transformation to permit analysis, and adjusted for age, BCL11A, and XmnI variant status. The box-plot below shows the overall effect of any of the non-synonymous exonic variants in FOXO3 on baseline HbF compared to wild-type. Each individual was heterozygous for a variant. FOXO3 is involved in multiple cellular processes, including cell cycle arrest, removal of reactive oxygen species, and regulation of erythroid differentiation. Erythroid maturation is altered by changes in post-translational maturation (PTM) such as removal of a phosphorylation site, as the in Ser553Phe variant identified in our analysis (Bakker, et al, JCB 2004). Changes in erythroid maturation kinetics may affect the amount of HbF produced. Variations in FOXO3 may represent another contributor to the heritability of HbF in patients with SCA. Further functional genomics analysis of FOXO3 may add to our understanding of gamma globin induction.Table 1Description of FOXO3 variants found to be associated with lower HbF in a cohort of pediatric sickle cell patients by WES and burden testing (T1).Chr 6LocationAmino Acid ChangeAltered PTMPolyPhen2 prediction%MAF108882607Asp66Asnnone knownPossibly damaging0.3108882830Ala140Valnone knownBenign0.6108984883Asp283Asnnone knownDamaging0.3108985057Ala121Thrnone knownBenign0.3108985280Pro415Leunone knownDamaging0.6108985679Arg548HismethylationDamaging0.3108985694Ser553PhephosphorylationBenign0.3 [Display omitted] Disclosures:Off Label Use: Hydroxyurea is not FDA approved for use in pediatric sickle cell patients.

Early Clinical Manifestations, Presence Of a Single Bantou Haplotype and High Baseline Reticulocyte Count Predict Severity In a Sickle Cell Anemia Newborn Cohort

IntroductionSickle cell anemia (SCA) is a monogenic disease with multigenic expressivity. The ability to identify high-risk SCA-children would permit accurate prognostication and better genetic counselling, and facilitate the planning of clinical trials. Miller et al. (CSSCD) reported in 2000 that early dactylitis, baseline Hb level<7g/dl, and leukocytosis correlated significantly with adverse outcomes later in life. However, Quinn et al. reported in 2007 in the Dallas newborn cohort that none of the events before age 3 predicted death or stroke after age 3. Patients and MethodsThis study revisits this issue using the Créteil Newborn Cohort (1986-2011). The dates and nature of the 1st SCA-related manifestations and further clinical events were prospectively recorded: vaso-occlusive crises (VOC) including dactylitis, acute chest syndromes (ACS), strokes, splenic sequestrations and acute anemia < 6g/dl. Alpha-genes, beta-globin haplotypes, G6PD activity, CD36 platelet expression were determined and baseline biological parameters (WBC, PMN, platelets, Hb, Hct, MCV, reticulocytes, HbF%, LDH) were recorded between 1-2 years of age, a minimum of 3 months away from a transfusion, one month from a painful episode and before any intensive therapy. The criteria of severity retained for this study were death or events requiring intensive therapy, defined as hydroxycarbamide (HC) prescribed for patients having experienced at least 3 VOC/year or 2 ACS or with baseline Hb<7g/dl; transfusion program (TP) for those with recurrent splenic sequestrations or strokes or abnormal velocities on Doppler (TCD); stem cell transplantation (HSCT) for those with indication of intensive therapy and genoidentical donor. Cox regression was used to analyze the predictive factors of severity. ResultsThis newborn cohort consists of 363 patients (55 SC,28 Sb+,262 SS,15 Sb0,3 SDPunjab). Median age at last visit was 6.4 years, providing 2827 patient-years of follow-up. As no SC or Sb+ patient died or experienced events requiring intensive therapy, analysis of severity criteria was only performed on SS, Sb0, SDPunjab, referred as SCA-patients (n=280). Alpha-thalassemia was present in 41.6%, Bantou/Bantou beta haplotype in 43.4% (vs 5.9% in the CSSCD cohort), Benin/Benin in 22.9%, Senegal/Senegal in 10.2%, “other” in 23.4%. G6PD deficiency was observed in 11.4% and CD36 non-expression in 7%. Kaplan-Meier estimates of survival by age 5 and 18 were 97.6% (95%CI:95.6-99.6%). One-hundred-fifty-one/280 SCA-patients (54%) required intensive therapy at the median age of 3.8yr (0.1-20): 69 HC, 136 TP, and 41 HSCT (patients may have successively received HC, TP and HSCT). Cumulative risk of severity criteria was 45% by age 5 and 70% by age 10. Univariate Cox analysis showed that Bantou presence (52.6%), CD36 non-expression, early SCA-1st manifestation (<1 and <3 year of age), baseline high level of WBC, reticulocytes and LDH, low levels of Hb and Hct were significantly associated with severity whereas platelets, HbF, MCV, alpha genes number and G6PD were not. Multivariate Cox analysis retained as significant and independent predictive risk factors for severity: high reticulocyte level (>400x109/L) (HR=2.82,95%CI:1.70-4.69,p<0.001); the presence of a single Bantou haplotype (HR=1.76,95% CI:1.18-2.60,p=0.007); and early (<1 year of age) first SCA-clinical manifestation (HR=2.35,95%CI:1.55-3.56,p<0.001). ConclusionThis study confirms Miller's report, showing that the risk level can be estimated by age 2 in SCA-patients, a result potentially useful for prospective trials (HC/HSCT). Moreover, we show for the first time that the presence of a single Bantou haplotype has predictive value for severity in this cohort where its frequency is much higher than in the CSSCD cohort. [Display omitted] Disclosures:Bernaudin:Novartis: Research Funding.

