XmnI Polymorphism Research Articles

Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in β-thalassemia and sickle cell disease patients.

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.

Pharmacogenomics of Drugs Used in β-Thalassemia and Sickle-Cell Disease: From Basic Research to Clinical Applications.

In this short review we have presented and discussed studies on pharmacogenomics (also termed pharmacogenetics) of the drugs employed in the treatment of β-thalassemia or Sickle-cell disease (SCD). This field of investigation is relevant, since it is expected to help clinicians select the appropriate drug and the correct dosage for each patient. We first discussed the search for DNA polymorphisms associated with a high expression of γ-globin genes and identified this using GWAS studies and CRISPR-based gene editing approaches. We then presented validated DNA polymorphisms associated with a high HbF production (including, but not limited to the HBG2 XmnI polymorphism and those related to the BCL11A, MYB, KLF-1, and LYAR genes). The expression of microRNAs involved in the regulation of γ-globin genes was also presented in the context of pharmacomiRNomics. Then, the pharmacogenomics of validated fetal hemoglobin inducers (hydroxyurea, butyrate and butyrate analogues, thalidomide, and sirolimus), of iron chelators, and of analgesics in the pain management of SCD patients were considered. Finally, we discuss current clinical trials, as well as international research networks focusing on clinical issues related to pharmacogenomics in hematological diseases.

A survey of intracranial blood flow velocity in thalassemia intermedia in Khuzestan Province, Iran

Background: Beta-thalassemia intermedia (BTI) is a type of hemoglobinopathy with an increased risk of cerebrovascular accidents, and transcranial cerebral Doppler ultrasonography (TCD) through determining the mean cerebral blood flow velocity (CBFV) can serve to predict the risk of a developing stroke. This study aims to compare patients with beta-thalassemia intermedia and healthy individuals in terms of the cerebral blood flow velocity.
 Materials and Methods: This research was a case control study on 35 BTI patients and 25 healthy subjects. The patients were categorized into three age groups including 7-10, 11-15 and 16-20 years old. The mean CBFVs were compared between the two groups. The factors of age, gender, serum ferritin level, hemoglobin level, spleen size, thrombocytosis, and thalassemia genotypes were evaluated for their effects on CBFVs.
 Results: Mean CBFVs were significantly higher in all the intracranial arteries of BTI patients compared to normal subjects (p-value < 0.05). The hemoglobin levels showed a negative correlation between the left and right vertebral arteries of BTI patients in terms of blood flow velocity (p-value < 0.05). The mean CBFVs in the left vertebral and basilar arteries were negatively correlated to age in BTI. There was no correlation among ferritin level, thrombocytosis, splenomegaly, splenectomy, XmnI polymorphism, and cerebral blood flow velocity in the BTI patients group (p-value > 0.05).
 Conclusion: This study showed that cerebral blood flow velocities of BTI patients were higher than normal control group. In addition, CBFVs were not affected by factors such as gender, serum ferritin, platelet count, size of spleen and XmnI genotype, however, there was negative correlation between age and hemoglobin level with CBFVs.

Genetic predictions of life expectancy in southern Thai patients with β0-thalassemia/Hb E.

The types of β-thalassemia mutations, α-thalassemia interactions, and Hb F-associated SNPs have been described in association with variable disease phenotypes. This study aimed to determine the updated spectrum of β-thalassemia mutations and evaluate the contribution of primary and secondary genetic modifiers and SNPs to disease severity, age at onset, and predicted life expectancy in southern Thai β-thalassemia patients. A total of 181 β-thalassemia patients were enrolled and 135 β0-thalassemia/Hb E patients without α-thalassemia interactions were divided into three categories according to disease severity, age at onset, and predicted life expectancy. A total of 16 β-thalassemia mutations were identified in this study, and the three most common β-thalassemia mutations accounted for 61.4% of all mutations. It was also found that the XmnI polymorphism and rs2071348 were associated with age at onset and the predicted life expectancy. More than 82% of β0-thalassemia/Hb E patients with CC genotype (XmnI) were 3 years old or younger at onset. Additionally, >90% of the higher predicted life expectancy in β0-thalassemia/Hb E patients had the T allele of XmnI. Therefore, genetic prediction for age at onset and life expectancy is beneficial and practical during prenatal diagnosis or newborn screening for better genetic counseling and optimal management.

