ALBERT

Description: Abstract 4281 Introduction: Quantification of HbA2 is a well established screening test for beta-thalassemia trait (BTT). However, there is a small subset of individuals heterozygous for beta-thalassemia mutations who have HbA2 levels less than or equal to 3.5%. Some reports have suggested that iron deficiency in BTT patients causes HbA2 to be lower than expected, while others have found no significant relationship between iron deficiency and the level of HbA2. These conflicting reports have led to confusion amongst clinicians as to the reliability of HbA2 measurement when screening for BTT in iron deficient individuals. Methods: From a database of 444 individuals with heterozygous beta-thalassemia, confirmed by molecular testing, we assessed the variability in HbA2. Individuals were classified as “iron deficient” or “non-iron deficient” based on their serum ferritin, using two definitions of iron deficiency (serum ferritin

Description: Abstract 2068 HbF interferes with deoxygenated HbS polymerization, and is a major genetic modifier of sickle cell anemia severity. In this study, we attempted to identify genetic factors responsible for HbF production in a small group of African American sickle cell anemia patients who have markedly elevated HbF levels. Initially, patients with HbF of more than 11% as determined by HPLC were selected. The following were excluded: age less than 5; MCV greater than 100; presence of HPFH 1 and 2 based on specific gap-PCR tests; other β-globin gene deletions as determined by multiplex ligation-dependent probe amplification (MLPA). We further excluded rare HBG1 and 2 promoter HPFH mutations by nucleotide sequencing. In the end, a unique group of 20 patients were identified for further studies. Their mean age was 16.3 ± 8.3 years; Hb 9.0 ± 1.3 g/dL; MCV 87.9 ± 7.3 fL; and Hb F 17.2 ± 4.8% (range 11–29%). A control group of 30 African American patients were chosen. They had similar age, Hb, and MCV, but their HbF was 5.0 ± 2.5% (range 0.5–8.8%). These patients were examined for the 3 known major HbF quantitative trait loci: the Xmn1 restriction site C/T polymorphism at NT -158 upstream of HBG2; the BCL11A polymorphism on Chr2p16; the HBS1L-MYB intergenic polymorphism on Chr6q23. These 3 HbF quantitative trait loci collectively account for 20–50% of HbF variance in different populations. We found a significant association between high HbF and BCL11A and HBS1L-MYB intergenic region QTLs in these patients, but these account for only 20% of HbF variance (Table). These results were further validated in 590 patients of similar age from the Cooperative Study of Sickle Cell Disease, 57 patients with HbF 20.6 ± 8.2% and 533 patients with HbF 3.1 ± 1.5% (Table). To further explore other possible causes of elevated HbF, we sequenced 8.6 kb DNA fragment between HBG1 and HBD in 15 high HbF and 15 control patients. This DNA fragment includes the 7.2 kb Corfu deletion that is associated with elevated HbF levels and also binding sites for BCL11A. Twenty SNPs were found. The minor allele frequencies were consistently higher in the high HbF group, but the difference was found to be statistically significant only in 4 SNPs, 3 SNPs between positions 49213 and 49994 and 1 SNP at position 54541 (P = 0.001 to 0.04), suggesting that polymorphisms in this region might contribute to HbF expression in African American sickle cell anemia patients. The G→A polymorphism at position 49876 creates a C/EBP binding site which is not present in the major allele. The G→A polymorphism at position 49994 eliminates an AP-1 and NF-E2 binding sites, which are present in the major alleles. All 3 factors are erythroid transcription factors. The possible functional roles of these minor alleles found in significantly higher frequencies in the high HbF patients need to be further investigated. