HBS1L-MYB Intergenic Region Research Articles

Genetic Modifiers of Sickle Cell Anemia Phenotype in a Cohort of Angolan Children.

The aim of this study was to identify genetic markers in the HBB Cluster; HBS1L-MYB intergenic region; and BCL11A, KLF1, FOX3, and ZBTB7A genes associated with the heterogeneous phenotypes of Sickle Cell Anemia (SCA) using next-generation sequencing, as well as to assess their influence and prevalence in an Angolan population. Hematological, biochemical, and clinical data were considered to determine patients' severity phenotypes. Samples from 192 patients were sequenced, and 5,019,378 variants of high quality were registered. A catalog of candidate modifier genes that clustered in pathophysiological pathways important for SCA was generated, and candidate genes associated with increasing vaso-occlusive crises (VOC) and with lower fetal hemoglobin (HbF) were identified. These data support the polygenic view of the genetic architecture of SCA phenotypic variability. Two single nucleotide polymorphisms in the intronic region of 2q16.1, harboring the BCL11A gene, are genome-wide and significantly associated with decreasing HbF. A set of variants was identified to nominally be associated with increasing VOC and are potential genetic modifiers harboring phenotypic variation among patients. To the best of our knowledge, this is the first investigation of clinical variation in SCA in Angola using a well-customized and targeted sequencing approach.

Hemoglobin EE disease in young Laotian children: Hematologic features and the contributions of genetic variations to Hb F expression.

The wide variation in hemoglobin (Hb) F levels has been observed in patients with Hb EE disease. This study aimed to describe hematologic features and determine the effect of genetic variants on Hb F expression in young children with Hb EE disease. Hematologic features and Hb profiles of Laotian children aged 6-23 months, who originally enrolled in the Lao-Zinc study, were retrospectively reviewed. Only children with Hb EE disease, as indicated by DNA analysis, were included in this current analysis. Genetic variants, including the G γ-XmnI polymorphism (C>T) of the HBG2 gene, the HBS1L-MYB intergenic region on chromosome 6, and the BCL11A on chromosome 2 as well as the mutations occurring on the Krüppel-like factor 1 (KLF1) gene, were examined. In total, 205 children were diagnosed as having Hb EE disease with Hb F ranged from 1.2 to 43.7%. Most of the children had mild to moderate anemia with a remarkable microcytosis. Analysis of the genetic variants revealed an extremely high frequency of the G γ-XmnI (93.7%). Applying multiple regression analysis adjusted for age, sex, and α-thal gene, a positive relation was observed for the rs4671393 (coefficient=3.87, p= .005) and the rs2297339 (coefficient=2.48, p= .046), but not the G γ-XmnI. A statistically non-significant relation was noted for the rs9399137 and the -154 (C>T) KLF1 mutation. Our findings provide insight into complex situation of Hb F variability in young children with Hb EE disease; and this can guide to appropriate care and counseling to affected families.

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.

Association of BCL11A and HBS1L-MYB Polymorphisms in Sickle Cell Anaemia Subjects of Different Age Groups in Nigeria

Haematological features and clinical severity of Sickle Cell Anaemia (SCA) are influenced by age, gender, genetic and community factors. BCL11A is cytogenetically located on short arm of chromosome 2 at position 16.1 while it is located at base pair 60,451,167 to 60,553,498 on chromosome 2 at the molecular level. Polymorphisms in the HBS1L-MYB Intergenic region were associated with F-cell levels and accounted for 19.4% of the F-cell variance in normal Europeans. This study highlights possible effects of age in Haemoglobin F Induction through possible association of BCL11A and HBS1L-MYB genes polymorphism in SCA Subjects population in Oshogbo metropolis. Thirty – Six SCA subjects were recruited with ages ranging between 1 and 40years divided into five groups of eight subjects each except age ranges 21 to 25 and 25 to 40 years which were six subjects each, while ten haemoglobin A subjects served as control for all age ranges. Red Cell indices and gene analysis such as HCT, RBC Count, Haemoglobin concentration, Hb F level, BCL11A and HBS1L-MYB genes were studied using standard methods. Results shows no statistically significant difference in fetal haemoglobin between control and subjects (P>0.05). There was a statistical decrease in haemoglobin level in the test subjects compared to controls. Also, the HBS1L-MYB protein expression was not significantly different in Haemoglobin A subjects and the SCA subjects, but there was a statistically significant decrease in BCL11A gene expression in SCA Subjects compared to Haemoglobin A Subjects (P<0.05). A statistically significant decrease in BCL11A gene expression was obtained in ages 16 to 20 and 21 to 40years age groups (p<0.05). There was a positive correlation between Fetal Haemoglobin and haematocrit and haemoglobin levels (r=0.04341, p=0.2227); (r=0.01705, p=0.4479), respectively. The correlation between Fetal haemoglobin and BCL11A and HBS1L-MYB showed statistically significant negative correlations (r= - 0.1220, p=0.0368), r= - 0.1260, p=0.0336 respectively). Conclusively, BCL11A and HBS1L-MYB genes promote induction of fetal haemoglobin in SCA subjects compared with controls recruited for this study.

