Β-thal Mutations Research Articles

Clinical, laboratory, and molecular characteristics of a cohort of children with hemoglobinopathy S/beta-thalassemia

IntroductionHemoglobinopathy Sβ-thalassemia (HbSβ-thal) has a wide range of clinical and laboratory severity. There is limited information on the natural history of HbSβ-thal and its modulating factors. We described the molecular, hematological, and clinical characteristics of a cohort of children with HbSβ-thal and estimated its incidence in Minas Gerais, Brazil. MethodsLaboratory and clinical data were retrieved from medical records. Molecular analysis was performed by HBB gene sequencing, PCR-RFLP, gap-PCR, and MLPA. ResultsEighty-nine children were included in the study. Fourteen alleles of β-thal mutations were identified. The incidence of HbSβ-thal in the state was 1 per 22,250 newborns. The most common βS-haplotypes were CAR and Benin. The most frequent βthal-haplotypes were V, II, and I. Coexistence of 3.7 kb HBA1/HBA2 deletion was present in 21.3 % of children. β-thalassemia mutations were associated with several clinical and laboratory features. In general, the incidence of clinical events per 100 patient-years was similar for children with HbSβ0-thal, IVS-I-5 G>A, and IVS-I-110 G>A. Children with HbSβ+-intermediate phenotypes had a more severe laboratory and clinical profile when compared with those with HbSβ+-mild ones. βS-haplotypes and α-thalassemia did not meaningfully influence the phenotype of children with HbSβ-thal. ConclusionThe early identification of β-thalassemia alleles may help the clinical management of these children.

A stepwise haematological screening and whole-exome sequencing reveal multiple mutations from SUPT5H causing an elevation of Hb A2 from a cohort of 47336 individuals.

Though an increase in Hb A2 is one of the most key markers of β-thal carriers, a few independent cases are reported to show elevated Hb A2 levels caused by mutations in other genes beyond β-globin gene. We reviewed the haematological indices of 47336 individuals to analyse the phenotype-genotype correlation and identified 1439 individuals (3.04%) positive in the elevation of Hb A2 . Globin and KLF1 genes analysis was performed, and further whole-exome sequencing was carried to dissect the genetic causes of those positive samples without β-thalassemic or KLF1 mutations. Of these 1439 individuals with elevated Hb A2 , 1381 had a molecular defect in globin genes, and most were β-thalassemic mutation; 10 had a molecular defect in KLF1 gene. Finally, among the 38 individuals without β-thalassemic or KLF1 mutations, 7 were identified to carried a loss-of-function mutation in SUPT5H. This study has provided a mutation spectrum of SUPT5H in a cohort screening leading to the elevation of Hb A2 . According to the previous observations that individuals with a combination of β-thal mutation and a SUPT5H variant might present moderate β-thaelassemia, these findings emphasized the importance of comprehensive molecular diagnosis to prevent birth defects of β-thaelassemia caused by rare mutations from modifier genes.

Prevalence and molecular characterization of alpha and beta-Thalassemia mutations among Hakka people in southern China.

Our aim was to investigate molecular features of thalassemia for proper clinical consultation and prevention in Heyuan. In our research, a total of 25,437 positive screening subjects were further subjected to a genetic analysis of α-thalassemia (α-thal) and β-thalassemia (β-thal). The deletion of α-thal mutation was tested by Gap-PCR, while the non-deletion of α-thal and β-thal mutation were identified by the PCR-reverse dot blot (PCR-RDB) technique. Nested PCR detected Hkαα/-- SEA and Hkαα/αα. Among the 25,437 positive screening subjects, 44.09% (11216/25437) subjects were bearers of thalassemia variations, and 30.85% (7847/25437) subjects showed α-thal changes alone. Among the 23 genotypes with α-thal mutation alone, the three common genotypes were --SEA/αα(68.34%), -α3.7/αα(16.44%), and -α4.2/αα(6.38%). Of the 11.50% (2924/25437) subjects and 29 genotypes with β-thal mutation alone, the three common genotypes were βCD41-42/βN(36.22%), βIVS-II-654/βN(30.88%), and β-28/βN(13.47%). Additionally, of the 1.75% (445/25437) subjects and 55 genotypes showed both α- and β-thal mutations. We also identified 269 cases of Hb H and six patients of Hkαα. Furthermore, the common genotypes of α-thal and β-thal mutations were consistent with allele frequencies of mutations. Our study establishes molecular features of thalassemia among Hakka people in Heyuan. It will be useful for developing strategies to prevent thalassemia.

Serum GDF15 in β-Thalassemia: A Quantitative Marker of Ineffective Erythropoiesis?

