BCL11A Rs1427407 Research Articles

Foetal haemoglobin elevation, unfavourable prognosis, and protective role of genetic variants HBG2 rs7482144, HBS1L-MYB rs9399137 and BCL11A rs4671393 in children with ALL

Foetal haemoglobin elevation, unfavourable prognosis, and protective role of genetic variants HBG2 rs7482144, HBS1L-MYB rs9399137 and BCL11A rs4671393 in children with ALL

A Population-Oriented Genetic Scoring System to Predict Phenotype: A Pathway to Personalized Medicine in Iraqis With β-Thalassemia

To assess the roles of genetic modifiers in Iraqi β-thalassemia patients, and determine whether a genotype-based scoring system could be used to predict phenotype, a total of 224 Iraqi patients with molecularly characterized homozygous or compound heterozygous β-thalassemia were further investigated for α-thalassemia deletions as well as five polymorphisms namely: rs7482144 C > T at HBG2, rs1427407 G > T and rs10189857 A > G at BCL11A, and rs28384513 A > C and rs9399137 T > C at HMIP. The enrolled patients had a median age of 14 years, with 96 males and 128 females. They included 144 thalassemia major, and 80 thalassemia intermedia patients. Multivariate logistic regression analysis revealed that a model including sex and four of these genetic modifiers, namely: β+ alleles, HBG2 rs7482144, α-thalassemia deletions, and BCL11A rs1427407 could significantly predict phenotype (major versus intermedia) with an overall accuracy of 83.9%. Furthermore, a log odds genetic score based on these significant predictors had a highly significant area under curve of 0.917 (95% CI 0.882–0.953). This study underscores the notion that genetic scoring systems should be tailored to populations in question, since genetic modifiers (and/or their relative weight) vary between populations. The population-oriented genetic scoring system created by the current study to predict β-thalassemia phenotype among Iraqis may pave the way to personalized medicine in this patient’s group.

Development of a Genetic Risk Score for Albuminuria in Pediatric Patients with Sickle Cell Anemia

Introduction Sickle cell anemia (SCA) causes progressive, age-dependent, kidney damage. Albuminuria is an early clinical biomarker for chronic kidney disease (CKD) and is associated with progressive glomerular injury. Identifying patients at risk for albuminuria is critical for early institution of renoprotective interventions to prevent progressive kidney injury. Developing a genetic risk score (GRS) to predict albuminuria is one approach to identifying high risk patients. Our prior data demonstrates that Apolipoprotein-L1 ( APOL1 G1/G2) is associated with early development of albuminuria in children with SCA (PMID:30890594). However, this gene variant does not account for all kidney disease progression. Multiple other gene variants that modify SCA-severity have been associated with CKD in adults with SCA. We hypothesized that developing a genetic risk score (GRS) will allow for identification of pediatric patients at increased risk for development and progression of kidney disease. We aimed to develop a novel pediatric GRS that can identify pediatric SCA patients at high risk for albuminuria. Methods Gene allele variants in APOL1 G1/G2, HMOX1, BCL11A and α-thalassemia (α -3.7 allele), were identified in 324 African American patients with SCA (HbSS or HbS 0-thalassemia genotypes) enrolled in the Sickle Cell Clinical Research and Intervention Program (NCT02098863), a lifespan, longitudinal cohort study. The first urine albumin to creatinine ratio (ACR) collected after birth was analyzed as a continuous variable and dichotomized by a threshold of ACR ≥30mg/g creatinine as albuminuria. We defined a GRS 3 based on established risk alleles in three genes: APOL1 (G1/G2 ), BCL11A (rs1427407), and α -3.7(PMID:27658436). High risk was defined as patients with APOL1 (G1/G1, G2/G2 or G1/G2), without α -3.7 and with wildtype BCL11A (G/G); low-risk was defined as patients negative for APOL1 G1/G2 and with α -3.7 (either α -3.7/αα or α -3.7/α -3.7) and the BCL11A T allele (either G/T or T/T). All other combinations were defined as intermediate risk. By adding HMOX1 (rs743811), C allele (TT/TC vs CC), we further refined GRS 3 as GRS 4 using the summation of the number of high-risk alleles of kidney outcomes for four variants and created four risk categories. The generalized linear regression model was used to associate additive dose-effect models for GRS 3 and GRS 4 with continuous ACR and albuminuria, adjusted for age, sex, hydroxyurea treatment, 5 principal components, and, if significant, the age-genotype interaction. ACR was log-transformed to approximate normality. As we included variants with known associations, we used a one-sided p-value for testing associations of individual variants, GRS 3, and two-sided p-values for GRS 4. P-values <0.05 were considered significant. Results In 324 individuals with SCA, those with albuminuria (34, 10.5%) appeared to be older than those without albuminuria (290, 89.5%) (mean 9.8 years [standard deviation ±4.4 years] vs. 8.6 [± 4.0], p=0.053). There were no significant differences between these groups by sex (female 58% vs. 49%, p=0.37), proportion on hydroxyurea (62% vs. 59%, p=0.85) or fetal hemoglobin (15.6 [± 10.61] vs. 14.8% [± 10.0], p= 0.81). All four individual genetic variants were associated with the first ACR (all p ≤ 0.028) and albuminuria (all p≤ 0.03). GRS 3 was significantly associated with the first ACR (estimate=1.4, standard error [SE]=0.5, p=0.0035) and albuminuria (odds ratio [OR]=3.0, 95% CI: 1.2-7.5, p= 0.008). After adding HMOX1 to GRS 3, GRS 4 was more significantly associated with the first ACR (estimate=0.9, SE=0.3, p=0.0005) and albuminuria (OR=2.0, 95% CI: 1.2-3.4, p=0.003). Summary We developed a GRS 3 based on three genetic variants associated with albuminuria in pediatric patients with SCA and stratified patients into different risk categories. Addition of a fourth genetic variant to create GRS 4improved the model. Our findings will need to be validated in larger longitudinal studies to further examine the association between GRS 4 and development of albuminuria and chronic kidney disease. Genetic prediction strategies using GRS has the potential to identify high risk SCA patients for whom renoprotective measures could prevent the development of albuminuria.