High Altitude Genetic Adaptation In Tibetans Does Not Include Increased Hemoglobin-Oxygen Affinity

Increased hemoglobin-oxygen affinity has been shown to be an adaptive response to hypoxia in many high altitude animals such as the Andean goose, guinea pig and llamas (Reynafarje C. 1975; Hebbel R. 1978). It has been reported that people living in a high altitude, hypoxic environment have also developed a similar adaptation. Native Tibetans are known to have lived at an average of 3000-5000 meters on the Tibetan Plateau for more than 20,000 years, and have undergone genetic adaptations that have enabled them to thrive in this reduced oxygen environment. Most Tibetans are thus protected from polycythemia and other features of chronic mountain sickness. Several studies have reported higher arterial oxygen saturations among Tibetans as part of their genetic adaptation (Beall C. 1994; Moore L. 2001; Niermeyer S. 1995), thereby concluding that they have higher hemoglobin-oxygen affinity. Further, recent genomic studies have reported that beta-globin haplotypes (HBB and HBG2) have been selected in Tibetans, suggesting the presence of hemoglobin variants as a beneficial factor of Tibetan adaptation (Yi. 2010). However, some of the reports of increased hemoglobin-oxygen affinity are based on single readings of arterial oxygen measurements. Hemoglobin-oxygen affinity is more optimally measured by deriving the P50 value, which is the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen. A decreased P50 can be due to mutated globin genes resulting in high oxygen affinity hemoglobins, low 2,3 BPG, high pH or low temperature. The hemoglobin-oxygen dissociation is optimally derived by hemoximeter measurements of the percent saturation of hemoglobin at various partial pressures of oxygen. The resultant curve has a sigmoid shape due to the cooperative binding of oxygen to the four globins in the hemoglobin tetramer; this cooperative interaction can be enumerated as a Hill coefficient “n”. If a hemoximeter is not readily available, the P50 can be estimated from the venous blood gas using the measured pO2, hemoglobin oxygen percent saturation O2%, and pH (Lichtman M. 1976); however the Hill coefficient “n” cannot be derived by this method.To definitely establish whether the Tibetan adaptation to high altitude hypoxia involves increased hemoglobin-oxygen affinity, we conducted the following study of direct and indirect oxygen-hemoglobin affinity among Tibetans living at two different altitudes.We enrolled 14 healthy ethnic Tibetans and one closely related Nepalese Sherpa. There were 8 males and 7 females ages ranging 35-75 years. The first group consisted of 5 ethnic Tibetans living in Srinagar, India (1,600 meters), on whom venous blood gases were done and the P50 was derived using pH, PO2 and O2 saturation using the formula described by Lichtman and colleagues. Three were born in Tibet and two were offspring of Tibet-born parents. The second group consisted of 10 volunteers (9 Tibetans and one Nepalese Sherpa) residing in Salt Lake City, UT, (1,300 meters) whose peripheral blood was evaluated by Hemox Analyzer for obtaining P50 values and “n” Hill coefficients for hemoglobin oxygen binding. All the ethnic Tibetans in Salt Lake City were born in Tibet except for one, and the Nepalese Sherpa was born in Nepal.The results are depicted in Table. The P50 measured by venous blood gases on the Tibetan volunteers from Srinagar, India and those measured by Hemox Analyzer on the 10 volunteers from Salt Lake City, UT were normal, with values in the normal range (22-28 mmHg). No hemoglobin variants were detected by high pressure liquid chromatography in these 15 Tibetan volunteers.TableSubject IDP50 (mmHg)“n” Hill CoefficientS 0526.38n/aS 0825.95S 1326.55S 1523.68S 2722.72U 1926.962.97U 2025.162.83U 2124.202.89U 2225.462.84U 2322.502.89U 2424.062.87U 2524.282.83U 2622.352.82U 2723.292.79U 2825.992.75We report no evidence for the presence of high hemoglobin-oxygen affinity in Tibetans as a constituent of their genetic adaptation. Our data rule out the existence of hemoglobin variants and aberrant 2,3 BPG metabolism as possible features of Tibetan high-altitude adaptation; however acquired transient metabolic alterations at high altitudes, cannot be excluded to account for possible changes in hemoglobin-oxygen affinity but these are not evolved persistent features of Tibetan genetic adaptation. Studies of Tibetans living in these extreme hypoxic environment (>4,000m) are now planned. Disclosures:No relevant conflicts of interest to declare.