Introduction: Ineffective erythropoiesis (IE) is a crucial hallmark of β-Thalassemia (β-Thal) and sets the goal for treatment of both Transfusion-Dependent (TDT) and Non Transfusion-Dependent Thalassemia (NTDT) patients. The Growth Differentiation Factor (GDF) group has a relevant role in the molecular regulation of IE. Specifically, GDF11 contributes to the inhibition of RBC maturation and it is targeted by activin traps, such as luspatercept. However, its role is still debated; i.e., in a mouse model of β-Thal, the absence of GDF11 alone is not sufficient to mitigate IE (Guerra A et al., Blood, 2019). GDF15 increases from early until late phases of erythroid differentiation and negatively regulates erythroid cell development in-vitro, modulating maturation and apoptosis. (Ranjbaran R et a.l, Exp Cell Res, 2020). A few clinical studies found elevated serum GDF15 levels in β-Thal (Tanno T et al., Nat Med, 2007; Huang Y et al., Int J Med Sci, 2019), but data are sparse and a clear correlation with the severity of the pathology is still missing.Methods: We run an observational study at our institution. At routine checks, patients were asked to consent to a specific blood sample for GDF15, whereas hemoglobin (Hb), serum erythropoietin (Epo), ferritin (Ftn), iron, and transferrin saturation (TSat) were measured as part of clinical practice. In a small subset of patients, a consent for serial sampling was added. Serum GDF15 was measured by ELISA (DuoSet DY957, R&D Systems). Demographics were collected from clinical records. Statistical analysis was performed using Statistica 10 (Statsoft).Results: GDF15 levels were measured in 458 individuals: 267 TDT, 77 NTDT, 45 β-Thal trait carriers (BTC), and 69 healthy (H) subjects. Median (IQR) levels of GDF15 were significantly different among diagnoses (P<0.0001), and specifically measured 0.22 (0.16-0.34) in H, 0.48 (0.28-0.96) in BTC, 1.35 (0.40-5.46) in NTDT and 5.95 (3.19-10.52) ng/mL in TDT. (FIG 1A). In TDT patients, a mild but highly consistent negative correlation was observed between Hb and GDF15 levels (R=-0.31, P=0.002). GDF15 levels correlated positively with Epo (R=0.60, P<0.0001), TSat (R=0.32, P=0.0003) and serum iron (R=0.27, P=0.003). In addition, they correlated with length of transfusion interval in splenectomized patients (R=0.65, P<0.001). In a small subset of longitudinal data in TDT, the transfusion cycle had a strong and uniform effect on GDF15 levels. FIG 1B shows a single individual (female, 38 years old, splenectomized): mean (±SD) GDF15 levels were 5.27 (± 1.99) and 2.10 (±0.97) ng/mL pre-transfusion and 8 days post-transfusion, respectively (P<0.01). After 21 days post-transfusion, they were 3.74 (±0.76), showing a trend of variation opposite to Hb. In another individual with a prenatal diagnosis of severe TDT (homozygous for IVS I:110), GDF15 levels were high at 5 months and showed a progressive decline after the start of regular transfusion therapy. After reaching the 10 mg/dL threshold for pre-transfusion Hb, GDF15 levels were approximately halved compared to 5 months of age (6.2 vs 2.7 ng/mL) (FIG 1C). In NTDT patients, GDF15 correlated positively with TSat (R=0.40, P<0.0001), serum iron (R=0.39, P<0.0001) and Ftn (R=0.25, P=0.01). Among TDT and NTDT patients, 45 and 19 were paediatrics, respectively. No significant differences were observed at different ages. In BTC, GDF15 correlated negatively with Hb levels (R=-0.43, P=0.002) and positively with Epo (R=0.78, P=0.02), TSat (R=0.77, P<0.0001), serum iron (R=0.62, P<0.0001) and Ftn (R=0.59, P<0.0001). No significant correlations were observed for H subjects.Discussion: GDF15 levels correlated with the severity of β-Thal phenotype, showing a 26-fold (TDT), a 6-fold (NTDT) and a 2-fold (BTC) increase compared to controls. In TDT patients, higher GDF15 levels correlated with lower Hb and higher Epo, which are typically observed as a result of IE in thalassemia. In addition, GDF15 correlated with markers of altered iron metabolism, such as TSat and serum iron. In individual patients, GDF15 showed strong and consistent variation with treatment. GDF15 was also associated with quantitative markers of disease in NTDT and BTC patients. This is to our knowledge the larger sample of patients carrying β-thal mutations in which GDF15 levels were measured and correlated with the severity of the disease. These results show that GDF15 may be a suitable and useful quantitative marker of IE. [Display omitted] DisclosuresPiga: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Research Funding. Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria.