English

High levels of hemoglobin F (HbF) could reduce the severity of sickle cell disease (SCD) by inhibiting hemoglobin S (HbS) polymerization. HbF expression is modulated by the Quantitative Trait Loci (QTLs) located on the HBS1L-MYB intergenic region (HMIP) (rs28384513) and intron 2 of the B-cell lymphoma/leukemia 11A (BCL11A) gene (rs4671393, rs1427407). To assess the impact of QTLs of HbF on clinical and biological parameters associated with the severity of sickle cell disease in a Senegalese pediatric population, 301 children with SCD not treated with hydroxyurea were recruited. The numbers of hospitalizations and VOC were estimated over 2 years. HbF levels were determined by HPLC. Three QTLs of HbF were genotyped by High Resolution Melting (HRM), two single nucleotide polymorphisms (SNPs) of BCL11A and one SNP of HMIP. Data analysis was performed using SPSS. The mean frequency of hospitalizations was 0.84 ± 1.29. The mean number of vaso-occlusive crises (VOC) episodes was 2.73 ± 1.98. The mean Hb concentration was 7.76 ± 1.05 g/dl. The mean HbS was 82.28 ± 4.78% and the mean HbF was 9.49 ± 5.12%. BCL11A (rs1427407) was associated with fewer hospitalizations. Both BCL11A SNPs were associated with increased HbF levels. BCL11A SNPs were associated with increased HbF levels, decreased HbS levels, and decreased hospitalizations (rs1427407). However, no association was noted between these SNPs and the number of VOC episodes. Thus, HbF QTLs are not the only genetic factors modulating the clinical severity of sickle cell disease, which suggests the involvement of other genetic factors such as alpha-thalassemia.   Key words: sickle cell disease, hemoglobin F, quantitative trait loci of HbF, HBS1L-MYB intergenic region, B-cell lymphoma/leukemia, vaso-occlusive crises.

Fetal hemoglobin modulates neurocognitive performance in sickle cell anemia✰,✰✰

Fetal hemoglobin (HbF) is a modifier of the clinical and hematologic phenotype of sickle cell anemia (SCA). Three quantitative trait loci (QTL) modulate HbF expression. The neurocognitive effects of variants in these QTL have yet to be explored. We evaluated the relation between 11 SNPs in the three HbF QTL: BCL11A, MYB, the HBB gene cluster, and full-scale intelligence (IQ) in SCA. The prospective longitudinal cohort study, Sickle Cell Clinical Research and Intervention Program, was used as a discovery cohort (n=166). The genotypes for 11 SNPs were extracted through whole genome sequencing and were analyzed using an additive model. A polygenic score for HbF (PGSHbF) integrating the numbers of low HbF alleles from 11 SNPs was analyzed as a continuous variable. The Cooperative Study of Sickle Cell Disease (n=156) and the Silent Cerebral Infarction Transfusion (n=114) Trial were used as two independent replication cohorts. Benjamini and Hochberg approach was used to calculate false discovery rate adjusted p-value (pFDR). HbF was positively associated with IQ (minimum raw p=0·0018) at pFDR<0·05. HbF mediated the relationship between two BCL11A SNPs, rs1427407 and rs7606173, HBS1L-MYB: rs9494142, and PGSHbF with IQ (minimum raw p=0·0035) at pFDR<0·05. As the major modulator of the severity of SCA, HbF also influences neurocognition, which is done through mediation of its QTL. These findings have implications for early identification of neurocognitive risk and targeted intervention.

Genetic modifiers of fetal hemoglobin affect the course of sickle cell disease in patients treated with hydroxyurea.

The course of sickle cell disease (SCD) is modified by polymorphisms boosting fetal hemoglobin (HbF) synthesis. However, it has remained an open question how these polymorphisms affect patients who are treated with the HbF-inducing drug hydroxyurea/ hydroxycarbamide. The German SCD registry offers the opportunity to answer this question, because >90% of patients are treated according to national guidelines recommending the use of hydroxyurea in all patients above 2 years of age. We analyzed the modifying effect of HbF-related genetic polymorphisms in 417 patients with homozygous SCD >2 years old who received hydroxyurea. HbF levels were correlated with higher total hemoglobin levels, lower rates of hemolysis, a lower frequency of painful crises and of red blood cell transfusions. The minor alleles of the polymorphisms in the γ-globin promoter (rs7482144), BCL11A (rs1427407) and HMIP (rs66650371) were strongly associated with increased HbF levels. However, these associations did not translate into lower frequencies of vaso-occlusive events which did not differ between patients either carrying or not carrying the HMIP and BCL11A polymorphisms. Patients on hydroxyurea carrying the γ-globin promoter polymorphism demonstrated substantially higher hemoglobin levels (P<10-4) but also higher frequencies of painful crises and hospitalizations (P<0.01) when compared to patients without this polymorphism. Taken together, these data indicate that the γ-globin, HMIP and BCL11A polymorphisms correlate with increased HbF in SCD patients on hydroxyurea. While HbF is negatively correlated with the frequency of painful crises and hospitalizations, this was not observed for the presence of known HbF-boosting alleles.