Effect Of Genetic Modifiers Of Baseline Fetal Hemoglobin On Hydroxyurea Response In Children With Sickle Cell Disease

Hydroxyurea is a safe and effective therapy for sickle cell disease (SCD), with the majority of its benefit correlating with the amount of fetal hemoglobin (HbF) produced at maximum tolerated dose, or MTD. There is substantial individual variability in HbF response to hydroxyurea, with baseline HbF levels accounting for approximately 40% of the observed variability in final HbF at MTD. Several genetic modifiers of baseline, or endogenous, HbF levels have previously been identified by genome wide association studies. These include certain beta-globin haplotypes, and polymorphisms at the BCL11A and HBS1L-MYB gene loci. The effect of these known genetic modifiers of baseline HbF on drug response has been investigated in a small (n=38) cohort of pediatric patients treated with hydroxyurea in several centers with different treatment guidelines. In this limited group, no association was found between the BCL11A or HBS1L-MYB variants and the change in HbF at MTD (ΔHbF; final HbF minus baseline HbF). Co-inheritance of alpha thalassemia has been reported to have a negative effect on HbF response to hydroxyurea. To independently verify these findings in a larger sample size, we tested the effect of four BCL11A single nucleotide polymorphisms (SNPs), three HBS1L-MYB SNPs, the XmnI polymorphism, and co-inheritance of α-thalassemia on ΔHbF at MTD in a cohort of 171 pediatric SCA patients, treated prospectively and uniformly on HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980) protocols. These patients represent the most accurate hydroxyurea phenotypes available, as all were meticulously titrated to MTD, and had complete laboratory data demonstrating compliance, such as absolute neutrophil count and absolute reticulocyte count within the therapeutic range. In our cohort, the BCL11A SNPs rs1427407, rs4671393 and rs11886868 were significantly associated with baseline HbF (Table 1). We saw no association between baseline HbF and any of the HBS1L-MYB SNPs, XmnI, α-thalassemia or BCL11A SNP rs7599488. In contrast to other reports, we found that coinheritance of α-thalassemia did not affect hydroxyurea treatment response (p=0.088). We found that BCL11A SNPs rs1427407, rs4671393 and rs11886868 were significantly associated with reduced ΔHbF following hydroxyurea treatment (Table 1), where individuals with the BCL11A SNPs had a smaller ΔHbF compared to individuals without the polymorphisms, as shown by the negative baseline β-value. For example, one BCL11A rs1427407 SNP is associated with a ΔHbF 3.46 percentage points lower than individuals without the SNP at MTD, with an additive, dose effect of the SNP at the second allele; homozygous individuals have higher baseline HbF, lower ΔHbF compared to heterozygotes or wild-type individuals (Figure 1). The other variants HBS1L-MYB SNPs, XmnI, α-thalassemia or BCL11A SNP rs7599488 did not significantly impact ΔHbF. None of the tested polymorphisms, including the BCL11A SNPs, were associated with a significant difference in final HbF levels. Individuals with higher baseline HbF due to BCL11A polymorphisms demonstrate a statistically significant lower rise in HbF in response to hydroxyurea than individuals without these polymorphisms. Identification of more variants associated with baseline and ΔHbF through next generation sequencing will help elucidate whether the negative effect of high baseline on ΔHbF is a BCL11A specific effect, or a manifestation of a general threshold effect, that there is a maximum amount of HbF an individual is able to achieve through hydroxyurea induction.Table 1Association between BCL11A SNPs and response to hydroxyurea.GeneSNP IDBaseline β-value (lnHbF)Baseline p-valueΔHbF β-value (%HbF)ΔHbF p-valueBCL11Ars14274070.3569.05x10-5-3.461.02x10-3BCL11Ars46713930.272.17x10-3-2.627.21x10-3BCL11Ars118868680.131.61x10-4-3.007.86x10-4BCL11Ars75994880.080.30-0.780.33 [Display omitted] Disclosures:Off Label Use: Hydroxyurea is not FDA approved for use in pediatric sickle cell patients.