Spectrum of β-Thalassemia Mutations in Some Areas of Guangxi Zhuang Autonomous Region of Southern China: A Study on a Pediatric Population Aged 0–15 Years

β-Thalassemia (β-thal), one of the most common form of single-gene inheritable blood diseases in the world, is highly prevalent in southern China, especially in the Guangxi Zhuang Autonomous Region. To update the β-thal mutation spectrum in this region, we performed hematological and genetic analyses on 888 β-thal major (β-TM), β-thal intermedia (β-TI) and β-thal carrier patients, aged 0–15 years old, from different parts of Guangxi Province. We identified 55 genotypes and 18 β-thal mutations. The codons 41/42 (–TTCT) (HBB: c.126_129delCTTT) (43.97%), codon 17 (A>T) (HBB: c.52A>T) (25.43%), −28(A>G) (HBB: c.-78A>G) (8.18%), IVS-II-654 (C>T) (HBB: c.316-197C>T) (7.85%) and codon 26 (G>A) (HBB: c.79G>A) (5.02%) were the five most common, accounting for more than 90.0%. The results of our study are providing an up-to-date β-thal mutation spectrum in the 0–15-year-old pediatric population, which will help genetic counseling and prevention of β-TM in mainland China’s most endemic region, Guangxi Zhuang Autonomous Region.

Diagnosis of Sickle Cell Disease and HBB Haplotyping in the Era of Personalized Medicine: Role of Next Generation Sequencing.

Hemoglobin genotype and HBB haplotype are established genetic factors that modify the clinical phenotype in sickle cell disease (SCD). Current methods of establishing these two factors are cumbersome and/or prone to errors. The throughput capability of next generation sequencing (NGS) makes it ideal for simultaneous interrogation of the many genes of interest in SCD. This study was designed to confirm the diagnosis in patients with HbSS and Sβ-thalassemia, identify any ß-thal mutations and simultaneously determine the ßS HBB haplotype. Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. Haplotyping was based on the alleles on five haplotype-specific SNPs. The patients studied included 159 HbSS patients and 68 Sβ-thal patients, previously diagnosed using high performance liquid chromatography (HPLC). There was considerable discordance between HPLC and NGS results, giving a false +ve rate of 20.5% with a sensitivity of 79% for the identification of Sβthal. Arab/India haplotype was found in 81.5% of βS chromosomes, while the two most common, of the 13 β-thal mutations detected, were IVS-1 del25 and IVS-II-1 (G>A). NGS is very versatile and can be deployed to simultaneously screen multiple gene loci for modifying polymorphisms, to afford personalized, evidence-based counselling and early intervention.

The Spectrum of β-Thalassemia Mutations in the Population Migration in Lebanon: A 6-Year Retrospective Study

β-Thalassemia (β-thal) is highly prevalent among the Mediterranean populations. In Lebanon, the carrier rate of the disease is estimated to be around 2.0–3.0%. In this retrospective study, we determined the spectrum of β-thal mutations in a total of 170 individuals from a sample of 140 Lebanese, Iraqi and Syrian refugee families in Lebanon, over a period from 2012 to 2018. Twenty-eight different β-globin gene mutations were identified. The most prevalent mutations were IVS-I-110 (G>A) (HBB: c.93-21G>A), IVS-II-1 (G>A) (HBB: c.315+1G>A), IVS-I-6 (T>C) (HBB: c.92+6T>C) and IVS-I-1 (G>A) (HBB: c.92+1G>A), accounting for the majority of mutations found in HBB mutations analysed in 250 alleles. Ten different β-globin gene mutations that were not previously described in Lebanon were identified in our study. These mutations include the IVS-II-848 (C>A) (HBB: c.316–3C>A), codons 9/10 (+T) (HBB: c.30_31insT), codon 15 (–T) (HBB: c.46delT), −86 (C>G) (HBB: c.-136C>G), Cap +22 (G>A) (HBB: c.-29G>A), −28 (A>C) (HBB: c.−78A>C), codon 7 (GAG>TAG) (HBB: c.22G>T), codon 26 (GAG>TAG) (HBB: c.79G>T), codons 41/42 (–TTCT) (HBB: c.126_129delCTTT), and codons 82/83 (–G) (HBB: c.250delG). Of these, six mutations [codons 9/10, codon 15 (–T), −86, codon 7, codon 26, codons 82/83) were identified in Lebanese samples only; one mutation (IVS-II-848) was identified in both Lebanese and Iraqis; and three mutations (Cap +22, −28, codons 41/42) were identified in Iraqi samples only. Further studies will help better delineate the spectrum of β-thal mutations among different ethnic groups, and provide crucial prevention strategies.

Molecular Spectrum of β-Thalassemia Mutations in Central to Eastern Thailand

The aim of this study was to determine the molecular spectrum of β-thalassemia (β-thal) mutations in eastern Thailand. We identified β-thal mutations using allele specific-polymerase chain reaction (ASPCR) and direct DNA sequencing. We found 18 different β-thal mutations in a total of 191 unrelated subjects. Six common β-thal mutations comprised 86.91% of all the mutations, including codons 41/42 (–TTCT) (HBB: c.126_129delCTTT) (35.60%), codon 17 (A>T) (HBB: c.52A>T) (18.85%), −28 (A>G) (HBB: c.-78A>G) (15.71%), IVS-II-654 (C>T) (HBB: c.316-197C>T) (6.28%), IVS-I-1 (G>T) (HBB: c.92+1G>T) (5.76%) and codon 19 (A>G) (HBB:(c.59A>G) (4.71%). In addition, a novel 60 kb deletion in two unrelated cases was characterized and initially suspected to originate from eastern Thailand. Moreover, we demonstrated the molecular spectrum of recent β-thal mutations in Thailand, and data from this study were compared with five reference laboratory centers in Thailand. This study is the first to identify the comprehensive molecular spectrum of β-thal mutations in eastern Thailand, information that may be essential for screening, genetic counseling and prenatal diagnosis (PND) in this region.