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5171 Infants who are heterozygous for a number of (γδβ)0-thalassemia deletions are known to present with neonatal hemolytic anemia (Koenig et al, Am J Hematol 84:603, 2009). The breakpoints for most of these large deletions have not been identified. Molecular diagnoses of these deletions therefore can be challenging. We now report an infant girl of Irish/Scottish descent with self-limited fetal and neonatal hemolytic anemia, in whom we have defined the extent of the large deletion removing the entire β-globin gene cluster. At 32-week of gestation, fetal tachycardia prompted percutaneous umbilical cord blood sampling, which revealed a hematocrit of 14%. Umbilical cord transfusion was undertaken followed by Caesarean section delivery. Brisk hemolysis continued in the first few days of life (reticulocyte count of 22 – 24%), with no other causes of hemolysis identified. In the following weeks, the infant was transfused on three occasions. After 2-month of age, she became transfusion-independent with a stable microcytic anemia. The infant's mother also had a history of hemolytic anemia requiring transfusion in the neonatal period, and subsequently became transfusion independent with a microcytic anemia. The mother and several of her family members were extensively investigated, and shown to have a novel (γδβ)0-thalassemia deletion of over 100 kb (Pirastu et al, J Clin Invest 72:602, 1983). Multiplex ligation-dependent probe amplification (MLPA) was carried out in the genomic DNA from the present neonate. The infant was heterozygous for a large deletion spanning at least from 5′ to the HS 5 of the LCR to 3′ of the β-globin gene. Sequential gap-PCR reactions and nucleotide sequencing were done. The deletion was characterized with its 5` breakpoint at nt 5,376,341 and 3` breakpoint at nt 5,178,572 (GenBank NT_009237). The deletion measures 197,770 bp, removing the β-globin LCR, all of the β-like globin genes, and several olfactory receptor genes. A diagnostic gap-PCR test was established for detection of this deletion. This case illustrates the syndrome of neonatal hemolytic anemia caused by large deletions removing the entire β-globin gene cluster. MLPA is a useful tool to screen for these deletions. The pathophysiology of these self-limited and sometimes severe fetal and neonatal hemolytic anemias is presently not understood. We speculate that expression of α-hemoglobin stabilizing protein (AHSP) and/or the proteolytic capacity to degrade excess α-globin chains within erythroid cells might be diminished during fetal and neonatal development, accounting for increased red cell membrane damage and hemolysis in affected fetuses and neonates. Disclosures: No relevant conflicts of interest to declare.

Description: Homozygous α0-thalassemia (deletion of all 4 α-globin genes) results in Hb Bart’s (γ4 tetramers). The high oxygen affinity of Hgb Bart’s leads to severe hypoxia inutero with resulting profound edema (hydrops) and congestive heart failure. Almost universally fetal death occurs prior to diagnosis, therefore not allowing the opportunity for treatment. Advancement of maternal-fetal medicine and neonatal ultrasound has led to the possibility of in utero diagnosis and treatment with inrauterine transfusions. Also, cases of premature infants surviving and undergoing chronic transfusion therapy have been reported. Hematopoietic stem cell transplant (HSCT) has become increasingly used in the cure of beta thalassemia, and provides the potential to cure a0thalassemia as well. To date, 3 reported cases of a0thalassemia have been successfully treated with HSCT. Two children underwent matched sibling bone marrow transplant at 20 and 21 months of age and one underwent a 5/6 HLA matched sibling umbilical cord blood transplant (UCBT). One child had received intrauterine transfusions. We report a case of an infant with a0thalassemia successfully treated with intrauterine transfusion therapy followed by unrelated donor UCBT. The child’s mother, of Cambodian descent, presented to the obstetrician at 23 weeks with concerns of decreased fetal movement. Fetal ultrasound revealed