Genetic modifiers of sickle cell anemia in the BABY HUG cohort: influence on laboratory and clinical phenotypes

The recently completed BABY HUG trial investigated the safety and efficacy of hydroxyurea in infants with sickle cell anemia (SCA). To investigate the effects of known genetic modifiers, genomic DNA on 190 randomized subjects were analyzed for alpha thalassemia, beta-globin haplotype, polymorphisms affecting endogenous fetal hemoglobin (HbF) levels (XmnI, BCL11A, and HBS1L-MYB), UGT1A1 promoter polymorphisms, and the common G6PD A(-) mutation. At study entry, infants with alpha thalassemia trait had significantly lower mean corpuscular volume, total bilirubin, and absolute reticulocyte count. Beta-globin haplotypes associated with milder disease had significantly higher hemoglobin and %HbF. BCL11A and XmnI polymorphisms had significant effects on baseline HbF, while UGT1A1 promoter polymorphisms significantly influenced baseline serum bilirubin. At study exit, subjects randomized to placebo still exhibited laboratory effects of alpha thalassemia and other modifiers, while those assigned hydroxyurea had treatment effects that exceeded most genetic influences. The pain phenotype was influenced by HbF modifiers in both treatment groups. These data document that genetic polymorphisms do modify laboratory and clinical phenotypes even in very young patients with SCA. The hydroxyurea effects are more potent, however, indicating that treatment criteria should not be limited to certain genetic subsets, and supporting the use of hydroxyurea for all young patients with SCA.

Distribution of beta-globin haplotypes among the tribes of southern Gujarat, India

The present study was carried out in Indo-European speaking tribal population groups of southern Gujarat (India) to elucidate the allelic and haplotypic content of β-globin system in individuals with HbAA genotypes. 6 neutral restriction sites of the β-globin system were analysed and various statistical parameters were estimated to draw meaningful interpretations. All the 6 sites were found to be polymorphic and most were in Hardy–Weinberg Equilibrium in the studied group. Haplotypes were constructed using two different combinations of the 6 restriction sites analysed. Analysis of the 5 sites revealed a set of three predominant haplotypes, ‘+−−−−’, ‘−++−+’ and ‘−+−++’; and haplotypes ‘+−−’, ‘++−’ and ‘+++’ were found to be the most frequent when the 3 sites were used to construct the haplotypes. Haplotypic heterozygosity levels (>83%) observed in the present study group were comparable to those observed in African and Afro-American populations and greater than other world populations. All the ancestral haplotypes, +−−−−−, −++−+, −+−++ and −−−−+ were found in the study group. The distribution pattern of various haplotypes was consistent with the global pattern. The paucity of comparable data from other Indian populations restricted one from making interpretations about the study group's relationships with other Indian populations but the results were indicative of older population histories or experience of gene flow by the study group and their affinities with populations of southern India.