Frequencies of Beta Thalassemia Mutations Show Different Pattern in Bannu Region than Other Parts of Pashtun Population in Khyber Pakhtunkhwa Province Pakistan.

β-Thalassemia arises as result of mutations in HBB gene, influencing the globin production which results in hypochromic and microcytic anaemia. The present study was aimed to investigate the occurrence of six common β-thal mutations, its inheritance pattern, frequency, and consanguinity in parents of Bannu region Khyber Pakhtunkhwa (KP) province, Pakistan. Conducting such studies may impart important information about thalassemia prevention like prenatal diagnosis (PND), carrier screening and genetic counselling which may be helpful in controlling the suspected births. During the study, 250 blood samples were retrieved from different families comprising of one transfusion dependent child and sporadic patients from different areas of Bannu region. The collected blood samples were investigated to see if there is any common mutations which may trigger β-Thalassemia employing amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) approach. Amongst the studied mutation in District Bannu, frame shift codons (FSC) 8/9 (+ G) (HBB: c.27_28insG) was observed to be the most common mutation followed by Codons 41/42 (- TTCT), IVS-I-5(G > C) and FSC 5 (- CT) having frequencies of 42, 26, 19 and 13 respectively. The results obtained by the present study were found different from previous studies demonstrated from other Pashtun regions of KP, showing heterogeneity in frequencies of known mutations. These observations may help in implementing parental meetings about disease recurrence in future, large scale mutation screening, and prenatal diagnosis in the whole Pashtun ethnicity including District Bannu.

Evaluation of the High Resolution Melting Approach for Detection of β-Thalassemia Gene Mutations

β-Thalassemia (β-thal), an autosomal recessive hemoglobinopathy, is one of the most common genetic disorders in Pakistan. Awareness of this disease, genetic counseling, extended family carrier screening and prenatal diagnosis (PND) are helpful in prevention and control. Currently, direct DNA sequencing and multiple amplification refractory mutation system-polymerase chain reaction (MARMS-PCR) are the methods used to detect β-thal mutations, the latter being the most widely used. This study aimed to evaluate PCR-high resolution melting (PCR-HRM) analysis for the detection of most common β-thal mutations that are found in Pakistan. This study was designed to identify the β-thal mutations using PCR-HRM analysis in a total of 90 samples [blood and chorionic villus sampling (CVS)]. These samples were first screened for routine mutations by MARMS-PCR and then evaluated by PCR-HRM analysis. The results of PCR-HRM analyses were further confirmed by direct DNA sequencing and all analyses interpreted the same results in all 90 samples. Eleven cases (36.6%) were detected to carry IVS-I-5 (G>C) (HBB: c0.92 + 5G>C), six cases (20.0%) with frameshift codons (FSC) 41/42 (–TTCT) (HBB: c.126_129delCTTT), five cases (16.0%) were diagnosed with codon 15 (G>A) (HBB: c.47G>A), three cases (10.0%) were found with codon 30 (G>C) (HBB: c.93G>C), one case was diagnosed with FSC 16 (–C) (HBB: c.51delC), one with IVS-I-1 (G>T) (HBB: c0.92 + 1G>T) and one with codon 5 (–CT) (HBB: c.17_18delCT). The PCR-HRM analysis represents a less tedious and more useful method for the detection of β-globin gene mutations.

The Diagnosis of HbS Genotypes and Identification of β-Thalassemia Mutations in Patients with Hbsβ-Thalassemia Using Next Generation Sequencing