Fetal hemoglobin-boosting haplotypes of BCL11A gene and HBS1L-MYB intergenic region in the prediction of clinical and hematological outcomes in a cohort of children with sickle cell anemia.

Single nucleotide polymorphisms (SNPs) of BCL11A gene and HBS1L-MYB intergenic region (named HMIP-2) affect both fetal hemoglobin (HbF) concentration and clinical outcomes in patients with sickle cell anemia (SCA). However, no previous study has examined the interaction among these SNPs in the regulation of HbF. We examined whether HbF-boosting haplotypes combining alleles of functional SNPs of BCL11A and HMIP-2 were associated with clinical outcomes and hematological parameters, and whether they interact to regulate HbF in a cohort of Brazilian children with SCA. The minor haplotype of BCL11A ("TCA", an allele combination of rs1427407, rs766432, and rs4671393) was associated with higher HbF, hemoglobin and lower reticulocytes count compared to reference haplotype "GAG". The minor haplotype of HMIP-2 ("CGC", an allele combination of rs9399137, rs4895441, and rs9494145) was associated with higher HbF and hemoglobin compared to reference haplotype "TAT". Subjects carrying minor haplotypes showed reduced rate of clinical complications compared to reference haplotypes. Non-carriers of both minor haplotypes for BCL11A and HMIP-2 showed the lowest HbF concentration. Subjects carrying only the minor haplotype of BCL11A showed significantly higher HbF concentration than non-carriers of any minor haplotype, which showed no significant difference compared to subjects carrying only the minor haplotype of HMIP-2. Interestingly, subjects carrying both minor haplotypes of BCL11A ("TCA") and HMIP-2 ("CGC") showed significantly higher HbF levels than subjects carrying only the minor haplotype of BCL11A. Our novel findings suggest that HbF-boosting haplotypes of BCL11A and HMIP-2 can predict clinical outcomes and may interact to regulate HbF in patients with SCA.

Common Genetic Variants of Fetal Hemoglobin Modify Hematological Phenotypes in MDS/MPN/Myeloma Patients Receiving Cytotoxic Drugs

Genetic studies identify common variants within the HBS1L-MYB intergenic region (HMIP), BCL11A, and Xmn1-HBG2 as associated with elevated fetal hemoglobin (HbF) levels and other clinically important human hematological traits. Recent studies suggest HbF is a predictor of outcome in MDS/AML patients receiving decitabine. We assessed effects of HbF genetic variants on hematological traits in Myeloproliferative Neoplasm (MPN), Myelodysplastic Syndrome (MDS) and myeloma on HbF-inducing therapy to determine potential for variants predicting treatment response. Seven common HbF variants at HMIP, BCL11A, Xmn1-HGB2 loci were genotyped in 89 patients with MPN on Hydroxyurea (HU), myeloma on Lenalidomide, and MDS on Azacytidine. HbF genetic association was seen with rs9494142 (HMIP) in MPN on HU (p = 0.04) and rs1427407 (BCL11A) in myeloma on Lenalidomide (p = 0.002). HMIP variants rs9494142 and rs6920211 influenced baseline platelets (p = 0.04) and hemoglobin treatment response (p = 0.02). rs1427407 (BCL11A) was significantly associated with increased platelets (p = 0.04) negating thrombocytopenic tendency of Lenalidomide. These HbF variants showed significantly discordant minor allele frequencies in MDS/MPN/myeloma compared to wider European population data. This small study finding together suggest the implication of these variants in treatment response and disease biology in MDS/MPN/myeloma warranting larger prospective genotype-phenotype association studies.

