Severe Drug-Induced Hemolysis in a Patient with Compound Heterozygosity for Hb Peterborough (HBB: c.334G>T) and Hb Lepore-Boston-Washington (NG_000007.3: g.63632_71046del).

Unstable hemoglobins (Hbs) are often overlooked in the differential diagnoses of drug-induced hemolysis. Hb Peterborough [ß111(G13)Val→Phe; HBB: c.334G>T] is a rare unstable Hb variant, predominantly found in individuals of Italian descent, due to a structural defect involving a single amino acid substitution (phenylalanine for valine at position 111 of the ß-globin chain). Unstable Hb variants are often inherited in the heterozygous state with Hb A (α2ß2) and rarely in compound heterozygosity with other Hb variants. The presence of another variant Hb often alters the phenotype, occasionally resulting in more severe disease. Using a combination of molecular techniques; multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing, we identified a compound heterozygosity for Hb Peterborough and Hb Lepore-Boston-Washington (Hb LBW) [δ87, ß116; NG_000007.3: g.63632_71046del] in a middle-aged gentleman with a history of chronic microcytic anemia and splenomegaly, presenting with severe drug-induced hemolysis, which was managed conservatively. The clinical history and presentation reflect the dual pathology due to the presence of two variant Hbs and their associated phenotypes. In this article, we discuss the phenotype resulting from the interaction of Hb Peterborough and Hb LBW and emphasize the importance of molecular testing in the diagnosis of rare Hb variants.

Ten Years of Routine α- and ß-Globin Gene Sequencing in UK Hemoglobinopathy Referrals Reveals 60 Novel Mutations.

We review and report here the genotypes and phenotypes of 60 novel thalassemia and abnormal hemoglobin (Hb) mutations discovered following the adoption of routine DNA sequencing of both α- and ß-globin genes for all UK hemoglobinopathy samples referred for molecular investigation. This screening strategy over the last 10 years has revealed a total of 11 new ß chain variants, 15 α chain variants, 19 ß-thalassemia (ß-thal) mutations and 15 α(+)-thalassemia (α(+)-thal) mutations. The large number of new thalassemia alleles confirms the wide racial heterogeneity of mutations in the UK immigrant population. Eleven of the new variants ran with Hb A on high performance liquid chromatography (HPLC), demonstrating the value of routine sequencing of both α- and ß-globin genes for all hemoglobinopathy investigations. The new ß chain variants are: Hb Bury [ß22(B4)Glu → Asp (HBB: c.69A > T)], Hb Fulwood [ß35(C1)Tyr → His (HBB: c.106T > C)], Hb Little Venice [ß42(CD1)Phe → Cys (HBB: c.128T > G)], Hb Cork [ß57(E1)Asn → Ser (HBB: c.173A > G), Hb Basingstoke [ß118(GH1)Phe → Ser (HBB: c.356T > C)], Hb Howden [ß20(B2)Val → Ala (HBB: c.62T > C)], Hb Wilton [ß41(C7)Phe → Leu (HBB: c.126C > A)], Hb Belsize Park [ß120(GH3)Lys → Asn (HBB: c.363A > T)], Hb Hampstead Heath [ß2(NA2)His → Gln;ß26(B8)Glu → Lys (HBB: c.[6C > G;79G > A])], Hb Grantham [ß85(F1)Phe → Cys (HBB: c.257T > G)] and Hb Calgary [ß64(E8)Gly → Val (HBB: c.194G > T). The new α chain variants are: Hb Edinburgh [α70(E19)Val → Gly (HBA2: c.212T > G)], Hb Walsgrave [α116(GH4)Glu → Val (HBA2: c.350A > T)], Hb Wexham [α117(GH5) and 118(H1) insertion Ser (HBA1: c.354-355insTCA)], Hb Coombe Park [α127(H10)Lys → Glu (HBA2: c.382A > G)], Hb Oxford [α17(A15)Val → Asp (HBA2: c.53T > A)], Hb Bridlington [α32(B13)Met → Thr (HBA1: c.98T > C), Hb Wolverhampton [α81(F2)Ser → Tyr (HBA2: c.9245C > A)], Hb Little Waltham [α13(A11)Ala → Asp (HBA2: c.41C > A)], Hb Derby [α61(E10)Lys → Arg (HBA1: c.185A > G)], Hb Uttoxter [α74(EF3)Tyr → Asp (HBA2: c.223G > T)], Hb Harehills [α124(H7)Ser → Cys (HBA1: c.374C > G)], Hb Hekinan II [α27(B8)Glu → Asp (HBA1: c.84G > T)], Hb Manitoba IV [α102(G9)Ser → Arg (HBA1: c.307A > C), Hb Witham [α139(HC1)Lys → Arg (HBA2: c.419A > G) and Hb Farnborough [α9(A7)Asn → Asp (HBA1: c.28A > G). In addition, 10 more paralogous α-globin chain variants have been discovered. The novel ß-thal alleles are: HBB: c.-138C > G, HBB: c.-121C > T, HBB: c.-80T > G, HBB: c.18_19delTG, HBB: c.219_220insT, HBB: c.315 + 2_315 + 13delTGAGTCTATGGG, HBB: c.316-70C > G, HBB: c.345_346insTGTGCTG, HBB: c.354delC, HBB: c.376-381delCCAGTG, HBB: c.393T > A, HBB: c.394_395insA, HBB: c.375_376insA, HBB: c.*+95_*+107delTGGATTCTinsC, HBB: c.* + 111_*+112delAA, HBB: c.*+112A > T, HBB: c.394C > T, HBB: c.271delG and HBB: c.316-3C > T. The novel α (+ )-thal alleles are: HBA1: c.95+1G > C, HBA1: c.315C > G [Hb Donnington, α104(G11)Cys → Trp], HBA1: c.327delC, HBA1: c.333_345del, HBA1: c.*+96G > A, HBA2: c.2T > G, HBA2: c.112delC, HBA2: c.143delA, HBA2: c.143_146delACCT, HBA2: c.156_157insG, HBA2: c.220_223delGTGG, HBA2: c.305T > C [Hb Bishopstown, α101(G8)Leu → His], HBA2: c.169_170delAA, HBA2: c.1A > T and HBA2: c.-3delA.