The Authority of Gene Modifiers in β-Thalassemia Major and Its Relationship to the Pathophysiology of the Disease

β-Thalassemia, an inherited red blood cell disorder, presents a significant health problem worldwide and is caused by defects in the β-globin gene, resulting in the reduction or absence of β-globin chain synthesis. That leads to blood transfusion dependency with its terrible complications. Polymorphisms at position -158 of XmnI-HBG2 on chromosome 11 and BCL11A site on chromosome 2p16 might be linked with elevated hemoglobin F (HbF) appearance, which may, in turn, improve β-thalassemia sternness. This study aims to walk around the amending effects of XmnI and BCL11A loci on HbF levels in Egyptian β-thalassemia patients. Material and Methods. A prospective case-control study of 70 multi-transfused β-thalassemia major patients and 22 controls was performed in the Paediatric hematology unit of Assiut university hospital from June 2019 till April 2021. PCR-RFLP was used to detect single nucleotide polymorphisms at XmnI and BCL11A site loci. Results. XmnI Polymorphism was detected in 9 of 70 patients and associated with higher mean HbF levels (53.48%) than patients without polymorphism (mean Hb level was 42.23%)(P-Value=0.035). The frequency of CT heterozygous genotype was 8 (11.4%), TT homozygous genotype was (1.4%), while the wild genotype CC was detected in 61 (87.1%) of the cases. While BCL11A Polymorphism detected in 21 of 70 patients did not affect either Hb or HbF levels (P-Value =0.26). The TT genotype frequency was 49 (70%), and TC heterozygous genotype was detected in 21 (30 %) of patients. The CC genotype was absent. Conclusion: XmnI -158Gγ polymorphism, but not BCL11A polymorphism, has a modifying effect on both Hb and HbF levels in Egyptian β-thalassaemia major patients. Our research goal is to investigate the frequency of XmnI and BCL11A polymorphism in thalassemia major and investigate the HbF level difference according to the XmnI and BCL11A polymorphism of phenotype group in thalassemia major.

Investigating the Efficacy and Safety of Thalidomide for Treating Patients With ß-Thalassemia: A Meta-Analysis.

At present, the main therapies for ß-thalassemia patients include regular blood transfusion and iron chelation, associating with a number of limitations. Thalidomide, a fetal hemoglobin (HbF) inducer that promotes γ-globin gene expression, has been reported to be effective for ß-thalassemia. Thus, this meta-analysis was conducted to assess the efficacy and safety of thalidomide for treating patients with ß-thalassemia. We searched the related studies from eight databases published from inception until December 1, 2021. The R 4.0.5 language programming was used to perform meta-analysis. After screening of retrieved articles, 12 articles were included that enrolled a total of 451 patients. The Cochrane Collaboration risk assessment tool was used to evaluate the quality and the bias risk of the randomized controlled trials (RCTs), and non randomized trials were assessed using Newcastle-Ottawa Scale (NOS). After treatment with thalidomide, the pooled overall response rate (ORR) was 85% (95% confidence interval (CI): 80–90%), and the pooled complete response rate (CRR) was 54% (95% confidence interval: 31–76%). Compared with the placebo group, the thalidomide group had higher odds of overall response rate (odds ratio = 20.4; 95% CI: 6.75–61.64) and complete response rate (odds ratio = 20.4; 95% CI: 6.75–61.64). A statistically significant increase in hemoglobin level and HbF level after treatment, while there was no statistically significant difference in adult hemoglobin (HbA) level, spleen size, and serum ferritin. According to the results of ORR and CRR, transfusion-dependent thalassemia (TDT) patients showed remarkable efficacy of thalidomide, 83 and 52% respectively. So we analyzed 30 transfusion-dependent thalassemia patients from three studies and found that the most frequent ß-globin gene mutations were CD41-42 (-TCTT), while response to thalidomide did not show any statistically significant relationship with XmnI polymorphism or CD41-42 (-TCTT) mutation. About 30% of patients experienced mild adverse effects of thalidomide. Collectively, thalidomide is a relatively safe and effective therapy to reduce the blood transfusion requirements and to increase Hb level in patients with ß-thalassemia.