Fetal hemoglobin regulating genetic variants identified in homozygous (HbSS) and heterozygous (HbSA) subjects from South Mexico.

Hemoglobin S is caused by a nucleotide change in HBB gene (HBB:c.20A>T, p.Glu6Val), is presented in diverse forms: simple carriers (HbSA), homozygotes (HbSS) also known as sickle cell anemia, and compound heterozygotes with other β-hemoglobinopathies. It is worldwide distributed, in Mexico, is frequently observed in the southern states Guerrero, Oaxaca and Chiapas. Elevated fetal hemoglobin (HbF) is associated with mild phenotype; single-nucleotide variants (SNVs) in modifier genes, such as BCL11A, HBG2, HBBP1 pseudogene and HBS1L-MYB intergenic region, upregulate HbF synthesis. The aim of this study was to identify HbF regulating genetic variants in HbSS and HbSA Mexican subjects. We studied 39 individuals (HbSS = 24, 61%, HbSA = 15, 39%) from Chiapas (67%) and Guerrero (33%), peripheral blood was collected in ethylenediamine tetraacetic acid (EDTA) for molecular and hematological studies, DNA was isolated by salting-out technic and genotyping was performed through allelic discrimination by real time polymerase chain reaction (RT-PCR) using Taqman® probes for 15 SNV (in BCL11A: rs6706648, rs7557939, rs4671393, rs11886868, rs766432, rs7599488, rs1427407; HBS1L-MYB: rs28384513, rs7776054, rs9399137, rs4895441, rs9402686, rs1320963; HBG2: rs7482144; and HBBP1: rs10128556). The obtained data were analyzed using IMB SPSS v.22.0 software. All minor alleles were observed in frequencies over 0.05, the most frequent was rs9402686 (0.82), while the less frequent was rs101028556 (0.08). In HbSS group, the mean fetal hemoglobin was 11.9 ± 5.9% and was significantly elevated in BCL11A rs11886868 wildtype homozygotes and in carriers of HBS1L-MYB intergenic region rs7776054 (p = 0.04 and p = 0.03, respectively). In conclusion, in HbSS Mexican patients, two SNVs were observed related to increased HbF; BCL11A rs11886868 and HBS1L-MYB rs7776054.

P1475: THE ROLE OF BCL11A AND HBS1L-MYB SINGLE NUCLEOTIDE POLYMORPHISMS IN EGYPTIAN SICKLE CELL DISEASE PATIENTS

Background: Sickle cell disease (SCD) is a debilitating blood disorder with a highly variable phenotype. Inter-individual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. The only currently FDA-approved HbF inducing drug for sickle cell disease (SCD) is hydroxyurea (HU). Genome wide association studies (GWAS) have identified three major quantitative trait loci (QTL) (intergenic region of HBS1L-MYB, BCL11A gene and XmnI site) associated with HbF. Several single nucleotide polymorphisms (SNPs) with persistence of HbF and amelioration of disease severity of β-thalassemia (β-thal) and SCD have been identified. Aims: The aim of the current study is to find the influence of these genetic modifiers, especially BCL11A (rs4671393) and HBS1L-MYB intergenic region (rs9399137) SNPs on the level of HbF, severity of SCD and response to hydroxyurea in Egyptian sickle cell disease patients. Methods: The present study included 100 patients with SCD; 56 patients were diagnosed as homozygous sickle cell anemia (SS) patients, while 44 patients were diagnosed as sickle beta thalassemia patients (Sβ). Genotyping for the genes was done by Real-Time PCR (RT-PCR) technique. Results: BCL11A (rs4671393) SNP showed higher hemoglobin and HbF levels when induced with HU, with significantly higher increment in HbF level post HU therapy as well as a milder clinical phenotype in our Sβ patients’ group. In our SS patients’ group it showed a lower incidence of pneumonia as well as a more effective response to HU treatment. HBS1L-MYB intergenic region (rs9399137) SNPs showed lower reticulocytic counts and serum ferritin levels in our Sβ patients’ group. Summary/Conclusion: In conclusion, our study shows that BCL11A (rs4671393) SNP showed higher hemoglobin and HbF levels when induced with HU, with significantly higher increment in HbF level post HU therapy as well as a milder clinical phenotype in our Egyptian study.