Impact of isolated germline JAK2V617I mutation on human hematopoiesis.

The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis.

Prenatal diagnosis of hemoglobinopathies by pyrosequencing: a more sensitive and rapid approach to fetal genotyping.

Prenatal diagnosis of the hemoglobinopathies by fetal DNA analysis is currently performed in most countries, either by DNA sequencing, restriction enzyme polymerase chain reaction (RE-PCR) or the amplification refractory mutation system (ARMS). These methods are time consuming and prolong the turnaround time for diagnosis. We here describe a method utilizing pyrosequencing for the prenatal diagnosis of 12 common nondeletional α- and ß-globin gene mutations in the UK population. In particular, it replaced the diagnosis of sickle cell disease by RE-PCR and for the diagnosis of ß-thalassemia (ß-thal) by Sanger DNA sequencing. We have genotyped 148 chorionic villi and 29 uncultured amniotic fluid DNA samples by pyrosequencing and found 100% concordance with the fetal diagnosis result obtained by ARMS-PCR or DNA sequencing. Pyrosequencing was more robust, revealing an 83% decrease in diagnostic failures using uncultured amniocyte DNA samples, and also quantitative, revealing one case of allelic imbalance due to maternal DNA contamination. Overall, we found pyrosequencing to be simpler, more robust, quicker, and less expensive than conventional sequencing and RE-PCR, making it a good choice for rapid and cost-effective prenatal diagnosis of thalassemia and sickle cell disease.

The role of the polycomb complex in silencing alpha-globin gene expression in nonerythroid cells.

Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human alpha- and beta-globin genes are silenced by fundamentally different mechanisms. The alpha-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the beta-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the alpha-cluster by sequences within the CpG islands associated with their promoters; the beta-globin promoters do not lie within such islands. Chromatin associated with the alpha-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The alpha- and beta-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.

A large deletion in the human alpha-globin cluster caused by a replication error is associated with an unexpectedly mild phenotype.

We have characterized a newly identified 16.6 kb deletion which removes a significant proportion of the human alpha-globin cluster including the psizeta1, alpha(D), psialpha1 and alpha2-globin genes but leaves the duplicated alpha1 gene intact. This complicated rearrangement results from a combination of slippage and strand switching at sites of microhomology during replication. Functional analysis shows that expression of the remaining alpha1 gene is increased, rather than down-regulated by this deletion. This could be related to its proximity to the remote upstream alpha-globin regulatory elements or reduced competition for these elements in the absence of the dominant alpha2-globin gene. The finding of a very mild phenotype associated with such an extensive deletion in the alpha-globin cluster implies that much of the DNA removed by the deletion is likely to be functionally unimportant. These findings suggest that other than the upstream regulatory elements and promoter proximal elements there are unlikely to be additional positive cis-acting sequences in the alpha-globin cluster.

Hb Bart's in cord blood: an accurate indicator of alpha-thalassemia.

We quantified Hb Bart's (gamma4) levels by high performance liquid chromatography (HPLC) in 103 fresh cord blood samples from Homerton Hospital, East London, UK. The alpha-globin gene arrangement was determined by Southern blot hybridization and genomic sequence analysis of the alpha-globin genes. The cord blood Hb Bart's levels ranged from 0.5 to 11.9% of total hemoglobin (Hb) and were arranged into three categories: i) levels below 1.5%; ii) levels between 1.5 and 5.7%; iii) levels above 6.1%. These corresponded to a normal alpha-globin genotype, a single deleted/inactivated alpha-globin gene and two deleted/inactivated alpha-globin genes, respectively. The study identified the 3.7 kb and 20.5 kb alpha-thalassemia (thal) deletions, three non deletional alpha-thal mutations and a novel alpha-globin gene rearrangement. Hb Bart's screening of fresh umbilical cord blood is an effective method to evaluate globin chain imbalance. This strategy could be utilized to screen populations for the incidence of alpha-thal and also to identify rare or new molecular lesions that reduce alpha-globin gene expression.