The conventional diagnosis of sickle cell disease (SCD) depends on RBC morphology, Hb electrophoresis and/or high-performance liquid chromatography (HPLC). However, these methods are fraught with ambiguities especially in areas where HbSβ-thalassemia is common. The distinguishing laboratory feature between patients with HbSS and HbSb-thalassemia is the level of HbA2. Unfortunately, when HbA2 is estimated with HPLC in patients with Sβthalassemia, it tends to be spuriously high because it co-migrates with glycated HbS. HbSβ-thalassemia accounts for 30 - 40% of the SCD patients being followed in Kuwait. Patients with a presumptive diagnosis of HbSS and/or Sβ-thalassemia, by HPLC were referred to the Hemoglobin Research Laboratory of Department of Pediatrics, Kuwait University for confirmatory molecular diagnosis. The DNA samples collected from 1994 to 2018 were recently subjected to NGS. DNA was isolated from peripheral leukocytes using the phenol-chloroform method. The Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. For theHBBlocus, all β-globin mutations and variants were confirmed by arrayed primer extension (APEX) or Sanger sequencing methods. In addition, genotyping of modifier SNPs inBCL11Aon chromosome 2 and theHBS1L-MYBintergenic region on chromosome 6 was carried out to identify their influence on HbF expression among our patients. In all, 126 SNPs were genotyped. We hereby, report the false positive and false negative rates for the diagnosis of Sb-thalassemia with HPLC compared to NGS. We also report the spectrum of β-thal mutations among our patients with Sβ-thalassemia. The DNA samples were from 232 patients aged from 1 to 59 with a mean of 12.7 ± 11.2 years. Analysis of the initial HPLC diagnosis showed that 27.7% were reported to have Sβ-thalassemia, while after NGS assessment, 29.4% were found to truly fit the diagnosis. Three individuals who were diagnosed as HbSS turned out to be HbAS and 1 each turned out to be HbAA and β-thalassemia trait respectively. Twelve patients that were originally thought to be Sβ-thalassemia turned out to be HbSS, i.e. a false positive rate of 5%, while 30 who were thought to be SS, turned out to be Sβ-thalassemia, i.e. a false negative rate of 13%. A total of 12 mutations were identified in 61 Sβ-thalassemia patients. Of these, the most common were the β0 IVS-1 del 25 and the IVS-II-1 (G/A) in 9 (14.8%) each. The most common β+ mutation was the IVS-I-110 (G/A) in 8 (13.1%), followed by the IVS-I-5 (G/C) in 7 (11.5%). While the Sβ0-thalassemia patients had no HbA on HPLC, the Sβ+ were associated with varying concentrations of HbA, ranging from 0 for those carrying the IVS-I-5 (G/C) mutation to a mean of 15.5 +/- 6.2% in the patients with IVS-I-110 (G/A) and 25.1% in the Sβ++patient with IVS-I-6 (C/T). Indeed, HPLC is far from deal in detecting HbSβ-thalassemia with significant rates of false positivity and negativity. NGS is very versatile; it can interrogate thousands of genes simultaneously, making it ideal for use in SCD. The HbS genotype, β-thalassemia mutation, haplotype and different modifier polymorphisms can be determined in one run. It is therefore very useful for personalized diagnosis that can document prognostic factors, making for purposeful counseling and follow-up from an early age. Disclosures No relevant conflicts of interest to declare.

β-Globin Gene Mutations in Pediatric Patients with β-Thalassemia in the Region of Çukurova, Turkey

β-Thalassemia (β-thal) is one of the most common genetic disorders in Turkey. In this study, we investigated the mutations and frequency of β-thal at the molecular level in pediatric β-thal patients in the Çukurova region. The β-thal mutations of 52 cases were analyzed. An automated blood cell counter was used for hematological data. Cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) methods were used for hemoglobin (Hb) typing. Amplification refractory mutation system (ARMS), restriction fragment length polymorphism (RFLP), gap-polymerase chain reaction (gap-PCR) and DNA sequencing analysis methods were used to determine genomic features. In this study, we found that 36 subjects carried homozygous mutations [IVS-I-110 (G>A) (HBB: c.93-21G>A) (58.3%), codon 8 (–AA) (HBB: c.25_26delAA) (5.6%), −30 (T>A) (HBB: c.-80T>A) (5.6%), IVS-I-6 (T>C) (HBB: c.92+6T>C) (5.6%) and IVS-II-1 (G>A) (HBB: c.315+1G>A) (5.6%)]. We found that 13 subjects carried compound heterozygosities for IVS-I-110/IVS-I-6 (15.4%) and IVS-I-110/frameshift codon (FSC) 44 (–C) (HBB: c.135delC) (15.4%). We observed that the Syrian subject also carried a compound heterozygosity for IVS-I-6/IVS-I-25 (–25 bp) (HBB: c.93_21del). We determined that the most frequently observed β-thal mutation in the Çukurova region, where various types of hemoglobinopathies have been observed, is the IVS-I-110 mutation. As the prevalence of the disease will affect the region where the immigrant population is dense, population screening and prenatal diagnosis (PND) should be increased and the public should be made aware of the consequences.