a thickened placenta and hydrops fetalis. Based on the family’s nationality and history of previous fetal loss, the suspicion of a0thalassemia was raised. Umbilical cord blood sampling revealed a hypochromic, microcytic anemia with target cells. The hemoglobin electrophoresis demonstrated Hgb Barts and Portland. Subsequent genotyping confirmed deletion of all 4 α-globin genes. Options presented to the family included termination of pregnancy as well as the option of intrauterine transfusion followed by either chronic transfusion therapy with iron chelation or the possibility of HSCT. The patient received three intrauterine transfusions prior to delivery at 32 weeks gestation. A poor physical profile, non-reassuring heart rate and breech position led to the premature delivery. Apgars were 1 at one minute, 6 at five minutes, and 9 at ten minutes. The baby was admitted to the NICU and required mechanical ventilation for two days. The hospital course was relatively uneventful and included red blood cell transfusion. After discharge, he was maintained with intermittent transfusions until 6 months of age. Following informed consent, he was conditioned for transplant with busulfan, cytoan and rabbit ATG. Since the infant lacked an HLA matched sibling, he received a 5/6 HLA matched unrelated donor umbilical cord blood unit delivering 11.8 × 107 nucleated cells/kg. Neutrophil engraftment (ANC〉500) was achieved on day + 15. FK506 and methotrexate 5mg/m2 on days 1, 3 and 6 was utilized for GVHD prophylaxis. His course was complicated by moderate venoocclusive disease, small subdural hemorrhage, and CMV and adenovirus viremia. He has not had any evidence of graft versus host disease. Initial chimerism (RFLP) showed approximately 63% donor derived cells, and subsequent testing showed increasing chimerism and 100% T-cell engraftment. He clinically is doing very well post transplant. This case demonstrates that intrauterine transfusion followed by unrelated donor UCBT is feasible for the treatment of a0thalassemia.

Description: High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p

Description: Abstract 1022 Beta-thalassemia is caused by β-globin gene mutations that result in either markedly decreased (β+) or absent (β0) β-globin chain production. Patients who are either homozygous or compound heterozygous for β-thalassemia mutations are severely anemic and require chronic RBC transfusions. Concomitant inheritance of an α -thalassemia mutation or increased HbF (α 2γ2) expression can ameliorate the disease severity. Surprisingly, many patients homozygous for the codon 8 (–AA) β0-thalassemia mutation are mildly anemic with over 95% HbF [Altay & Gürgey, Ann NY Acad Sci 612:81, 1990]. We now report one such patient, a 20-year old man of Iraqi ancestry who was found to have splenomegaly but was otherwise well. His hemoglobin was 12.4 g/dL, MCV 77 fL, reticulocyte count 3.2%. Hemoglobin analysis by HPLC revealed HbF 98%, HbA2 2%. Multiplex ligation-dependent probe amplification (MLPA) of his β-globin gene cluster confirmed that he did not have a large deletion as found in some patients with hereditary persistence of fetal hemoglobin (HPFH), nor γ-globin gene quadruplication. In addition, he was heterozygous for the α 2 IVSI donor splice site 5 bp deletion (–GTGAG), the α Hph thalassemia mutation. To determine the genetic basis for his persistently high HbF expression, his γ-globin gene promoters were sequenced and no HPFH point mutation was found. We next assessed the status of his 3 major HbF quantitative trait loci (QTL). He was homozygous for SNP rs7482144 C〉T minor allele (Xmn I polymorphism) at 158 bp 5' to the Gγ-globin gene on chr 11p15; homozygous for rs9399137 minor allele in the HBSIL-MYB intergenic polymorphism (HMIP) on chr 6q23, homozygous for the 3-bp deletion which is in linkage disequilibrium with rs9399137 minor allele [Farrell et al, Blood 117:4935, 2011]; and heterozygous for rs766432 minor allele in the 2nd intron of BCL11A on chr 2p16. Thus