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.

A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

Fetal hemoglobin (HbF) modifies the severity of sickle cell disease (SCD) and β-thalassemia. One of the major quantitative trait loci (QTL) associated with HbF level is the HBS1L-MYB intergenic polymorphisms (HMIP) on chr6q23. MYB regulates proliferation and maturation of erythroid cells, and modulates gene expression within the HBB gene cluster. A distal enhancer located at ~84 kb upstream of MYB has been shown by GWAS (Farrell et al , Blood 2011), insertional mutation (Suzuki et al, Mol Cell Biol 2013), long-range interaction by 3C analysis (Stadhouders et al, J Clin Invest 2014), and gene editing with Cas9 nucleases (Canver et al, Nat Genet 2017). This enhancer encompasses a 3-bp deletion polymorphism (rs66650371), which is surrounded by binding sites for erythroid-specific transcription factors, and is likely the functional motif to account for most of the effect upon HbF level by this HbF QTL. Furthermore, ENCODE datasets annotated RNA polymerase 2 occupancy and a 50-bp RNA transcript adjacent to rs66650371, and led us to hypothesize that this transcript is part of a long noncoding RNA (lncRNA). LncRNAs are greater than 200 nucleotides long, are transcribed throughout the genome, and have broad functionality.We characterized a novel 1283 bp lncRNA (HMI-LNCRNA) which was transcribed from this MYB enhancer and contained binding sites for TAL1/E47, GATA, and RUNX1. Its expression in 25 human tissues and cells was examined by qPCR. The thymus and testis had by far the highest expression. Increased expression was seen in erythroblasts from cultured cord blood CD34+ cells, followed by fetal liver, bone marrow, spleen and K562 cells. Other non-hematopoietic tissues had mostly minimal expression. The expression pattern of HMI-LNCRNA displayed similar features of most lncRNAs, including being located almost entirely within nuclei in erythroid cells, suggesting that it might regulate gene expression; and being less abundant than the protein-coding gene, MYB . HMI-LNCRNA expression was significantly higher in erythroblasts from cultured adult peripheral blood CD34+ cells, which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells, which expressed much more HBG . Similarly, HMI-LNCRNA expression was higher in HUDEP-2 cells, an immortalized human adult-like erythroid cells that expressed only HBB, compared to HUDEP-1 cells that expressed predominantly HBG .Transduction of HUDEP-2 cells with HMI-lncRNA shRNA led to downregulation of HMI-LNCRNA expression to 50% of normal level, and resulted in 200-fold increase in HBG mRNA but no change in HBB or HBS1L mRNA. HBG mRNA expression in these cells amounted to 20% of the total HBB and HBG mRNA levels. The changes in HBG mRNA levels were associated with corresponding changes in HBG protein levels documented by Western blot analysis and immunofluorescence staining using anti-HBG antibody. Similar changes in mRNA and protein levels of HBG were seen when HUDEP-2 cells transduced with HMI-lncRNA shRNA were allowed to mature in culture for up to 7 days. There was no change in the expression of BCL11A and other transcription factors known to modulate HBG expression in HUDEP-2 cells transduced with HMI-lncRNA shRNA. FACS analysis of CD71 and CD235 expression showed that 55-65% of HUDEP-2 “naïve” cells or cells transduced with scrambled shRNA were double positive for both markers, while 85% of HUDEP-2 cells transduced with HMI-lncRNA shRNA were double positive, suggesting that downregulation of HMI-LNCRNA promoted maturation of HUDEP-2 cells.HMI-LNCRNA is likely an enhancer RNA (eRNA), and facilitates the interaction between the distal enhancer with the MYB promoter. Our experimental results indicate that it has an important role in regulating HBG expression. Its downregulation can result in markedly increased HBG expression. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in SCD and β-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