Significance of genetic modifiers of hemoglobinopathies leading towards precision medicine

Hemoglobinopathies though a monogenic disorder, show phenotypic variability. Hence, understanding the genetics underlying the heritable sub-phenotypes of hemoglobinopathies, specific to each population, would be prognostically useful and could inform personalized therapeutics. This study aimed to evaluate the role of genetic modifiers leading to higher HbF production with cumulative impact of the modifiers on disease severity. 200 patients (100 β-thalassemia homozygotes, 100 Sickle Cell Anemia), and 50 healthy controls were recruited. Primary screening followed with molecular analysis for confirming the β-hemoglobinopathy was performed. Co-existing α-thalassemia and the polymorphisms located in 3 genetic loci linked to HbF regulation were screened. The most remarkable result was the association of SNPs with clinically relevant phenotypic groups. The γ-globin gene promoter polymorphisms [− 158 C → T, + 25 G → A],BCL11A rs1427407 G → T, − 3 bp HBS1L-MYB rs66650371 and rs9399137 T → C polymorphisms were correlated with higher HbF, in group that has lower disease severity score (P < 0.00001), milder clinical presentation, and a significant delay in the age of the first transfusion. Our study emphasizes the complex genetic interactions underlying the disease phenotype that may be a prognostic marker for predicting the clinical severity and assist in disease management.

BCL11A POLYMORPHISMS ASSOCIATED WITH INCREASED FETAL HEMOGLOBIN IN RESPONSE TO HYDROXYUREA TREATMENT IN A COHORT OF PEDIATRIC PATIENTS WITH SICKLE CELL ANEMIA

Fetal Hemoglobin (HbF) is a major modifier of clinical events in patients with sickle cell anemia (SCA). Hydroxyurea (HU) improves their clinical course by raising HbF level. There is variability in the response to HU treatment among SCA patients. Genome-wide association studies (GWAS) have identified the BCL11A gene and HBS1L-MYB intergenic region (HMIP-2) as the main regulators of baseline HbF production, and polymorphisms in both loci have been associated with HbF levels. However, it is unknown whether genetic polymorphisms involved in regulation of baseline HbF can modulate HbF in response to HU treatment. Therefore, we examined whether polymorphisms in BCL11A (rs11886868, rs766432, rs4671393, rs1427407, rs7557939 and rs7599488) and HMIP-2 (rs9399137, rs9402686, rs4895441 and rs9494145) were associated with HbF changes in a retrospective cohort of 110 pediatric patients diagnosed with SCA (HbSS) by the Newborn Screening Program of Minas Gerais, and followed up at Center of Hematology and Hemotherapy of Minas Gerais (HEMOMINAS), Belo Horizonte. Participants have been on HU treatment for at least one year. We assessed “delta HbF”for each patient as the “final HbF relative concentration”(last available test during HU treatment and at least three months after blood transfusion) “minus baseline HbF”(last available test before HU treatment and after the age of 5 years, and at least 3 months after blood transfusion). Genotypes were assessed by Taqman assays and real-time PCR or PCR-RFLP. Univariate associations were performed using Mann-Whitney U Test. For the 110 enrolled SCA patients (59/53.6% were females), we calculated the average age at start of HU (10.0±2.8 years), average HU dose (24.40±4.41 mg/kg/day) and the average HU treatment time (4.23±2.01 years). Mean of baseline HbF was 12.01±6.05% and final HbF concentration 18.97±8.75%. While we found no significant differences at final HbF or delta HbF among genotypes of HMIP-2 polymorphisms, carriers of the minor alleles of rs9402686, rs4895441 and rs9494145 showed higher baseline HbF when compared to wild-type homozygous patients (all p 0.05). Moreover, carriers of the minor allele T (GT or TT genotypes) of rs1427407 showed higher baseline HbF (p = 0.002) and delta HbF (p = 0.049) when compared to GG homozygous patients. Similarly, carriers of the minor allele C (AC or CC genotypes) of rs766432 showed higher delta HbF when compared to AA homozygous patients (p = 0.009). Finally, carriers of the minor allele T (CT or TT genotypes) of rs7599488 showed higher final HbF (p = 0.003) and delta HbF (p = 0.002) when compared to CC homozygous patients. BCL11A is a negative regulator of HbF expression. BCL11A polymorphisms were associated with increased HbF after HU treatment in pediatric patients with SCA, suggesting they modulate HbF levels in response to HU. Understanding genetic factors underlying HU response may clarify the interpatient variability on HbF induction upon HU treatment and help to guide therapy.

Response of Patients with Transfusion-Dependent β-Thalassemia (TDT) to Betibeglogene Autotemcel (beti-cel; LentiGlobin for β-Thalassemia) Gene Therapy Based on HBB Genotype and Disease Genetic Modifiers

Response of Patients with Transfusion-Dependent β-Thalassemia (TDT) to Betibeglogene Autotemcel (beti-cel; LentiGlobin for β-Thalassemia) Gene Therapy Based on HBB Genotype and Disease Genetic Modifiers

Novel Genetic Loci That Influence Fetal Hemoglobin Expression in Children with Sickle Cell Anemia

