Transient neonatal hemolytic anemia due to the novel gamma globin gene mutation HBG2:C.290T>C, p.Leu97Pro (hemoglobin Wareham).

Unstable gamma globin variants can cause transient neonatal hemolytic anemia. We have identified a novel variant in a newborn who presented with jaundice and anemia requiring phototherapy and red blood cell transfusion. The patient was found to be heterozygous for the mutation HGB2:c.290T>C, p.Leu97Pro, which we have termed hemoglobin (Hb) Wareham. This substitution is expected to generate an unstable hemoglobin with increased oxygen affinity based on the homologous mutation previously described in the beta globin gene, which is termed as Hb Debrousse. The patient fully recovered by 9 months of age as expected with the transition from fetal to adult hemoglobin.

Editing aberrant splice sites efficiently restores ß-globin expression in ß-thalassemia.

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.

A Mild Phenotype of Severe ß+ Thalassemia in a 16-Month-Old Boy.

ß thalassemia is characterized by a deficient production of functional ß-globin chains and a relative excess of α-globin chains. An extremely diverse clinical spectrum-asymptomatic to transfusion-dependent-is primarily due to homozygosity or compound heterozygosity for the very large number of ß-thalassemia-causing mutations, along with interacting mutations that affect the α-globin and γ-globin genes and their expression. We report a case of a 16-month-old boy who was initially diagnosed with iron deficiency anemia until he was later found to be homozygous for a severe ß-thalassemia genotype with a mild hematologic phenotype. This was likely as a result of his ability to produce high levels of fetal hemoglobin.

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 ß-thalassemia.

Hb Presbyterian (HBB: c.327C>G) in a Nicaraguan Family.

Hemoglobin (Hb) is the protein responsible for oxygen transportation. It is a tetrameric protein comprising two α- and two ß-globin subunits. In the literature, a large number of mutations in the α- and ß-globin genes have been documented. Among these mutations, Hb Presbyterian (HBB: c.327 C>G), is a naturally occurring mutant exerting low oxygen affinity. The C to G exchange (AAC>AAG) at codon 108 of the ß-globin gene results in the substitution of asparagine by lysine. Here, we document the identification of HBB: c.327 C>G in a 6-year-old female patient and her father from Nicaragua and Cuba, respectively. The presence of the abnormal Hb was confirmed by cellulose acetate electrophoresis, high performance liquid chromatography (HPLC) and genomic DNA sequencing. The ß-globin gene sequences for both, father and daughter, disclosed the heterozygous mutation at codon 108 to be Hb Presbyterian or HBB: c.327 C>G. The mutant Hb was previously reported in four families from North America, Germany, Japan and Spain, respectively. This is the fifth family carrying HBB: c.327 C>G described to date and the first report from Latin America.

A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells.

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, ß-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.

A candidate transacting modulator of fetal hemoglobin gene expression in the Arab-Indian haplotype of sickle cell anemia.

Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) ß-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.

Diverse hematological phenotypes of ß-thalassemia carriers.

Most ß-thalassemia carriers have mild anemia, low mean corpuscular volume and mean corpuscular hemoglobin, and elevated hemoglobin α2 (HbA2 ). However, there is considerable variability resulting from coinheritance with α- and/or δ-globin gene mutations, dominant inheritance of ß-thalassemia mutations, highly unstable variant globin chains, large deletions removing part or all of the ß-globin gene cluster, loss of heterozygosity of the ß-globin gene cluster during development, or concomitant erythroid enzyme or membrane protein abnormalities. Recognition of the specific abnormality and correct diagnosis can allay anxiety and unnecessary investigation, help formulate treatment programs, and deliver appropriate genetic and family counseling.

The genetic basis of asymptomatic codon 8 frame-shift (HBB:c25_26delAA) ß(0) -thalassaemia homozygotes.

Two 21-year old dizygotic twin men of Iraqi descent were homozygous for HBB codon 8, deletion of two nucleotides (-AA) frame-shift ß(0) -thalassaemia mutation (FSC8; HBB:c25_26delAA). Both were clinically well, had splenomegaly, and were never transfused. They had mild microcytic anaemia (Hb 120-130 g/l) and 98% of their haemoglobin was fetal haemoglobin (HbF). Both were carriers of Hph α-thalassaemia mutation. On the three major HbF quantitative trait loci (QTL), the twins were homozygous for G>A HBG2 Xmn1 site at single nucleotide polymorphism (SNP) rs7482144, homozygous for 3-bp deletion HBS1L-MYB intergenic polymorphism (HMIP) at rs66650371, and heterozygous for the A>C BCL11A intron 2 polymorphism at rs766432. These findings were compared with those found in 22 other FSC8 homozygote patients: four presented with thalassaemia intermedia phenotype, and 18 were transfusion dependent. The inheritance of homozygosity for HMIP 3-bp deletion at rs66650371 and heterozygosity for Hph α-thalassaemia mutation was found in the twins and not found in any of the other 22 patients. Further studies are needed to uncover likely additional genetic variants that could contribute to the exceptionally high HbF levels and mild phenotype in these twins.

Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.

Hereditary Persistence of Fetal Hemoglobin Caused by Single Nucleotide Promoter Mutations in Sickle Cell Trait and Hb SC Disease.

Hereditary persistence of fetal hemoglobin (HPFH) can be caused by point mutations in the γ-globin gene promoters. We report three rare cases: a child compound heterozygous for Hb S (HBB: c.20A > T) and HPFH with a novel point mutation in the (A)γ-globin gene promoter who had 42.0% Hb S, 17.0% Hb A and 38.0% Hb F; a man with Hb SC (HBB: c.19G > A) disease and a point mutation in the (G)γ-globin gene promoter who had 54.0% Hb S, 18.0% Hb C and 25.0% Hb F; a child heterozygous for Hb S and HPFH due to mutations in both the (A)γ- and (G)γ-globin gene promoters in cis [(G)γ(A)γ(ß(+)) HPFH], with 67.0% Hb A, 6.5% Hb S and 25.0% Hb F.

The first report of a homozygous codons 9/10 (+T) ß-thalassemia mutation in a Turkish patient.

For the first time in Turkey, we report a thalassemic patient with a homozygous codons 9/10 (+T) genotype. Currently, the patient is 3 years and 2 months old and received an initial transfusion at the age of 18 months. After being alloimmunized following this transfusion, he required frequent transfusions, every week to every other week. Although alloimmunization was controlled after methyl-prednisolone, intravenous immunoglobulin, plasmapheresis and rituximab, the transfusion requirements continued related to hypersplenism. Subsequent to splenectomy, transfusion requirements disappeared with average hemoglobin (Hb) levels around 11.0 g/dL. The mother underwent prenatal diagnosis (PND) when she became pregnant for the third time; this revealed a heterozygous codons 9/10 fetus.

Hb Youngstown [ß101(G3)Glu → Ala; HBB: c.305A > C]: An unstable hemoglobin variant causing severe hemolytic anemia.

Hb Youngstown is a rare hemoglobin (Hb) variant caused by substitution of glutamic acid with alanine at amino acid residue 101 of the ß-globin chain as a result of an A > C transversion on the ß-globin gene nucleotide sequences [ß101(G3)Glu → Ala; HBB: c.305A > C]. We now report three patients from two different families, one from South Africa and the other from Costa Rica, who are heterozygous for this Hb variant. All three carriers had marked hemolysis, consistent with Hb Youngstown being a highly unstable variant. The substitution of glutamic acid, a large and negatively charged amino acid, with alanine, a small and non polar amino acid, in the interface of the α1- and ß2-globin subunits might interfere with the transition between the oxy- and deoxyHb, and lead to Hb instability and hemolytic anemia.

Novel dominant ß-thalassemia: Hb Boston-Kuwait [codon 139/140(+T)].

Dominant ß-thalassemias exhibit a hybrid phenotype of unstable hemoglobin and ineffective erythropoiesis. Most arise from heterozygous ß-globin gene mutations in exons 3 or 2 and present in adulthood as thalassemia intermedia. We report a novel, de novo ß-globin mutation presenting in a toddler with features of thalassemia major and chromaturia. Hemoglobin Boston-Kuwait is an elongated ß-chain variant (163 amino acids) that results from a frameshift mutation caused by a thymidine insertion in codons 139/140. Hematopoietic stem cell transplant provided a successful alternative therapy for this severe form of dominant ß-thalassemia.

Identification of the first mutation in a BRE motif of the ß-globin gene and its inheritance with two other α-globin gene mutations in a Lebanese family.

A 7-year old boy presented with a history of recurrent respiratory infections and hypochromic microcytic anemia. Iron profiles were normal thereby prompting genetic analysis of α- and ß-globin mutations. The first mutation in a BRE motif of the ß-globin gene in the proband, sibling and the mother was identified. The proband and his sibling also inherited common α-globin mutations from the father and mother. In all cases, no serious thalassemia disease was detected.