Study of Xmn-I polymorphism in β-thalassemic children in Egypt

Background β-thalassemia (βT) has a wide spectrum of clinical severity that may be attributed to the wide variations in βT gene mutations. β-Globin mutations with the Xmn-I site might be associated with elevated fetal hemoglobin levels, which in turn may affect the severity of βT phenotype. Aim To investigate the frequency of Xmn-I polymorphism (−158 C>T) among Egyptian children with βT, and to examine the relationship between Xmn-I polymorphism and βT phenotypes. Patients and methods This cross-sectional study was conducted on 112 βT patients (55 males and 57 females) with a mean age of 8.34±3.71 years (2–16 years). Laboratory investigations included complete blood count, hemoglobin electrophoresis, β-globin mutation, identification of Xmn-I polymorphism by two methods: PCR–restriction fragment length polymorphism and amplification refractory mutation system. Results All patients (76 with βT major and 36 with βT intermedia) were negative for the Xmn-I SNP whether in homozygous (+/+) or heterozygous (+/−) state. Conclusion This study demonstrated that Xmn-I polymorphism was not detected in the studied patients and this supports the low frequency of this polymorphism in other Egyptian studies.

Drug Repurposing: Hydroxyurea Therapy Improves the Transfusion-Free Interval in HbE/Beta-Thalassemia-Major Patients with the XmnI Polymorphism.

Aims: HbE/β-thalassemia is the most prevalent form of severe β-thalassemia in Asian countries. Hydroxyurea (HU) is the most common drug used for the management of sickle-cell anemia but not thalassemia. In this study, we aimed to assess clinical HU response among the Bengali HbE/β-thalassemia patients with respect to the XmnI γGglobin polymorphism and elucidate the association between this polymorphism and HU response efficacy. Materials and Methods: We enrolled 49 transfusion-dependent patients with HbE/β-thalassemia. Fetal hemoglobin levels were measured using high-performance liquid chromatography and complete blood counts were determined pre- and post-HU therapy. Polymerase chain reaction-restriction fragment length polymorphism analyses were performed for genotyping the XmnI γGglobin polymorphism. Results: A total of 30 (61.22%) patients were found to be responders, whereas the remaining 19 (38.78%) were nonresponders. We found 33 patients with the heterozygous (C/T) and three with the homozygous mutant (T/T) genotype status. We obtained a statistically significant correlation (p < 0.001) between the XmnI polymorphism genotype and transfusion-free interval. Patients with the XmnI polymorphism were found to be good responders for HU therapy and showed increased hemoglobin levels. Conclusions: Our findings indicate that HU is a potential drug candidate for thalassemia management, particularly for HbE/β-thalassemia. These results hold implications in repurposing HU as an effective and efficient therapy for HbE/β-thalassemia.

Association of XmnI Polymorphism with Fetal Hemoglobin Level in Sudanese Patients with Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited blood disorder that affects red blood cells. The studyof various modulating factors, and genetic factors affecting the clinical severity of the SCD is an interestingresearch focus especially in communities with a distinct genetic background. The XmnI polymorphism isa common genetic variation that was reported in previous studies to increase fetal hemoglobin (HbF) level.This was a descriptive cross-sectional study, conducted in El-Obeid city in Northern Kordofan state, westernSudan, during the period from August to November 2016. The Xmn1 polymorphic site was determined bypolymerase chain reaction. Data was analyzed using SPSS software program version 20. P-value of 0.05and below was considered of significance. In present study HbF level among normal individuals AA , shownsignificant difference (p&lt;0.05) between presence of Xmnl +/+ and absence of Xmnl -/- site. in patients withSS , the HbF level was higher in those who had one or two Xmnl sites as compared to those with the siteabsent . In patients with sickle cell trait AS and AA, only the presence of the one and two Xmnl site (+/+)compared to the absence of the site (-/-) was associated with significant increase in the HbF level. There isa close link between the Xmn1 polymorphism site and HbF level. A wide range of HbF level was obtainedboth in the present and absence of this site. Further studies with a large sample size as well as analysis BShaplotypes among the patient with sickle cell anemia population are needed for better understand of possibleassociation .

A Pragmatic Scoring Tool to Predict Hydroxyurea Response Among β-Thalassemia Major Patients in Pakistan.