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p&amp;lt;0.05) in both datasets were then selected for two independent replication steps; these were designated either African-American (N=157) or African (N=269). Third, the SNVs that were significant in both dual discovery and at least one of the replication cohorts were then verified using an additional Caribbean cohort (N=149) with TaqMan techniques for genotyping specific variants. Through this multistep process, we searched for genomic loci with consistent HbF associations across multiple cohorts. Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p&amp;lt;10-8) in the discovery analysis, and 1,048 additional variants were identified with nominal HbF association (p&amp;lt;0.001). We found that 173 of these novel variants had sustained association in at least one of the replication cohorts (p&amp;lt;0.05). We selected 20 variants with the strongest and most consistent associations with HbF from the discovery and replication analyses for further verification (Table 1). Expected HbF associations with BCL11A (rs1427407) and HBS1L-MYB (rs4895441) were identified. Among other 18 novel candidate variants, the rs77737207 variant (allele frequency ~0.10) near the RUNX1T1 locus was strongly associated with lower HbF levels, while coding variant rs2279587 (allele frequency ~0.03) in the ITGA1 gene approached statistical significance (p&amp;lt;0.08) in the final verification cohort and was associated with higher levels of HbF. Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation. Both of these genes represent novel loci that may be involved in the regulation of HbF expression in children with SCA and should be investigated further using cellular and animal models. Disclosures No relevant conflicts of interest to declare.

High fetal hemoglobin\xa0level is associated with increased risk of cerebral vasculopathy in children with sickle cell disease in Mayotte

BackgroundUnderstanding the genetics underlying the heritable subphenotypes of sickle cell anemia, specific to each population, would be prognostically useful and could inform personalized therapeutics.The objective of this study was to describe the genetic modulators of sickle cell disease in a cohort of pediatric patients followed up in Mayotte.MethodsThis retrospective cohort study analyzed clinical and biological data, collected between January1st2007 and December 31st2017, in children younger than 18 years.ResultsWe included 185 children with 72% SS, 16% Sβ0-thalassemia and 12% Sβ + thalassemia. The average age was 9.5 years; 10% of patients were lost to follow up. The Bantu haplotype was associated with an increase in hospitalizations and transfusions. The alpha-thalassemic mutation was associated with a decrease of hemolysis biological parameters (anemia, reticulocytes), and a decrease of cerebral vasculopathy. The Single Nucleotide Polymorphisms BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were associated with the group of children with HbF > 10%. Patients with HbF > 10% presented a significant risk of early onset of cerebral vasculopathy.ConclusionsThe most remarkable result of our study was the association of SNPs with clinically relevant phenotypic groups. BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were correlated with HbF > 10%, a group that has a higher risk of cerebral vasculopathy and should be oriented towards the hemolytic sub-phenotype.

The association of HBG2, BCL11A, and HBS1L-MYB polymorphisms to thalidomide response in Chinese β-thalassemia patients

Thalidomide has been shown to reactivate fetal hemoglobin (HbF) production and reduce the need for blood transfusions in β-thalassemia patients. However, some patients show a minor response or no response to thalidomide. In view of its potential side effects, targeted prescription of thalidomide is imperative. We initially aimed to explore the relevance of HBG2 (rs7482144), BCL11A (rs11886868, rs4671393, rs766432 and rs1427407) and HBS1L-MYB (rs9399137, rs4895440 and rs4895441) single nucleotide polymorphisms (SNPs) in thalidomide response. Eight SNPs were investigated by PCR and DNA sequencing, and their roles in thalidomide response in Chinese β-thalassemia patients were assessed. Results demonstrated that minor alleles of four SNPs were associated with an increased main response risk (rs7482144: P = 0.015; rs9399137: OR = 4.911, P = 0.029; rs4895440: OR = 4.522, P = 0.040; and rs4895441: OR = 4.522, P = 0.040). For patients with non-transfusion-dependent thalassemia (NTDT), with an increase in the minor allele numbers of rs7482144 (P = 0.011), rs9399137 (P = 0.013), rs4895440 (P = 0.011) and rs4895441 (P = 0.011), Hb increments after treatment were increased significantly as well. The cumulative effects of patients carrying any combination of one or three significant minor alleles included a gradually increased main response risk compared to those without the significant minor alleles (P = 0.040–0.018, OR = 8.556–11.000). Furthermore, Hb increments after treatment correlated with cumulative numbers of minor alleles in the four significant SNPs among patients with NTDT (P = 0.001). It was demonstrated that SNPs in HBG2 and HBS1L-MYB contributed significantly to thalidomide response in Chinese patients with β-thalassemia and that the cumulative number of minor SNP alleles may serve as good predictors of treatment response in this population.

Multi-Locus Models to Address Hb F Variability in Portuguese β-Thalassemia Carriers

Hb F production is under the influence of major quantitative trait loci (QTL). The present study aims: i) to replicate the association with Hb F for representative genetic variants in the three major Hb F QTLs in a Portuguese sample of β-thalassemia (β-thal) carriers; and ii) to test different genetic multi-locus models to account for the genetic component of Hb F variation. A population sample of 79 Portuguese β-thal carriers (39 males, 40 females), aged between 2 to 70 years old, were genotyped for polymorphisms in the locus control region (LCR)-5’ hypersensitive site 4 (5’HS4) rs16912979, XmnI-HBG2 rs7482144, BCL11A rs1427407 and HMIP rs66650371, using standard biomolecular procedures. Univariate linear regression models were used to test for genetic associations with Hb F. The minor alleles of the individual variants BCL11A rs1427407 (T) (0.165), HMIP rs66650371 (3 bp del) (0.247) and XmnI-HBG2 rs7482144 (T) (0.196), were found to be significantly associated with increased levels of Hb F (p = 0.029, p = 0.002 and p = 0.0004, respectively), explaining about 6.0, 12.0 and 15.0% of Hb F variation, respectively. In a multiple linear regression approach, the three loci accounted for about 30.0% of Hb F variance. Two genetic risk scores (GRS), rationalizing the number of minor alleles into a single genetic variable, explained about 30.0 and 32.0% of the Hb F variation. In conclusion, we replicated in β-thal carriers previously reported associations with Hb F. Multi-locus models combining three representative variants of Hb F influencing QTLs can explain a larger amount of Hb F variability.