A genome-wide meta-analysis yields 46 new loci associating with biomarkers of iron homeostasis

Iron is essential for many biological functions and iron deficiency and overload have major health implications. We performed a meta-analysis of three genome-wide association studies from Iceland, the UK and Denmark of blood levels of ferritin (N = 246,139), total iron binding capacity (N = 135,430), iron (N = 163,511) and transferrin saturation (N = 131,471). We found 62 independent sequence variants associating with iron homeostasis parameters at 56 loci, including 46 novel loci. Variants at DUOX2, F5, SLC11A2 and TMPRSS6 associate with iron deficiency anemia, while variants at TF, HFE, TFR2 and TMPRSS6 associate with iron overload. A HBS1L-MYB intergenic region variant associates both with increased risk of iron overload and reduced risk of iron deficiency anemia. The DUOX2 missense variant is present in 14% of the population, associates with all iron homeostasis biomarkers, and increases the risk of iron deficiency anemia by 29%. The associations implicate proteins contributing to the main physiological processes involved in iron homeostasis: iron sensing and storage, inflammation, absorption of iron from the gut, iron recycling, erythropoiesis and bleeding/menstruation.

Association between BCL11A, HSB1L-MYB, and XmnI γG-158 (C/T) gene polymorphism and hemoglobin F level in Egyptian sickle cell disease patients.

Sickle cell disease (SCD) is a monogenic disease characterized by multisystem morbidity and highly variable clinical course. Inter-individual variability in hemoglobin F (HbF) levels is one of the main modifiers that account for the clinical heterogeneity in SCD. HbF levels are affected by, among other factors, single nucleotide polymorphisms (SNPs) at the BCL11A gene and the HBS1L-MYB intergenic region and Xmn1 gene. Our aim was to investigate HbF-enhancer haplotypes at these loci to obtain a first overview of the genetic situation of SCD patients in Egypt and its impact on the severity of the disease. The study included 100 SCD patients and 100 matched controls. Genotyping of BCL11A (rs1886868 C/T), HBS1L-MYB (rs9389268 A/G) and Xmn1 γG158 (rs7842144 C/T) SNPs showed no statistically significant difference between SCD patients and controls except for the hetero-mutant genotypes of BCL11A which was significantly higher in SCD patients compared with controls. Baseline HbF levels were significantly higher in those with co-inheritance of polymorphic genotypes of BCL11A + HSB1L-MYB and BCL11A + Xmn1. Steady-state HbF levels, used as an indicator of disease severity, were significantly higher in SCD-Sβ patients having the polymorphic genotypes of HSB1L-MYB. Fold change of HbF in both patient groups did not differ between those harboring the wild and the polymorphic genotypes of the studied SNPs. In conclusion, BCL11A, HSB1L, and Xmn1 genetic polymorphisms had no positive impact on baseline HbF levels solely but had if coexisted. Discovery of the molecular mechanisms controlling HbF production could provide a more effective strategy for HbF induction.

Association of HMIP1 C-893A polymorphism and disease severity in patients with sickle cell anemia.

IntroductionSickle cell anemia (SCA) is a Mendelian disorder with a heterogeneous clinical course. The reasons for this phenotypic diversity are not entirely established, but it is known that high fetal hemoglobin levels lead to a milder course of the disease. Additionally, genetic variants in the intergenic region HBS1L-MYB promote high levels of fetal hemoglobin into adulthood.ObjectiveIn the present study, we investigated the HMIP1 C-839A (rs9376092) polymorphism, located at the HBS1L-MYB intergenic region block 1, in SCA patients.MethodWe analyzed 299 SCA patients followed in two reference centers in Brazil. The HMIP1 C-839A (rs9376092) genotypes were determined by allele specific polymerase chain reactions. Clinical and laboratory data were obtained from patient interviews and medical records.ResultsThe median fetal hemoglobin levels were higher in patients with the HMIP1 C-839A (rs9376092) AA genotype (CC = 6.4%, CA = 5.6% and AA = 8.6%), but this difference did not reach significance (p = 0.194). No association between HMIP1 C-839A (rs9376092) genotypes and other clinical and laboratorial features was detected (p > 0.05).ConclusionIn summary, our data could not support the previously related association between the HMIP1 C-893A (rs9376092) polymorphism and differential fetal hemoglobin levels.