he had alleles associated with elevated HbF in all 3 QTL. No mutation was found in his KLF1 genes. Within HS2 in his β LCR is the motif (TA)9 (CA)2 (TA)2 CG (TA)10 which is found in Senegal β-globin haplotype 3 [Öner et al, Blood 79:813, 1992]. The Corfu deletion removes part of the δ-globin gene and ∼6 kb upstream flanking sequence, encompassing the 2-kb region reported to be necessary for γ-globin gene silencing [Sankaran et al, NEJM 365:807, 2011], and is often in LD with the IVSI-5 G〉A severe β+-thalassemia mutation. Corfu heterozygotes have slightly increased HbF, but Corfu homozygotes have HbF ∼95%. We sequenced 6.5 kb upstream of δ-globin gene in our patient, and found homozygosity throughout and only 2 nucleotide differences from the GRCh37/hg19 assembly sequence: C〉T (rs3759074) at 2,065 bp, and T〉G (rs7948416) at 718 bp upstream of δ globin gene transcription start site. No deletion was found. At the repressor protein BP1 binding site 530 bp upstream of the β-globin gene, our patient had the common reference sequence, (AC)2 (AT)7 T7. Among 13 subjects heterozygous for codon 8 (–AA) β0-thalassemia mutation, their Hb was 11.6 ± 1.8 g/dL, HbF 2.8 ± 2.6% [Öner et al, Hemoglobin 14:1, 1990]. We studied two unrelated codon 8 (–AA) heterozygotes. One was a 37-year old woman with Hb 9.8, HbA 88%, HbF 5.7%. She was homozygous for the Xmn I polymorphism, and heterozygous for the HbF QTL on chr 6q23 and 2p16. The other was a 24-year old woman with Hb 11.2, HbA 90%, HbF 7.5%. She was homozygous for the Xmn I polymorphism, and heterozygous for the HbF QTL on chr 2p16. These results support the hypothesis that determinant(s) in cis to the β-globin gene cluster, including the Xmn I QTL and related functional motif(s), in concert with HMIP and BCL11A QTL can sustain high-level γ-globin gene transcription in adults. Robust γ-mRNA accumulation and HbF expression occur only when β-mRNA is markedly decreased due to nonsense mediated decay in the codon 8 (–AA) homozygote, as has been shown in patients homozygous for the Corfu deletion [Chakalova et al, Blood 105:2154, 2005] and in experimental model system [Russell, Eur J Haematol 79:516, 2007]. Our findings could have implications for the therapeutic design to induce HbF expression in β-hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

Description: Hereditary persistence of fetal hemoglobin (HPFH) and (δβ)0 thalassemia are caused by deletions within the β-globin gene (HBB) cluster that remove elements that affect the expression of the γ-globin genes (HBG2 and HBG1, or HBG). These deletions are of different lengths and have different 5’ and 3’ breakpoints. The phenotypes associated with heterozygous carriers of (δβ)0 thalassemia and HPFH deletions are differentiated by levels of 5-15% HbF distributed heterocellularly in the former and 15-30% HbF distributed pancellularly in the latter. We found a novel 588.6 kb deletion that removed both the 3.5 kb fragment 5’ to HBD that is deleted in Corfu β thalassemia and contains a BCL11A binding site, and the known cis-acting elements downstream of HBB. The proband with this deletion had a HbF of 5.4% (Morrison et al, Blood, 2014 abstract 3452). To study the relative importance of 5’ and 3’ regulatory elements in HBG expression we studied 209 cases culled from the literature and from our laboratory where the 3.5 kb element 5’ to HBD and enhancers 3’ to HBB were deleted and HBG remained intact. We used a backwards stepwise regression statistical analysis to determine which deleted elements had the greatest effect on HbF levels. The combination of the deletion of 3.5 kb intergenic region 5’ to HBD, the presence of the HPFH-1 “3D” enhancer juxtaposed to HBG, and the deletion of the 3’ HS1 region accounted for 66.7% of the HbF variation in heterozygotes for HPFH and (δβ)0-thalassemia deletions. The HPFH-1 “3D” enhancer juxtaposed to HBG— the main difference between HPFH-1 and 2 compared with Spanish (δβ)0-thalassemia—was associated with an increase in HbF of 20.78% (p