Detection of BCL11A and HBS1L-MYB Genotypes in Sickle Cell Anemia

Sickle Cell Anemia (SCA) is one of the most common monogenic disorders worldwide. Molecular modifiers of clinical symptoms play an essential role in the amelioration of the effects of the disease. Single Nucleotide Polymorphisms (SNPs) of the BCL11A gene and within the HBS1L-MYB intergenic region, which are located outside the β-globin locus on chromosome 11, are considered to be genetic modifiers that are associated with elevated levels of foetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin, HbS. The work reported here aimed to detect the most common SNPs of BCL11A and HBS1L-MYB related to HbF in SCA patients and to estimate the frequency of occurrence of these genotypes. A total of 132 SCA patients whose condition was stable were recruited from Jeddah city, Saudi Arabia. SNPs at site locus rs4671393 on BCL11A, and at loci rs28384513 and rs9399137 on HBS1L-MYB were identified using TaqMan genotyping assay. Haematological parameters were analysed based on complete blood count and haemoglobin separation using the capillary electrophoresis technique. Highly significant differences in the diagnostic haematological parameters, including all blood-cell types and HbF, were observed between the study cohort and control groups. We also found that BCL11A rs4671393 genotypes of GG and AG were more likely to show increases in HbF levels than other genotypes. In addition, a strong relationship was found between HBS1L-MYB rs9399137 and rs28384513 genotypes in the cohort, whereas no significant association was observed between BCL11A rs4671393 variant and other variants. Our study highlights the importance of investigating genetic determinants that play roles in the amelioration of the severity of clinical symptoms and complications of SCA.

BCL11A rs1427407 Genotypes in Sickle Cell Anemia Patients Undergo to Stroke Problems in Sudan.

BackgroundSickle cell disease is an autosomal recessive condition that results from the presence of a mutated form of hemoglobin. Some genetic variants of BCL11A are amenable to therapeutic manipulation. The present study investigated the relationship of a BCL11A variant (rs1427407) and its plasma levels with vaso-occlusive crises and stroke complications among patients in Sudan with sickle cell disease.MethodsThis cross-sectional study was performed between June 2014 and October 2016. The subjects included 166 patients who were diagnosed with sickle cell disease and 35 healthy control subjects, who were grouped according to sex and age (<15 years, 15–25 years, and >25 years). All patients and/or their guardians provided informed consent. Blood samples were collected from the patients and controls under aseptic conditions.ResultsPlasma BCL11A levels were elevated in cases with vaso-occlusive crises that lasted for >3 years. In addition, plasma BCL11A levels were high in cases with the GG genotype (vs. GT and TT) at rs1427407. Furthermore, the BCL11A rs1427407 GG/GT genotypes increased the risk of vaso-occlusive crisis and stroke in the patients with sickle cell disease.ConclusionThe BCL11A variant (rs1427407) and its plasma levels were associated with vaso-occlusive crisis and stroke in patients with sickle cell disease.