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.

Role of XmnI polymorphism in HbF induction in HbE/β and β-thalassaemia patients

Background: Thalassaemia is one of the most common genetic blood disorders worldwide. Patients with β-thalassaemia major and HbE/β-thalassaemia are blood transfusion dependent. Foetal haemoglobin or HbF can play a role in disease manifestations in these patients and there is evidence that a homozygous state for XmnI polymorphic site, associated with increased expression of Gγ-gene, may play an important role among other factors in ameliorating the clinical severity of homozygous β-thalassaemia and thalassaemia intermedia. The aim of this review was to provide a comprehensive review of the role of XmnI polymorphic site for increased HbF production in HbE/β and β-thalassaemia patients.
 Methods: Published literatures were reviewed on the allelic frequency of Xmn1 polymorphism and its effect on HbF induction among thalassaemia patients of different countries.
 Results: In all β-thalassaemias, Hb F levels are relatively increased due to the selective survival of the erythroid precursors that synthesize relatively more γ-chains. The expression of HbF level is dominated by three different loci: HBG2: γ -158C>T, BCL11A, and HBS1L-MYB intergenic region. Genetic determinants influencing Hb F response can be within the β-globin complex or trans-acting. The published literature showed that the C>T substitution (rs7482144) at position –158 of the Gγ-globin gene, referred to as the XmnI-Gγ polymorphism, is a common sequence variant in all population groups, present at a frequency of 0.32 to 0.35. It was found in some studies, response to Hydroxyurea (HU) has been shown to be largely associated with the presence of the C>T polymorphism at -158 XmnI site (HBG2:c.- 53-158C>T) upstream of the Gγ-globin gene and HU therapy exerts a 2- to 9- fold increase in γ-mRNA expression in β-thalassaemia patients.
 Conclusion: A number of various study groups around the world suggests that XmnI polymorphism is an important key regulator of disease severity of HbE/β and β-thalassaemia patients.

The association of HBG2, BCL11A, and HMIP polymorphisms with fetal hemoglobin and clinical phenotype in Iraqi Kurds with sickle cell disease.

Fetal hemoglobin (HbF) is the major modifier for sickle cell disease (SCD) severity. HbF is modulated mainly by three major quantitative trait loci (QTL) on chromosomes 2, 6, and 11. Five SNPs in the three QTLs (HBG2, rs7482144; BCL11A, rs1427407 and rs10189857; and HBS1L-MYB intergenic region, rs28384513 and rs9399137) were investigated by multiplex PCR and reverse hybridization, and their roles in HbF and clinical phenotype variability in Iraqi Kurds with SCD were assessed. HBG2 rs7482144 with minor allele frequency (MAF) of 0.133 was the most significant contributor to HbF variability, contributing 18.1%, followed by rs1427407 (MAF of 0.266) and rs9399137 (MAF of 0.137) at 14.3% and 8.8%, respectively. The other two SNPs were not significant contributors. Furthermore, when the cumulative numbers of minor alleles in the three contributing SNPs were assessed, HbF% and hemoglobin concentration increased with increasing number of minor alleles (P<0.0005 and 0.001, respectively), while serum lactic dehydrogenase, reticulocytes, leukocytes, transfusion, and pain frequencies decreased (P=0.003, 0.004, <0.0005, <0.0005, and 0.017, respectively). It was demonstrated that SNPs in all three major HbF QTLs contribute significantly to HbF and clinical variability in Iraqi Kurds with SCD and that the cumulative number of minor alleles at contributing SNPs may serve as a better predictor of such variability in this population.