Molecular Heterogeneity of β-Thalassemia in the Kohat Region, Khyber Pakhtunkhwa Province, Pakistan

The present study was intended to report the incidence of the most frequently occurring β-thalassemia (β-thal) mutations in the Kohat region [Khyber Pakhtunkhwa (KP) Province, Pakistan], their inheritance pattern in patients, and consanguinity in the parents. Moreover, this study could provide valuable information regarding thalassemia diagnoses such as prenatal diagnosis (PND), genetic counseling and carrier screening for controlling the affected births in the population. During this study, 160 peripheral blood samples of affected patients, their parents and siblings were collected from 25 discrete families having at least one child needing regular blood transfusions from different areas of the Kohat region. β-Thalassemia mutations found in the population were screened via the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). A total of 320 alleles was evaluated for the presence of six β-thal mutations. Of these six β-thal mutations, the frameshift codons (FSC) 8/9 (+G) (HBB: c.27_28insG) was found to be the most frequent in the studied population, and more interestingly, followed by IVS-I-5 (G>C) (HBB: c.92+5G>C) and FSC 5 (–CT) (HBB: c.17_18delCT). The findings of the present study show differences with previous results from other regions of the Pashtun population, which demarcates the heterogeneity in mutations found in the Pashtun ethnicity. These observations may help in implementing parental meetings about disease recurrence in future, large scale mutation screening and PND for the population of the Kohat region and also the whole Pashtun ethnicity.

Hb S/β-Thalassemia in the REDS-III Brazil Sickle Cell Disease Cohort: Clinical, Laboratory and Molecular Characteristics

We described the clinical, laboratory and molecular characteristics of individuals with Hb S (HBB: c.20A>T)/β-thalassemia (Hb S/β-thal) participating in the Recipient Epidemiology and Donor Evaluation Study (REDS-III) Brazil Sickle Cell Disease cohort. HBB gene sequencing was performed to genotype each β-thal mutation. Patients were classified as Hb S/β0-thal, Hb S/β+-thal-severe or Hb S/β+-thal based on prior literature and databases of hemoglobin (Hb) variants. Characteristics of patients with each β-thal mutation were described and the clinical profile of patients grouped into Hb S/β0-thal, Hb S/β+-thal and Hb S/β+-thal-severe were compared. Of the 2793 patients enrolled, 84 (3.0%) had Hb S/β0-thal and 83 (3.0%) had Hb S/β+-thal; 40/83 (48.2%) patients with Hb S/β+-thal had mutations defined as severe. We identified 19 different β-thal mutations, eight Hb S/β0-thal, three Hb S/β+-thal-severe and eight Hb S/β+-thal. The most frequent β0 and β+ mutations were codon 39 (HBB: c.118C>T) and IVS-I-6 (T>C) (HBB: c.92+6T>C), respectively. Individuals with Hb S/β0-thal had a similar clinical and laboratory phenotype when compared to those with Hb S/β+-thal-severe. Individuals with Hb S/β+-thal-severe had significantly lower total Hb and Hb A levels and higher Hb S, white blood cell (WBC) count, platelets and hemolysis markers when compared to those with Hb S/β+-thal. Likewise, individuals with Hb S/β+-thal-severe showed a significantly higher occurrence of hospitalizations, vaso-occlusive events (VOE), acute chest syndrome (ACS), splenic sequestration, blood utilization, and hydroxyurea (HU) therapy.

Genetic Background of β-Thalassemia in Northeast Algeria with Assessment of the Thalassemia Severity Score and Description of a new β0-Thalassemia Frameshift Mutation (HBB: c.374dup; p.Pro126Thrfs*15)

β-Thalassemia (β-thal) is a genetic disorder representing a major health problem in Algeria. Our first objective was to determine the allelic frequencies and molecular spectrum of β-thal mutations in patients with major hemoglobinopathies [β-thal major (β-TM) and sickle cell disease] in three provinces of northeast Algeria. Our second objective was to assess if the clinical management of β-TM patients depended on their region of origin. Our last objective was to assess a population originating from Maghreb, the reliability of the thalassemia severity score (TSS) for patients with homozygous β-thal. Sanger HBB gene sequencing was performed on 59 patients with sickle cell disease and 60 with β-TM. For the latter patients, the genetic modifiers of the TSS were genotyped: α-thalassemia (α-thal) deletions and four Hb F-inducing polymorphisms (XmnI, rs1427407 and rs10189857 for BCL11A and rs9399137 for HMIP). Eleven different β-thal mutations were found but two of them (HBB: c.118C>T and HBB: c.93-21G>A) accounted for about 70.0% of the β-thal alleles. A relatively high proportion of Hb S (HBB: c.20A>T)/β-thal genotypes (27.0%) was found in our sickle cell disease cohort where a new frameshift β0-thal mutation (HBB: c.374dup; p.Pro126Thrfs*15) was identified. No difference was found in the three provinces. Of the 60 β-TM patients, those with a high or very high TSS were significantly younger at the age of first transfusion, thus assessing the reliability of this scoring system in a Maghrebin cohort. Trends for a lower age of splenectomy and high ferritin levels were also detected for the higher TSS categories.