Despite high prevalence and incidence of β-thalassemia in Pakistan, there is very limited work on the use of hydroxyurea (HU) in thalassemia patients in the country. This is the first insight regarding genetic profiling of BCL11A and HU responses in Pakistani β-thalassemia. It correlates single-nucleotide polymorphisms on BCL11A (rs4671393, rs766432) and HBG2 (XmnI), age at first transfusion, and β-globin mutations with HU response in β-thalassemia major (BTM). Of 272 patients treated with HU, 98 were complete responders, 55 partial responders, and 119 nonresponders. Our analysis shows that HU response was significantly associated with patients having IVSI-1 or CD 30 mutation (P<0.001), age at first transfusion >1 year (P<0.001), and with the presence of XmnI polymorphism (P<0.001). The single-nucleotide polymorphisms of BCL11A were more prevalent among responders, but could not show significant association with HU response (P>0.05). Cumulative effect of all 5 predicting factors through simple binary scoring indicates that the likelihood of HU response increases with the number of primary and secondary genetic modifiers (P<0.001). Predictors scoring is a pragmatic tool to foresee HU response in patients with BTM. The authors recommend a score of ≥2 for starting HU therapy in Pakistani patients with BTM.

Krüppel-like factor 1 (KLF1) gene single nucleotide polymorphisms in sickle cell disease and its association with disease-related morbidities.

Sickle cell disease has varied clinical symptoms, and patients having high fetal hemoglobin (HbF) have milder symptoms. Various genetic factors are known to modulate the HbF levels. Krüppel-like factor 1 (KLF1) is a transcription factor that regulates the beta-like globin gene expression. Any variation in KLF1 gene may alter the sickle cell disease phenotype. Xmn-I polymorphism is also known to regulate the gamma globin gene expression. Present studies were carried out to investigate the effect of KLF1 gene mutations and Xmn-I polymorphism on the sickle cell disease severity and to ascertain the genotype-phenotype correlation. One hundred and eighteen sickle cell disease patients having a median follow-up of 5years (3-10years) were recruited. Clinical details were recorded from their retrospective medical records. Xmn-I polymorphism were analyzed using PCR-RFLP method. Variations in KLF1 gene were identified using Sanger sequencing. Out of 118 patients, 24 had acute chest syndrome and 21 patients had more than 2 pain episodes per year. There were no significant differences in sickle cell disease-related morbidities in male and females barring leg ulcers. A total of 6 polymorphism were observed in KLF1 gene, out of which 3 are novel (c.-304G > C, c.*141A > G and c.*178A > G). No statistically significant association of any of SNPs identified in KLF1 gene or Xmn-I polymorphism was seen with HbF levels as well as the sickle cell disease-related morbidities. No association exists between fetal hemoglobin or sickle cell disease-related morbidities and Xmn-I polymorphism or with SNPs identified in KLF1 gene in the studied cohort.

Contribution of Xmn I Polymorphism in the Variation of Hemoglobin F in Thalassemia Intermedia Patients in Pakistan

Background: The −158 (C→T) nucleotide change, known as Xmn I polymorphism, occurs in G γ-globin gene promoter and may result in elevated fetal hemoglobin (HbF). However some studies did not find any association of HbF with the mutation. This study was taken to confirm the influence of Xmn-I polymorphism on HbF production in thalassemia intermedia patients in Pakistani population. Methods: Blood samples from 100 known thalassemia intermedia patients were collected and analyzed for Xmn -I polymorphism and levels of hemoglobin F. Results: High levels of HbF were found in Thalassemia intermedia patients being heterozygous and homozygous for Xmn -I polymorphism. Conclusion: Xmn I polymorphism (C-T) of the G γ-globin gene promoter is associated with increased expression of the G γ-globin gene, higher production of HbF and lesser clinical severity.

Association Between Genetic Polymorphisms and Hb F Levels in Heterozygous β-Thalassemia 3.5 kb Deletions

Single nucleotide polymorphisms (SNPs) in several genetic modifying factors have been related to Hb F levels, including Gγ XmnI polymorphism, B-cell lymphoma/leukemia 11 A (BCL11A), HBS1L-MYB intergenic polymorphism (HMIP) and a mutation in the Krüppel-like factor 1 (KLF1). This study aimed to determine whether genetic variability of these modifying factors affects Hb F levels in heterozygous β-thalassemia (β-thal) 3.5 kb deletion (NC_000011.10: g.5224302-5227791del13490bp). A total of 111 β-thal 3.5 kb deletion carriers with Hb F levels ranging from 0.9 to 18.4% was recruited for this study. Genotyping of SNPs including HBG2 rs7482144, HMIP rs4895441 and rs9399137, BCL11A rs4671393 and KLF1 rs2072596 was identified. Multiple regression analyses showed that only two SNPs (HMIP rs4895441 and rs9399137) influenced Hb F levels. Interestingly, a combination of these two SNPs was associated with higher Hb F levels. Our study is the first to demonstrate that the rs4895441, rs9399137 of HMIP are associated with elevated Hb F levels in the heterozygous β-thal 3.5 kb deletion.