A long noncoding RNA from the HBS1L-MYB intergenic region on chr6q23 regulates human fetal hemoglobin expression

The HBS1L-MYB intergenic region (chr6q23) regulates erythroid cell proliferation, maturation, and fetal hemoglobin (HbF) expression. An enhancer element within this locus, highlighted by a 3-bp deletion polymorphism (rs66650371), is known to interact with the promoter of the neighboring gene, MYB, to increase its expression, thereby regulating HbF production. RNA polymerase II binding and a 50-bp transcript from this enhancer region reported in ENCODE datasets suggested the presence of a long noncoding RNA (lncRNA). We characterized a novel 1283bp transcript (HMI-LNCRNA; chr6:135,096,362–135,097,644; hg38) that was transcribed from the enhancer region of MYB. Within erythroid cells, HMI-LNCRNA was almost exclusively present in nucleus, and was much less abundant than the mRNA for MYB. HMI-LNCRNA expression was significantly higher in erythroblasts derived from cultured adult peripheral blood CD34+ cells which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells which expressed much more HBG. Down-regulation of HMI-LNCRNA in HUDEP-2 cells, which expressed mostly HBB, significantly upregulated HBG expression both at the mRNA (200-fold) and protein levels, and promoted erythroid maturation. No change was found in the expression of BCL11A and other key transcription factors known to modulate HBG expression. HMI-LNCRNA plays an important role in regulating HBG expression, and its downregulation can result in a significant increase in HbF. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in sickle cell disease and β-thalassemia.

2SNP heritability and effects of genetic variants for neutrophil-to-lymphocyte and platelet-to-lymphocyte ratio.

Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are important biomarkers for disease development and progression. To gain insight into the genetic causes of variance in NLR and PLR in the general population, we conducted genome-wide association (GWA) analyses and estimated SNP heritability in a sample of 5901 related healthy Dutch individuals. GWA analyses identified a new genome-wide significant locus on the HBS1L-MYB intergenic region for PLR, which replicated in a sample of 2538 British twins. For platelet count, we replicated three known genome-wide significant loci in our cohort (at CCDC71L-PIK3CG, BAK1 and ARHGEF3). For neutrophil count, we replicated the PSMD3 locus. For the identified top SNPs, we found significant cis and trans eQTL effects for several loci involved in hematological and immunological pathways. Linkage Disequilibrium score (LD) regression analyses for PLR and NLR confirmed that both traits are heritable, with a polygenetic SNP-heritability for PLR of 14.1%, and for NLR of 2.4%. Genetic correlations were present between ratios and the constituent counts, with the genetic correlation (r=0.45) of PLR with platelet count reaching statistical significance. In conclusion, we established that two important biomarkers have a significant heritable SNP component, and identified the first genome-wide locus for PLR.

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants from genome-wide association studies largely cluster in regulatory DNA. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating mutagenesis libraries with single or combinatorial nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, a locus associated with red blood cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false positive regions, which emphasizes the importance of off-target analysis in design of saturating mutagenesis experiments. Taken together, these data establish a widely applicable high-throughput and high-resolution methodology to reliably identify minimal functional sequences within large regions of disease- and trait-associated DNA.

Genetic Variants at BCL11A and HBS1L-MYB loci Influence Hb F Levels in Chinese Zhuang β-Thalassemia Intermedia Patients

Increased Hb F levels can ameliorate the symptoms of β-thalassemia (β-thal). Due to the genetic heterogenicity of β-thal, the relationship between genetic variants in modifier genes and Hb F level has been studied in different populations. The Chinese Zhuang has the second largest population in China and has 6.78% prevalence of β-thal. However, the effects of these single nucleotide polymorphism (SNP) variants on the Hb F levels of β-thal intermedia (β-TI) patients in this population have not been reported. To explore the association between modifier loci (β-globin gene cluster, HBS1L-MYB intergenic region and BCL11A) and Hb F levels in Chinese Zhuang β-TI patients, 96 unrelated β-TI patients (50 males and 46 females) with different Hb F levels were recruited and genotyped by mass spectrometry. A total of 13 SNPs were confirmed to be in a significant relationship with Hb F levels in this population. Of these, high-risk genotypes of six Hb F-associated SNPs, rs9376090, rs7776054, rs9399137, rs9389268, rs9402685 in the HBS1L-MYB intergenic region and rs189984760 in the BCL11A locus, showed association with high Hb F levels, especially for SNPs in linkage disequilibrium. One novel Hb F-associated SNP, rs189984760, was identified in our study. Our findings will be of valuable reference for correlation between modifier genes and Hb F in Chinese Zhuang populations and may lead to better understand the modifying mechanisms for β-thal.