A Case Report of Compound Heterozygosity for β0/β+-Thalassemia Resulting from under Diagnosed β-Thalassemia Found in a Hb A'2 Sample

Hb A'2 (or Hb B2) (HBD: c.49G>C) is the most frequent δ chain variant that has been described in Africa but not in Thailand. We report here a 10-month-old Thai infant with compound heterozygosity for β0 codon 17 (A>T; HBB: c.52A>T) and β+ IVS II-654 (C>T; HBB: c.316-197C>T). Under diagnosed β-thalassemia (β-thal) in her father, who carries Hb A'2 and a heterozygous β0 codon 17 mutation, and the mother, who carries a heterozygous β+ IVS II-654 mutation, was noted. Although Hb A'2 does not cause any problems, heterozygosity for Hb A'2 can lead to under diagnosis of β-thal in Hb A'2 samples. This case highlights the importance of Hb A'2 in prenatal diagnosis (PND). Thus, molecular analysis for β-thal mutations should be carried out when a small peak presents at the retention time (RT) of 4.71 min. on high performance liquid chromatography (HPLC) and the summation level of this peak and Hb A2 was equal or higher than 4.0%.

The Spectrum of β-Thalassemia Mutations in Hamadan Province, West Iran

β-Thalassemia (β-thal) is one of the most common hemoglobinopathies worldwide and is caused by mutations on the β-globin (HBB) gene. The aim of the present study was to determine the mutation spectrum of the β-globin gene in β-thal carriers who were originally from Hamadan Province, Western Iran. Two hundred and eighty-two β-thal carriers participated in the study. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and direct sequencing were used for detection of different mutations. A total of 25 different mutations, including 21 β-thal mutations and four other hemoglobin (Hb) variants, in 280 β-thal carriers (99.3%) were detected in the present study. Three types of mutations including IVS-II-1 (G>A) (HBB: c.315+1G>A) (26.24%), codons 8/9 (+G) (HBB: c.27_28insG) (14.54%) and codons 36/37 (–T) (HBB: c.112delT) (12.76%) accounted for more than 50.0% of the identified mutations. Moreover, IVS-I-110 (G>A) (HBB: c.93-21G>A), codon 44 (–C) (HBB: c.135delC) and IVS-I (25 bp deletion) (HBB: c.93-21_del), had frequencies of 7.09, 7.09 and 5.67%, respectively. Allele frequencies of the remaining 19 mutations were less than 5.0%. This study is the first comprehensive study on a large sample size in Hamadan Province, Iran. In conclusion, the present study significantly increased the spectrum of HBB gene mutations in Hamadan Province compared with previous studies. Therefore, these results can be helpful in identifying β-thal carriers and at-risk fetuses through prenatal diagnosis (PND).

KLF1 Mutations Are Not Common in Israel but Can Explain Occasional Cases of Elevated HbA2 or Very Elevated Fetal Hemoglobin

Introduction: Mutations in KLF1 (Kruppel Like Factor1) have been noted to cause a number of different phenotypes of erythrocyte abnormalities, as KLF1 is known as a master regulator of many genes expressed in red blood cells. One important manifestation caused by KLF1 mutations is the upregulation of γ- or δ-globin genes, with associated microcytosis, thus mimicking beta thalassemia (β-thal) trait. Such findings cause difficulty in counseling couples referred for prenatal diagnosis of β-thal. KLF1 mutations have been reported to be more frequent in geographic regions where β-thal is common. Therefore we undertook to analyze for the presence of KLF1 mutations in Israeli individuals.Materials and Methods: We selected 100 individuals for analysis belonging to one of 4 different groups: 1. Individuals with isolated elevated HbA2 (n=14), or isolated elevation of HbF (n=13) or elevation of both HbA2 and HbF (n=6), who are not carriers of a β-thal mutation by sequence analysis. 2. Individuals with β-thal trait (n=19) who have a higher HbF (and/or HbA2) than is expected for β-thal trait, who do not have β-thal intermedia on clinical criteria (blood count, peripheral smear, spleen size). These individuals carried one of 8 mutations known to cause β-thal in our region 3. Two patients (pts) with a history of transfusions (one with massive splenomegaly and sickle trait, suspected to have coinherited CDA, and one with β-thal intermedia with HbF elevation unexpectedly high for his mutation (TATA box -28 A to C; HBB:c.-78A>C). This patient had HbF levels ranging from 23-33% and HbA2 ranging from 7.8-8.9% over years of followup from age 39-50 years. 4. Anonymous controls (n=46) who are pts with a hematological malignancy not suspected of carrying a hemoglobinopathy. KLF1 (exons 1, 2 and 3) was amplified using PCR (exon 1: 564 base pair product and exons 2 and 3: 1703 base pair product). PCR products were subjected to DNA sequence analysis using an Applied Biosystems ABI apparatus. The sequence obtained was analyzed using BLAST alignment and deviations from the published sequence were analyzed using the Mutation Taster program.Results: Two pts were found to carry substitutions with possible or proven clinical significance. One pt is heterozygous for a substitution at c.972C>A (codon 324 exon 3, E324D, aspartic acid to glutamine) which has not been previously reported. According to Mutation Taster this substitution may have clinical significance. This pt, who has normal hematological parameters, had isolated HbA2 elevation (4.1%) with no β-thal mutation identified. The second pt was found to be heterozygous for a substitution at c.901C>T (codon 301 exon 2, R301C, arginine to cysteine). This substitution has been reported by Gallienne et al, 2012, to be associated with elevated Hb F. This young pt was found to have 28% Hb F with a low HbA2 level of 1.4% and MCV of 63.8. His father also had 28% HbF. No β-thal mutation was identified in the pt or his father but the patient was found to be homozygous for a single alpha globin gene deletion. In addition to these 2 substitutions, many pts and controls carried known polymorphisms in KLF1. These polymorphisms are: c.304T>C, exon 2 residue 102 (S102P) (found in 24 pts and 19 controls); -148(G>A), in the upstream noncoding region (found in 11 pts and 8 controls); c.544T>C codon 182 exon 2 (F182L) (found in 3 pts and 4 controls); and c.115A>C, codon 39 in exon 2, M39L (found in 1 pt and 3 controls). One rare, previously unreported substitution was found in heterozygous form in the thal intermedia pt, located at c.259C>G codon 87 exon 2, P87A (proline to alanine). This was not found in the controls, however according to the location in the gene, and to Mutation Taster, this is not suspected to be a functional variant.Conclusions: Functional variants in KLF1 were rarely found in this group of patients. One out of 14 (7%) of individuals with isolated elevated HbA2 and 1/13 patients with isolated HbF elevation (7.7%) were identified as carrying heterozygous KLF1 functional variants. Two novel substitutions were found: E324D which is suspected to have clinical significance and P87A which is not presently suspected to be a functional variant. We conclude that KLF1 analysis may explain occasional individuals with high HbA2, or marked HbF elevation, in the absence of β-thal trait in our patient population. Larger studies are needed to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