XmnI Polymorphism in Sickle Cell Disease in North Morocco

Sickle cell disease is one of the most common severe monogenic disorders in the world. The –158 XmnI polymorphism (C>T) of the Gγ-globin gene promoter is known to be associated with increased expression of the Gγ-globin gene, thus, higher production of Hb F and lesser clinical severity. This study aims to determine the frequency of the XmnI polymorphism and its association with Hb F levels as a modulating factor of sickle cell disease severity in north Moroccan patients. Three hundred and eight subjects carrying the sickle cell mutation and 160 healthy individuals were recruited at the regional hospital of Larache, Morocco. The complete blood count and the Hb F levels were analyzed. The XmnI polymorphism was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and statistical analysis were done using the Statistical Package for Social Sciences software version 20. Our results estimated the allelic frequency of the XmnI polymorphism in our population at 15.8%. Out of 468 samples, 7.6% were homozygous [+/+] and 16.4% were heterozygous [+/−] for the XmnI polymorphism. This polymorphism was revealed at 20.6% in SS patients, 24.2% in AS carriers, 28.6% in Hb S (HBB: c.20A>T)/β-thalassemia (β-thal) patients and 22.5% in AA subjects. The north Moroccan sickle cell disease patients have shown a low frequency of the XmnI polymorphism. This was later found to be associated with high Hb F levels and mild clinical severity.

Association between Different Polymorphic Markers and β-Thalassemia Intermedia in Central Iran

β-Thalassemia intermedia (β-TI) is a clinical condition characterized by moderate, non transfusional anemia and hepatosplenomegaly. The main objective of this study was to determine the molecular basis of the clinical phenotype of β-TI in Iran. To elucidate the mild phenotype of many patients with β-TI, we screened for three prevalent β-globin gene mutations [IVS-II-1 (G>A) HBB: c.315+1G>A, IVS-I-110 (G>A) HBB: c.93-21G>A and IVS-I-5 (G>C) [HBB: c.92+5G>C], deletions on the α-globin genes, XmnI polymorphisms and restriction fragment length polymorphism (RFLP) haplotypes on the β-globin gene cluster in 50 β-TI patients. Fifty-eight percent of the patients (29 cases) were associated with the mentioned mutations. We showed that the HBB: c.315+1G>A mutation is linked to haplotype [+ – + +] (57.69%). This haplotype is in linkage disequilibrium with the XmnI polymorphism (NG_000007.3: g.42677C>T) and has been associated with increased expression of Hb F in β-TI patients. The XmnI polymorphism is defined in association with this prevalent mutation. Two patients had a single α-globin gene deletion [–α3.7 (rightward) deletion]. The main genetic factor in mild phenotype β-TI patients is the linkage of an XmnI polymorphism (NG_000007.3: g.42677C>T) with the HBB: c.315+1G>A (80.76%), which is associated with increased production of Hb F and coinheritance of haplotype [+ – + +] with β-TI, especially with the homozygous HBB: c.315+1G>A mutation. Molecular basis of β-TI could be explained by the involvement of different factors that tend to develop the disease phenotype.

Thalassemia Major and Intermedia Patients in East Java do not Show Fetal Hemoglobin Level Difference in Relation to XMNI Polymorphism.

Introduction:Thalassemia is a genetic disorder, which shows, varies phenotype due to genetic modifier. XmnI is one of the genetic modifiers which affect clinical severity in thalassemia. XmnI polymorphism may increase HbF production beyond fetal life, thus ameliorating the clinical phenotype.Aim:this study aimed to investigate the difference in HbF level and the relation of HbF level and XmnI polymorphism in Thalassemia Major (TM) and Thalassemia Intermedia (TI) patients.Methods:forty-eight beta thalassemia patients (28 males and 20 females), consists of 16 TM and 32 TI; mean age, 25.30 year old. Hemoglobin Fetal and HbA2 level were determined using High performance Liquid Chromatography (HPLC), and XmnI polymorphism was confirmed by PCR-RFLP. Statistical analysis was done using T-test, Mann-Whitney and Pearson Chi-square.Results:The frequency of heterozygote (+/-) XmnI polymorphism in TM and TI patients was 56.25% vs 71.87%, while the frequency of homozygote (-/-) in TM and TI was 43.75% vs 28.13% with p value >0.05. The insignificant difference also found in HbF level between XmnI +/- and -/- in TM and TI patients.Conclusion:This study revealed that thalassemia major and thalassemia intermedia patients in East Java showed similar XmnI polymorphism. These phenomena also showed by HbF level in relation to XmnI polymorphism in the phenotype groups (TM and TI).