Pulmonary Function Abnormalities in Adults with Sickle Cell Anemia

Sickle cell anemia (SCA) is an inherited red blood cell disorder that leads to hemoglobin polymerization when it deoxygenates. Lung function abnormalities are observed in up to 90% of adults with SCA (PMID: 16556694) and both the prevalence (PMID: 20644948) and rate of FEV1 decline (PMID: 18383325) are greater in SCA adults compared to the general population. Furthermore, low FEV1 is an independent predictor of early mortality in SCA (PMID: 26261241). Understanding the risk factors and mechanisms for abnormal lung function, which can impact blood oxygen and pH levels, is especially important in SCA.We evaluated the clinical, laboratory, and genetic risk factors for abnormal pulmonary function in a retrospective cohort of SCA patients. Pulmonary function test (PFT) abnormalities were categorized based on the American Thoracic Society/European Respiratory Society Guidelines. Alpha-thalassemia and BCL11A rs1427407 status were determined by PCR. Continuous and categorical variables were compared using the Kruskal-Wallis and Chi-square test, respectively, and Kaplan-Meier curves and Cox proportional hazards models were used to compare survival patterns. Median and interquartile ranges (IQR) are provided.Between 12/2004 and 12/2015, 90 SCA adult patients had PFTs performed as part of their routine medical care. The median age of the cohort was 37 years (IQR, 24-47years), 46% were male, 59% were on hydroxyurea, and 28% were cigarette smokers. Abnormal pulmonary function was observed in 56 (62%); 32 (35.6%) had obstructive, 15 (16.7%) had restrictive, and 9 (10%) had mixed lung defects. SCA patients with abnormal PFTs were more anemic (8.4 vs. 9.0g/dL, P=0.004), had higher LDH (441 vs. 306U/L, P=0.002) and urine albumin concentrations (180 vs. 29mg/g creatinine, P=0.004), and a lower hemoglobin F% (4.2% vs. 8.0%, P=0.004) compared to SCA with normal PFTs. SCA patients who inherited the BCL11A rs1427407 T allele had a lower prevalence of abnormal PFTs (G/G: 67%, G/T: 55%, T/T: 50%; P=0.2), but this was not statistically significant, and we did not observe any patterns between co-inheritance of α-thalassemia and abnormal PFTs (αα/αα: 60%, α-/αα: 64%, α-/α-: 60%; P=0.9). On logistic regression analysis, LDH (natural log OR 8.2, P=0.03) was independently associated with abnormal pulmonary function after adjusting for age, gender, and hydroxyurea use. SCA patients with abnormal pulmonary function had earlier mortality compared to those with normal pulmonary function (Figure 1) (log-rank P=0.03; age-adjusted HR 6.1, P=0.09).Next, we analyzed clinical and laboratory risk factors associated with obstructive or restrictive lung disease. Lower hemoglobin F% and a greater prevalence of tricuspid regurgitant velocity (TRJV) > 3.0m/s were associated with both restrictive and obstructive pulmonary function compared to normal pulmonary function (Table 1). In addition, lower hemoglobin concentration and higher LDH were associated with restrictive lung disease while less common treatment with hydroxyurea and higher urine albumin concentration were associated with obstructive lung disease compared to SCA patients with normal PFTs (Table 1). SCA patients with restrictive lung disease had a lower oxygen saturation, determined by pulse oximetry, compared to those with normal PFTs (P=0.02) while no difference in oxygen saturation was observed between those with obstructive versus normal PFTs (P=0.6) (Figure 2). Mortality risk was significantly increased for SCA patients with restrictive (age-adjusted HR 2.0, P=0.01) but not obstructive lung disease (age-adjusted HR 1.6, P=0.4) compared to those with normal PFTs (log rank P=0.006) (Figure 3).In conclusion, we demonstrate that increased hemolysis is associated with abnormal pulmonary function based on a lower hemoglobin concentration and lower hemoglobin F% and higher LDH. We also demonstrate that restrictive-type lung disease is associated with a lower oxygen saturation and increased mortality risk compared to those with normal PFTs. Future studies investigating the role of hemolysis in lung disease and therapies to improve pulmonary function, particularly in those with restrictive lung disease, are warranted in SCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HMOX1 and Acute Kidney Injury in Sickle Cell Anemia

▪Acute kidney injury (AKI) is associated with tubulointerstitial fibrosis and nephron loss and may lead to an increased risk for subsequently developing chronic kidney disease (CKD). In adults with sickle cell anemia (SCA), high rates of CKD have been consistently observed, although the incidence and risk factors for AKI are less clear.We evaluated the incidence of AKI, defined according to Kidney Disease Improving Global Outcomes (KDIGO) guidelines as a rise in serum creatinine by ≥0.3mg/dL within 48 hours or ≥1.5 times baseline within seven days, in 158 of 299 adult SCA patients enrolled in a longitudinal cohort from the University of Illinois at Chicago. These patients were selected based on the availability of genotyping for α-thalassemia, BCL11A rs1427407, APOL1 G1/G2, and the HMOX1 rs743811 and GT-repeat variants. Median values and interquartile range (IQR) are provided. With a median follow up time of 66 months (IQR, 51-74 months), 137 AKI events were observed in 63 (40%) SCA patients. AKI was most commonly observed in the following settings: acute chest syndrome (25%), an uncomplicated vaso-occlusive crisis (VOC)(24%), a VOC with pre-renal azotemia determined by a fractional excretion of sodium <1% or BUN-to-creatinine ratio >20:1 (14%), or a VOC with increased hemolysis, defined as an increase in serum LDH or indirect bilirubin level >1.5 times over the baseline value at the time of enrollment (12%).Compared to individuals who did not develop AKI, SCA adults who developed an AKI event were older (AKI: median and IQR age of 35 (26-46) years, no AKI: 28 (23 - 26) years; P=0.01) and had a lower estimated glomerular filtration rate (eGFR) (AKI: median and IQR eGFR of 123 (88-150) mL/min/1.73m2, no AKI: 141 (118-154) mL/min/1.73m2; P=0.02) by the Kruskal-Wallis test at the time of enrollment.We evaluated the association of a panel of candidate gene variants with the risk of developing an AKI event. These included loci related to the degree of hemolysis (α-thalassemia, BCL11A rs1427407), to chronic kidney disease (APOL1 G1/G2 risk variants), and to heme metabolism (HMOX1) . Using a logistic regression model that adjusted for age and eGFR at the time of enrollment, the risk of an AKI event was associated with older age (10-year OR 2.6, 95%CI 1.4-4.8, P=0.002), HMOX1 rs743811 (OR 3.1, 95%CI 1.1-8.7, P=0.03), and long HMOX1 GT-repeats, defined as >25 repeats (OR 2.5, 95%CI 1.01-6.1, P=0.04).Next, we assessed whether AKI is associated with a more rapid decline in eGFR and with CKD progression, defined as a 50% reduction in eGFR, on longitudinal follow up. Using a mixed effects model that adjusted for age and eGFR at the time of enrollment, the rate of eGFR decline was significantly greater in those with an AKI event (β = -0.51) vs. no AKI event (β = -0.16) (P=0.03). With a median follow up time of 66 months (IQR, 51-74 months), CKD progression was observed in 21% (13/61) of SCA patients with an AKI event versus 9% (8/88) without an AKI event. After adjusting for age and eGFR at the time of enrollment, the severity of an AKI event according to KDIGO guidelines (stage 1 if serum creatinine rises 1.5-1.9 times baseline, stage 2 if the rise is 2.0-2.9 times baseline, and stage 3 if the rise is ≥3 times baseline or ≥4.0 mg/dL or requires renal replacement therapy) was a risk factor for CKD progression (unadjusted HR 1.6, 95%CI 1.1-2.3, P=0.02; age- and eGFR-adjusted HR 1.6, 95%CI 1.1-2.5, P=0.03).In conclusion, AKI is commonly observed in adults with sickle cell anemia and is associated with increasing age and the HMOX1 GT-repeat and rs743811 polymorphisms. Furthermore, AKI may be associated with a steeper decline in kidney function and more severe AKI events may be a risk factor for subsequent CKD progression in SCA. Future studies understanding the mechanisms, consequences of AKI on long-term kidney function, and therapies to prevent AKI in SCA are warranted. DisclosuresGordeuk:Emmaus Life Sciences: Consultancy.