Beta-S globin haplotypes in patients with sickle cell anemia: one approach to understand the diversity in Brazil

Sickle cell anemia (SCA) is defined as a monogenic disease that characterizes thehomozygous state of hemoglobin S (Hb S). Hemoglobin S polymerizes under adverse conditionssuch as deoxygenation, acidosis or dehydration. Its polymers deform erythrocytes and leadto a diverse and complex pathophysiology(1,2). Beta-S globin haplotypes (βS haplotypes) areimportant in the comprehension of the clinical diversity of SCA patients. Five haplotypes havebeen associated with different ethnic groups. They are identified at specific restriction sites andnamed according to their region of origin and prevalence: Bantu, Benin, Senegal, Arabic-Indian(Saudi) and Cameroon(3). This study aimed to assess the frequencies of βS haplotypes in patientswith SCA from Rio de Janeiro State, Brazil. We analyzed DNA samples from 790 patients withsickle cell disease (SCD) and classified them according to their haplotypes through an analysisof six polymorphic sites reported by Sutton et al.(4). According to these authors, haplotypes thatdo not exhibit known digestion patterns should be classified as atypical. The polymorphic sites were evaluated by polymerase chain reaction (PCR) followed by restriction fragment lengthpolymorphism (RFLP)(4,5). Of the 790 patients, 527 had SCA with the following frequencies of genotyped βS haplotypes: 295 (56.0%) Bantu/Bantu; 128 (24.3%) Bantu/Benin; 44 (8.3%)Bantu/Atypical; 36 (6.84%) Benin/Benin; 10 (1.9%) Benin/Atypical; three (0.57%) Bantu/Cameroon; three (0.57%) Benin/Cameroon; three (0.57%) Atypical/Atypical; two (0.38%)Bantu/Saudi; two (0.38%) Bantu/Senegal and one (0.19%) Benin/Saudi. From the totalnumber of 1054 chromosomes analyzed, 769 (72.96%) were identified with the Bantu allele according to the allelic frequency evaluation; 214 (20.3%) Benin; 60 (5.7%) Atypical; six(0.57%) Cameroon; three (0.28%) Saudi and two (0.19%) Senegal. Data from genotype and allele frequencies are shown in Table 1. The high frequencies of Bantu and Benin haplotypesdemonstrate the influence of Western and South-Central African on the evolution of the Brazilian population(6). We identified atypical haplotypes which may have originated due togenetic mechanisms such as gene conversions and the intense miscegenation between ethnicgroups that inhabit Rio de Janeiro State and Brazil. We emphasize the need of identifying and characterizing these genetic variations in order to better understand the clinical aspects and phenotypic diversity found in Brazilian SCD patients. Table 1 Allelic and genotypic frequency of beta-S globin haplotypes

Gallstones in a Newborn-Cohort with Sickle Cell Anemia (SCA): Cumulative Risk and Predictive Factors