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.&#x0D; Methods: Published literatures were reviewed on the allelic frequency of Xmn1 polymorphism and its effect on HbF induction among thalassaemia patients of different countries.&#x0D; 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&gt;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&gt;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&gt;T polymorphism at -158 XmnI site (HBG2:c.- 53-158C&gt;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.&#x0D; 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.

PB2406 THE INFLUENCE OF XMNI AND BCL11A GENE MODIFIERS ON HEMOGLOBIN F LEVELS IN EGYPTIAN β‐THALASSEMIA MAJOR PATIENTS

Background:β‐thalassemia major (β‐TM) is an autosomal recessive disorder in which β‐globin chain synthesis is severely decreased or absent which lead to blood transfusion dependency with its catastrophic complications. Polymorphisms at position ‐158 of XmnI‐HBG2 on chromosome 11 and BCL11A site on chromosome 2p16 might be associated with elevated hemoglobin F (HbF) expression which may,in turn,improve β‐thalassemia severityAims:This study aims to explore the modifying effects of XmnI and BCL11A loci on HbF levels in Egyptian β‐thalassemia patientsMethods:A prospective case‐control study of 70 multi‐transfused β‐thalassemia major patients and 22 controls was conducted in Paediatric hematology unit of Assiut university hospital from January 2016 till April 2017. PCR‐RFLP was used for detection of single nucleotide polymorphisms at XmnI and BCL11A site loci.Results:XmnI Polymorphism detected in 9 of 70 patients and associated with higher mean HbF levels (53.48%) than patients without polymorphism (meanHbF level was 42.23%)(P‐Value = 0.035).The frequency of CT heterozygousgenotype was 8(11.4%), TT homozygous genotype was (1.4%) while the wild genotype CC detected in 61 (87.1%) of the cases. While BCL11A Polymorphism detected in 21 of 70 patients had no effect on either Hb or HbF levels (P‐Value = 0.26). The TT genotype frequency was 49 (70%) and TC heterozygous genotype was detected in 21 (30 %) of patients. The CC genotype was absent.Summary/Conclusion:The presence of XmnI polymorphism rather than BCL11A polymorphism has a modifying effect on HbF production and increased Hb levels between β‐TM patients and so, it helps in identifying patients’ benefits from HU therapy.image