Candidate Gene Associations with Vaso-Occlusive Crisis in Children with Sickle Cell Anemia Enrolled in the Multinational DOVE Trial

Introduction: Sickle cell disease (SCD) is complex, with marked inter-individual variability in disease severity. The clinical heterogeneity of SCD is largely influenced by genetic factors, including several well-established modifiers of fetal hemoglobin (HbF) levels within the beta globin locus, BCL11A, and HBS1L-MYB genes. However, little is known about genetic factors that may influence variability in treatment response, particularly in clinical trials assessing potential therapeutic agents for SCD. The DOVE (Determining Effects of Platelet Inhibition on Vaso-Occlusive Events) study was a multinational, phase 3, randomized, double-blinded, placebo-controlled trial evaluating the efficacy and safety of the antiplatelet agent prasugrel for the reduction of vaso-occlusive crisis (VOC) in children with sickle cell anemia (NCT01794000). Although the primary endpoint of reduction of rate of VOC did not reach statistical significance in the DOVE trial in the ITT population, a treatment effect of prasugrel was suggested in certain subgroups. The objective of this study was to evaluate candidate genetic variants with SCD severity and to assess impact of these variants on response to prasugrel, as measured by the frequency of VOC (composite of painful crisis and acute chest syndrome events) during the study period of up to 24 months.Methods: De-identified dried blood spot samples were collected for genetic analyses as per informed consent and study protocol. Genomic DNA was extracted from a 3 mm Whatman FTA card punch using Qiagen QIAamp DNA kit. A total of 313 (92%) DOVE patients (Hb SS or Hb S/β0 thalassemia) were genotyped using the TaqMan assay. After quality control, 253 patients and 28 variants were considered for genetic evaluation. These variants have been reported to be associated with SCD and/or its related comorbidities. All variants were analyzed for VOC in the prasugrel and placebo groups separately and for treatment by variant interactions in the overall study population. Variants were also assessed in the overall study population for associations with a hemolytic score, derived from 5 biomarkers of hemolysis and baseline hematologic markers including leukocyte, neutrophil, and platelet counts, and red cell mean corpuscular volume. All analyses were conducted using additive multivariate regression framework as the main model and were adjusted for age, race, geographic region, and hydroxyurea (HU) use at baseline. In addition, genotypic, dominant, and recessive models were evaluated as supportive analyses.Results: Two variants, rs7482144, located in the 5'- HBG2 gene promoter (-158 Xmn1) on chr 11 and rs1427407, located in the BCL11A gene on chr 2, were associated with VOC and a treatment interaction effect of prasugrel. HBG2 rs7482144, was associated with a significant decrease in frequency of VOC in prasugrel-treated patients relative to placebo (adjusted p=0.0065). The BCL11A rs1427407 variant was associated with an increase in VOC in prasugrel-treated patients relative to placebo (adjusted p=0.0205).No genetic associations were found with baseline composite hemolytic score in the overall population. However, significant associations with KLF-1, HBG2, and HBE variants were observed with individual hematologic markers. The KLF-1 variant (rs112631212) was associated with lower leukocyte (p<0.0001) and neutrophil (p=0.0002) counts in the subgroup of participants on HU; whereas the HBG2 (rs2070972; p=0.0116) and HBE (rs3834466 and rs7130110; p=0.0274 and p=0.0340 respectively) variants were associated with elevated platelet counts in the overall group.Conclusions: Given the study sample size, this study highlights the significance of HBG2 and BCL11A variants for the frequency of VOC in prasugrel-treated patients versus placebo. These variants have been reported in association with HbF/F-cells, an ameliorating factor for VOC that is only partly explained by endogenous levels of HbF. Our findings suggest that these variants may have contributed to the treatment-related effects of prasugrel on VOC outcomes in the DOVE trial. Identifying such genetic variants that are associated with differential response to treatment may help guide selection of patients for clinical trials based on predicted risk of VOC. DisclosuresHoppe:Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership, Other: Former employee and minor shareholder of Eli Lilly and Company. Foster:Eli Lilly and Company: Employment, Equity Ownership, Other: Employee and minor shareholder of Eli Lilly and Company. Heath:Eli Lilly and Company: Employment, Equity Ownership, Other: Employee and minor shareholder of Eli Lilly and Company. Pillai:Eli Lilly and Company: Employment, Equity Ownership, Other: Employee and minor shareholder of Eli Lilly and Company. Pallav:Eli Lilly and Company: Employment, Equity Ownership, Other: Employee and minor shareholder of Eli Lilly and Company.

Associations of α-thalassemia and BCL11A with stroke in Nigerian, United States, and United Kingdom sickle cell anemia cohorts.