Abstract 513 Background:The accelerated hemolysis in SCA increases bilirubin excretion and the susceptibility to pigment gallstone formation. Uridine diphosphate-glucuronosyltransferase (UGT1A1) gene polymorphism and alpha-thalassemia are factors known to modify this risk. However, their relative influence and other factors such as SCA disease severity and the use of ceftriaxone for acute febrile episodes remain to be explored in a newborn cohort. The goal of this study was to define the cumulative incidence and risk factors for the occurrence of gallstones in a SCA-newborn-cohort. Patients and Methods:Alpha-genes, beta-globin haplotypes, UGT1AI genotype, G6PD-enzymatic activity were recorded; baseline biological parameters were determined during the second year of life, away from crises and transfusions as previously described (Blood 2011,117, 4). Annual check-ups including abdominal sonography were performed as soon as the age of 1 year. Cholecystectomy was systematically performed in the 6 months following the discovery and confirmation of presence of gallstones. All hospitalizations and their causes were prospectively recorded. Patients were censored on the date of gallstone occurrence or last abdominal sonography; event rates/year (pain crises, ACS, febrile episodes) were calculated, and KM-estimated cumulative risk for gallstones and Cox regression were used to assess the predictive factors for gallstones. Results:SCA-patients (364: 353 SS, 8 Sb0, 3 SDPunjab; 182 F,182 M) born before February 2010 and followed until the age of 18–20 years were included in this study. Alpha-thalassemia was present in 46% patients (available in 323; 2 genes: n=33; 3 genes: n=116; 4 genes: n=172; 5 genes: n=2); beta-haplotypes (available in 291) were Car/Car in 42.6%, Ben/Ben in 19.9%, Sen/Sen in 9.6%, and others in 27.8%. G6PD activity (available in 309) was deficient in 11.6%. UGTA1 polymorphism (available in 175) showed Gilbert genotype in 19.8%, i.e., 7/7 in 13.4% and 7/8 in 6.4%. The frequency of the other genotypes was: 5/6: 6.4%; 5/7:7%; 5/8:0.6%; 6/6:23.3%, 6/7:34.3%; and 6/8:8.1%. Unconjugated bilirubin values at baseline were significantly correlated with the total number of UGTA1A1 repeats (r=0.415, p<0.001). Mean rates (SD) of pain crises, ACS and acute febrile episodes were 0.56 (0.68), 0.12 (0.18), 0.34 (0.54), respectively. Gallstones were observed in 98/364 patients at the median age of 9.2 (range: 2.5 to 17.9 years). The cumulative risk of gallstones was 5% by age 5, 20.7% by age 10 and 35.6 % by age 15 and was not significantly different in patients with Gilbert polymorphism (Log Rank p=0.4) but was significantly related to the number of alpha-genes (Log-Rank p=0.039). However, the risk was significantly increased in patients homozygous or heterozygous for (TA8) (Log Rank p=0.016). Univariate Cox regression analysis showed that the number of alpha-genes was a significant risk factor (p=0.014) whereas G6PD deficiency, beta-haplotypes and Gilbert genotype were not significant risk factors; however, the presence of at least one allele (TA8) was a significant risk factor (p=0.025). Among the baseline biological parameters, hemoglobin, WBC, neutrophils, platelets, MCV, and bilirubin were not significant factors whereas HbF level (p=0.028), reticulocyte count (p<0.001) and LDH (p=0.020) significantly increased the risk for gallstones. Among clinical events, pain crises, ACS and febrile episodes significantly increased the risk (p<0.001). Multivariate Cox analysis including all the significant risk factors in the univariate analysis retained the deletion of 2 alpha genes (HR=4.66; 95% CI:1.11-19.52,p=0.035) which decreases the risk, the presence of at least one allele (TA8) (HR=2.26:95% CI:1.07-4.78, p=0.032), which increases the risk and the baseline reticulocytes count per 1×109/L increase(HR:1.001;1.000-1.002;p=0.005), as independent and significant predictive factors for gallstones. Conclusion:This prospective SCA newborn-cohort study shows that gallstones may begin as soon as 2.5 years of age and that the cumulative risk reaches a high 35.6% by age 15. The deletion of 2 alpha genes, the presence of at least one UGT1A1 (TA8) allele and high baseline reticulocyte count are independent and significant predictive factors showing that the risks are multifacturial and highly and primarily dependent on hemolysis [Display omitted] Disclosures:No relevant conflicts of interest to declare.

TNF-alpha and IL-8: Serum levels and gene polymorphisms (−308G>A and −251A>T) are associated with classical biomarkers and medical history in children with sickle cell anemia

Sickle cell anemia (SCA) is a disorder characterized by a heterogeneous clinical outcome. In the present study, we investigated the associations between Tumor Necrosis Factor-alpha (TNF-alpha) −308G>A and Interleukin 8 (IL-8) −251A>T gene polymorphisms, medical history and classical biomarkers in children with steady-state SCA. In total, 210 SCA patients aged 2–21years and 200 healthy controls were studied. Gene polymorphisms, betaS-globin haplotypes and a 3.7-kb deletion in alpha2-thalassemia (α2-thal3.7kb) were investigated by PCR/RFLP analysis, and cytokine levels were determined by ELISA. Splenomegaly (p=.032) was more prevalent among children younger than 5years of age. The A allele of the TNF-alpha −308G>A gene polymorphism and the presence of α2-thal3.7kb were associated with an increase risk of splenic sequestration events (p=.001; p=.046), while the T allele of the IL-8 −251A>T gene polymorphism was considered to be a protective factor for splenomegaly events (p=.032). Moreover, the A allele of the TNF-alpha −308G>A gene polymorphism was associated with high TNF-alpha levels (p=.021), and the hemoglobin F and hemoglobin S haplotypes were correlated with serum levels of IL-8. The logistic regression analysis showed significant effects of the TNF-alpha and IL-8 gene polymorphisms, betaS-globin gene haplotypes and α2-thal3.7kb on the occurrence of splenic sequestration events. Our study emphasizes that the identification of new genetic and immunological biomarkers and their associations with classical markers is an important strategy to elucidate the underlying causes of different SCA phenotypes and their effects on patient outcome.