PB2402 BETA‐GLOBIN GENE CLUSTER HAPLOTYPES ASSOCIATED WITH HBF VARIATION IN β‐THALASSEMIA CARRIERS OF PORTUGUESE ORIGIN

Background:Genetic association studies showed that HbF levels variation in haemoglobinopathies, such as sickle cell disease and β‐thalassemia (β‐thal), are under the influence of single nucleotide polymorphisms (SNPs) in several genomic loci. The first described polymorphism influencing gamma globin gene expression was XmnI in HBG2 promoter −158C &gt; T (rs7482144). Several reports showed that β‐thal carriers with HbF raised levels have a significantly higher rs7482144 minor allele frequency than patients with low HbF levels. The functional significance of HBG2‐XmnI mutation has never been clearly defined and it is possible that it reflects a marker in linkage disequilibrium (LD) with other functional polymorphism elsewhere in the β‐globin gene cluster. It has been suggested that the role assigned to XmnI could be played by the rs16912979 A &gt; G polymorphism in the palindromic region of 5′HS4 β‐globin locus control region (Neishabury et al., Blood Cells Mol Dis. 2012;48:1‐5).Aims:The main goal of this work was to define haplotype backgrounds in β‐globin gene cluster that could explain HbF variation in a population sample of β‐thal carriers.Methods:We studied 79 Portuguese β‐thal carriers (39 males, 40 females), aged between 2 to 70 years old, with HbF levels ranging from 0.2% to 5.1%. HbA2 levels were 3.4 to 5.8% (mean 4.71). Six different HBB mutations were identified at heterozygous state, the two most commons were c.92+6T &gt; C [IVSI‐6 (T &gt; C)] (n = 30) and c.118C &gt; T [CD39 CAG &gt; TAG)] (n = 23). The remaining mutations were c.48G &gt; A [CD15 TGG &gt; TGA] (n = 11), c.92 + 1G &gt; A [IVS‐I‐1 (G &gt; A)] (n = 10), c.93‐21G &gt; A [IVSI‐110 G &gt; A] (n = 3) and c.20delA [CD 6 ‐A] (n = 2). HbA2 and HbF levels were determined by HPLC. The two SNPs rs16912979 and rs7482144 were genotyped by PCR‐RFLP and the HBB gene mutations were identified by Sanger sequencing. Associations of SNPs with HbF levels were assessed by linear regression models using PLINK. Informed consent was provided by all the participants.Results:The two SNPs HBG2‐XmnI rs7482144 and 5′HS4 rs16912979 were in moderate LD (r2 = 0.65): from a total of 41 rs16912979 G alleles, 73.2% were linked to an rs7482144 T‐allele. Linear regression, in the additive model, showed significant association between the XmnI rs7482144 minor T‐allele and increased levels of HbF (β = 0.648; P = 0.0004). A marginal association was found between the 5’HS4 rs16912979 minor G‐allele and HbF (β = 0.648; P = 0.039), however, no significant association was found after conditioning on XmnI rs7482144 (P = 0.192). The common mutation HBB: c.118C &gt; T [CD39] was found strongly associated with increased levels of HbF (β = 0.948; P = 6.4e‐5), remaining significant after conditional analysis on XmnI rs7482144 (P = 0.0002). Haplotypes combining the HbF‐associated alleles on SNPs rs16912979 and rs7482144 (haplotype GT) showed a significant association with raised levels of HbF (P = 0.0008), explaining 13.7% of HbF levels variation. Combining rs16912979, rs7482144 and mutation HBB: c.118T, three haplotypes were found significantly associated with HbF: GTT (β = 1.346; P = 0.00089), ACT (β = 0.784; P = 0.017) and GTC (β = 0.492; P = 0.032), explaining 13.4%, 7.2% and 5.9% of HbF levels variance, respectively.Summary/Conclusion:Our data suggest that in Portuguese β‐thal carriers the HBG2‐XmnI polymorphism and HBB: c.118C &gt; T [CD39] mutation are associated with HbF levels, showing independent effects. The marginally significant effect on HbF levels variation of SNP 5’HS4 rs16912979 appear to be mediated by the effect of XmnI polymorphism.

Compound Heterozygote of Hb S (HBB: c.20A>T)/Hb Westdale (HBB: c.380_396delTGCAGGCTGCCTATCAG): Report of Four Cases from Odisha State, India

We report four cases of compound heterozygotes for Hb S (HBB: c.20A>T) and a rare β0-thalassemia (β0-thal) mutation, Hb Westdale (HBB: c.380_396delTGCAGGCTGCCTATCAG), characterized by a 17 bp deletion between codons 126 to 131 in exon 3 of the β-globin gene of human hemoglobin (Hb) confirmed by direct β-globin gene sequencing. All four cases were from four unrelated families belonging to the Agharia caste, an endogamous ethnic community of the Sundargarh and Jharsuguda districts of Odisha State, India. Detailed observations indicated that all four cases of Hb S/Hb Westdale were clinically severe. On family screening, six family members were found to be heterozygous for Hb Westdale and were asymptomatic. Deletional α-thalassemia (α-thal) and XmnI polymorphism were studied for all the Hb Westdale cases. The Hb S/Hb Westdale cases had an early median age at onset of symptoms and presentation, more requirement of blood transfusions, splenomegaly and hepatomegaly and were found to be clinically more severe when compared with the Hb S-β-thal with IVS-I-5 (G>C) (HBB: c.92 + 5G>C) cases. Overall, the findings indicate that this rare and hitherto unreported compound heterozygosity of Hb S/Hb Westdale is a clinically significant hemoglobinopathy and its finding in a large endogamous community of Odisha State, India will have important implication in the epidemiology and understanding of the clinical spectrum of sickle cell disease in Indian context and prenatal diagnosis.