Alpha-thalassemia and the BCL11A rs1427407 T allele are commonly observed in sickle cell anemia (SCA) patients and are associated with reduced hemolysis and higher hemoglobin F levels, respectively. We investigated whether a high-risk genetic profile, defined as SCA patients who did not inherit either α-thalassemia or the BCL11A rs1427407 T allele, had stronger associations with clinical and laboratory variables than the individual genetic components in the University of Ibadan cohort (n=249). We then replicated our findings in SCA cohorts from the University of Illinois at Chicago (UIC)(n=260) and Walk-Treatment of Pulmonary Hypertension and Sickle cell disease with Sildenafil Therapy (Walk-PHaSST)(n=387). High-risk was associated with higher reticulocytes (15.0% vs. 7.8%, P=0.08) and stroke history (6% vs. 1%, P=0.02) than standard risk patients and these associations were more significant than the individual genetic components in the University of Ibadan cohort. These findings were replicated in high-risk patients from UIC and Walk-PHaSST for reticulocytes (UIC: 13.5% vs. 11.8%, P=0.03; Walk-PHaSST: 9.6% vs. 8.2%, P=0.0003) and stroke history (UIC: 32% vs. 22%, P=0.07; Walk-PHaSST: 14% vs. 7%, P=0.01). On combined analysis, high-risk had strong associations with increased markers of hemolysis (hemoglobin β= -0.29, 95%CI: -0.50 to -0.09; P=0.006; reticulocyte% β=2.29, 95%CI: 1.31 to 3.25; P=1x10-5) and stroke history (OR=2.0, 95%CI: 1.3 to 3.0; P=0.0002), but no association with frequent vaso-occlusive crises (≥3/year). A high-risk genetic profile is associated with increased hemolysis and stroke history in three independent cohorts. This profile may help identify patients to prioritize for hydroxyurea and for closer monitoring strategies for stroke.

Genetic Modifiers Identify a High Risk Group for Stroke in Three Independent Cohorts of Sickle Cell Anemia Patients

Two common genetic modifiers, α-thalassemia and the BCL11A rs1427407 T allele, are observed in approximately one-third of patients with sickle cell anemia (SCA) and are associated with reduced hemolysis and higher hemoglobin F (HbF) levels, respectively.We investigated the laboratory and clinical effects of α-thalassemia and the BCL11A rs1427407 T allele in the University of Ibadan cohort of SCA patients and replicated our findings in two independent SCA cohorts, University of Illinois at Chicago (UIC) and Walk-Treatment of Pulmonary Hypertension and Sickle cell disease with Sildenafil Therapy (Walk-PHaSST). Alpha-thalassemia status was determined by PCR in all 3 cohorts while the BCL11A rs1427407 genotype was determined by PCR in the Ibadan and UIC cohorts and imputed in the Walk-PHaSST cohort. Comparisons according to genotype were performed using the linear trend test for continuous variables and Cochran’s test of linear trend for categorical variables.Alpha-thalassemia was observed in 43% of 257 SCA patients from Ibadan and was associated with higher body mass index and lower white blood cell count (Table 1). The BCL11A rs1427407 T allele was observed in 46% of SCA patients from Ibadan and was associated with higher hemoglobin concentration. HbF levels by HPLC were available in 25 patients (12 with the BCL11A rs1427407 T allele) enrolled in a study of low-dose hydroxyurea; these levels were higher in patients with at least one rs1427407 T allele at baseline and progressively during therapy with hydroxyurea 10 mg/kg/day (repeated measures P=0.01).We defined a high risk genetic group as the absence of α-thalassemia in combination with absence of the BCL11A rs1427407 T allele. This high risk group was observed in 31% of SCA patients from the Ibadan cohort and was associated with a higher reticulocyte percentage (15.0% vs. 7.8%, P=0.08) and a higher prevalence for a history of stroke (6% vs. 1%, P=0.02). The association with stroke history persisted on logistic regression analysis after adjusting for age, gender, and hydroxyurea therapy (OR 9.4, 95%CI: 1.2-72.8; P=0.03). We then replicated the association of this high risk group with markers of hemolysis and with history of stroke in the UIC and Walk-PHaSST SCA cohorts. In the UIC cohort, the high risk group was observed in 34% (92/271) and was also associated with higher reticulocyte counts (13.5% vs. 11.9%, P=0.10) and higher prevalence for stroke history (33% vs. 22%; age, gender, HU-adjusted OR 1.7, 95%CI: 1.0-3.0; P=0.066). In the Walk-PHaSST cohort, this high risk profile was observed in 38% (149/394) and was associated with a higher reticulocyte percentage (9.7% vs. 8.4%, P=0.0005), lower hemoglobin concentration (8.4 vs. 8.8 g/dL; P=0.017), and higher prevalence for stroke history (15% vs. 6%; age, gender, HU-adjusted OR 2.6, 95%CI: 1.3-5.3; P=0.007).In conclusion, a high risk group of SCA patients, defined by the lack of the protective α-thalassemia and the BCL11A rs1427407 variants, is associated with a higher degree of hemolysis and a higher prevalence of stroke history on cross sectional analysis in three independent cohorts. This high-risk profile may help identify patients to prioritize for hydroxyurea therapy and for closer monitoring strategies for stroke. [Display omitted] DisclosuresHsu:Sancilio: Research Funding; Astra Zeneca: Consultancy, Research Funding; Purdue Pharma: Research Funding; Gerson Lehman Group: Consultancy; Eli Lilly: Research Funding; Centers for Medicare and Medicaid Innovation: Research Funding; Hilton Publishing: Consultancy, Research Funding; Mast Therapeutics: Research Funding; EMMI Solutions: Consultancy; Pfizer: Consultancy, Research Funding.