β-Thalassemia Mutations in Jamaica: Geographic Variation in Small Communities

Over the last 43 years, surveys of over 200,000 subjects in Jamaica have identified β-thalassemia (β-thal) mutations. In most, these genes were detected at birth in patients with sickle cell-β-thal and so the prevalence and distribution would not be influenced by subsequent clinical course. There were two newborn populations, 100,000 deliveries in the corporate area between 1973-1981 and 84,940 in south and western Jamaica between 2008-2016. A third population, which derived from the Manchester Project in central Jamaica, screened 16,612 secondary school children, aged predominantly 15-19 years, and identified 150 students with the β-thal trait and 11 with sickle cell [Hb S (HBB: c.20A>T)]- or Hb C (HBB: c.19G>A)-β-thal. The latter patients may have been subject to symptomatic selection, but this should not have affected those with β-thal trait. Of the 24 different molecular mutations, β0-thal genes accounted for 10.0-27.0% of these groups and most common was IVS-II-849 (A>G) (HBB: c.316-2A>G). Of the β+ mutations, seven subjects had severe genes with low levels of β chain synthesis but the majority were benign mutations in the promoter region. The –29 (A>G) (HBB: c.-79A>G) mutation dominated in the newborn study in Kingston, similar to experiences in Guadeloupe and African Americans but the –88 (C>T) (HBB: c.-138C>T) mutation was more common among school students in central Jamaica. Caribbean populations are genetically heterogeneous but variations within different parts of Jamaica is of potential importance for prenatal diagnosis and genetic counseling. This information may also be useful among the large Jamaican diaspora.

Rare β- and δ-Globin Gene Mutations in the Pathare Prabhus: Original Inhabitants of Mumbai, India

Genetic structure of the Indian population is influenced by waves of several immigrants from West Eurasia. Therefore, genetic information of various ethnic groups is valuable to understand their origins, the pattern of migration as well as the genetic relationship between them. No genetic data is available on Pathare Prabhu, which is a small indigenous Hindu community from Mumbai, Maharashtra State, India. The aim of this study was to screen the Pathare Prabhus for hemoglobinopathies, which is a major public health problem in India. Two hundred and fifty-seven unrelated Pathare Prabhus subjects were screened for various hemoglobinopathies. Complete blood counts (CBC) were done on an automated hematology counter. High performance liquid chromatography (HPLC) was used to identify β-thalassemia (β-thal) carriers. Molecular characterization of the β gene defects was done by reverse dot-blot hybridization, amplification refractory mutation system (ARMS) and DNA sequencing. Deletional α-thalassemia (α-thal) was detected by multiplex polymerase chain reaction (PCR). Hb A2-Saurashtra (HBD: c.301C>T) was identified by DNA sequencing; its modeling was also done. The prevalence of β-thal was 3.89%, while deletional α-thal was 5.4%. The initiation codon (ATG>ACG) (HBB: c.2T>C) was seen in eight individuals (80.0%), Hb D-Punjab (HBB: c.364G>C) and Hb A2-Saurashtra, was found in two and one individual, respectively. A community-specific β-thal mutation was found in Pathare Prabhus in significant proportions. This information is useful in developing an algorithm for a prenatal diagnosis (PND) program.