Why is the novel Hb Miguel Servet visualised by CE-HPLC newborn and not by the CE-HPLC ß-thalassaemia programme?

Screening of haemoglobinopathies is indicated for the detection of sickle cell anaemia; thus, neonates can benefit from early and adequate treatment that prevents neurological damage, reduces morbidity and mortality associated with the disease. These types of programmes sometimes lead to unexpected findings. We present a new haemoglobin (Hb) variant (Hb Miguel Servet) detected by newborn screening. During neonatal screening of haemoglobinopathies by cation-exchange high-performance liquid chromatography (CE-HPLC) newborn, an Hb variant was detected. An analysis at 8 months of age using capillary zone electrophoresis (CZE) confirmed the presence of this new Hb. The molecular characterisation was performed by automatic sequencing of the α and ß globin genes in an ABI PRISM 3100 Genetic Analyzer. Hb analysis by CE-HPLC ß-thalassaemia short programmedid not indicate the presence of abnormal Hbs. By CZE showed a peak in the zone 12 zone comprising 3.3% of the total Hb. A new analysis by CE-HPLC on a Tosoh G8-2 (Horiba) shown a peak, in the region of HbA1b, did not interfere with the quantification of HbA1c. Sequencing of the ß gene revealed the substitution of a guanine for a thymine (GGT >TGT) in codon 69 of the second exon, resulting in substitution of cysteine for the amino acid glutamine (HBB:c.208G>T). Hb Miguel Servet is a ß-chain globin variant detected by CE-HPLC newborn (BioRad), by CZE and by CE-HPLC-CE Tosoh G8-2 (Horiba), but no by CE-HPLC-CE ß-thalassaemia short programme (BioRad). In fact, for all the techniques that are visualised, what would be detected would be the glutathione variant of Hb (Miguel Servet).

Comprehensive analysis of mitochondrial and nuclear DNA variations in patients affected by hemoglobinopathies: A pilot study.

The hemoglobin disorders are the most common single gene disorders in the world. Previous studies have suggested that they are deeply geographically structured and a variety of genetic determinants influences different clinical phenotypes between patients inheriting identical ß-globin gene mutations. In order to get new insights into the heterogeneity of hemoglobin disorders, we investigated the molecular variations on nuclear genes (i.e. HBB, HBG2, BCL11A, HBS1L and MYB) and mitochondrial DNA control region. This pilot study was carried out on 53 patients belonging to different continents and molecularly classified in 4 subgroup: ß-thalassemia (ß+/ß+, ß0/ß0 and ß+/ß0)(15), sickle cell disease (HbS/HbS)(20), sickle cell/ß-thalassemia (HbS/ß+ or HBS/ß0)(10), and non-thalassemic compound heterozygous (HbS/HbC, HbO-Arab/HbC)(8). This comprehensive phylogenetic analysis provided a clear separation between African and European patients either in nuclear or mitochondrial variations. Notably, informing on the phylogeographic structure of affected individuals, this accurate genetic stratification, could help to optimize the diagnostic algorithm for patients with uncertain or unknown origin.

Using microarray analysis to identify genes and pathways that regulate fetal hemoglobin levels.

Increased levels of fetal hemoglobin (HbF: α2γ2) can ameliorate the clinical severity of the ß-hemoglobinopathies. Microarray analysis represents a powerful approach to identify novel genetic factors regulating the γ-globin gene. Gene expression profiling was previously performed on 14 individuals with high or normal HbF levels to identify the genetic factors that control γ-globin gene expression. To obtain more accurate and reliable results, our results were combined with public microarray dataset GSE22109 deposited in the Gene Expression Omnibus database. Annotation of case versus control samples was taken directly from the microarray documentation. The differentially expressed genes (DEGs) were obtained and were deeply analyzed by bioinformatics methods. Combined with our own chip expression data, potential genes HBE1, TFRC, and CSF2 were selected out for subsequent qRT-PCR validation. A total of 184 DEGs were identified from GSE22109 and the protein-protein interaction network was constructed. Gene set enrichment analysis showed that the hematopoietic cell lineage pathway overlaps in the two datasets. HBE1, CSF2, and TFRC were confirmed by qRT-PCR. Our results suggest novel candidate genes and pathways associated with the γ-globin gene expression.

Serious adverse events in African-American cancer patients with sickle cell trait and inherited haemoglobinopathies in a SEER-Medicare claims cohort.

African-American (AA) cancer patients have long-experienced worse outcomes compared to non-Hispanic whites (NHW). No studies to date have evaluated the prognostic impact of sickle cell trait (SCT) and other inherited haemoglobinopathies, of which several are disproportionately high in the AA population. In a cohort analysis of treated patients diagnosed with breast or prostate cancer in the linked SEER-Medicare database, the relative risk (RR) for ≥1 serious adverse events (AEs), defined as hospitalisations or emergency department visits, was estimated for 371 AA patients with a haemoglobinopathy (AA+) compared to patients without haemoglobinopathies (17,303 AA-; 144,863 NHW-). AA+ patients had significantly increased risk for ≥1 AEs compared to AA- (RR = 1.19; 95% CI 1.11-1.27) and NHW- (RR = 1.23; 95% CI 1.15-1.31) patients. The magnitude of effect was similar by cancer type, and in analyses of AA+ with SCT only. Our findings suggest a novel hypothesis for disparities in cancer outcomes.

Late Effects Screening Guidelines after Hematopoietic Cell Transplantation (HCT) for Hemoglobinopathy: Consensus Statement from the Second Pediatric Blood and Marrow Transplant Consortium International Conference on Late Effects after Pediatric HCT.

Allogeneic hematopoietic cell transplantation (HCT) can halt organ damage and eliminate symptoms in hemoglobin disorders, including sickle cell disease (SCD) and thalassemia major. Managing the residual manifestations of pre-HCT disease complications and the long-term effects of HCT requires systematic monitoring, follow-up and intervention when indicated. Late complications vary with age and disease status at HCT and with transplant variables such as preparative regimen, donor source and compatibility, and immune reconstitution. An international consensus conference sponsored by the Pediatric Blood and Marrow Transplant Consortium in May 2016 entitled "Late Effects Screening and Recommendations Following HCT for Immune Deficiency and Nonmalignant Hematologic Disorders" focused on follow-up after HCT for hemoglobinopathy. An earlier publication from experts who participated in this session described the pathophysiology and spectrum of complications that HCT recipients experience after HCT for SCD and thalassemia major. This companion publication summarizes the consensus reached by this group of experts about long-term follow-up guidelines after HCT for hemoglobinopathy. In addition, these guidelines might also be included in studies of novel curative therapies such as autologous HCT after hematopoietic progenitor stem cell gene modification.

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Genome and Epigenome Editing to Treat Disorders of the Hematopoietic System.

The possibility of editing complex genomes in a targeted fashion has revolutionized basic research as well as biomedical and biotechnological applications in the last 5 years. The targeted introduction of genetic changes has allowed researchers to create smart model systems for basic research, bio-engineers to modify crops and farm animals, and translational scientists to develop novel treatment approaches for inherited and acquired disorders for which curative treatment options are not yet available. With the rapid development of genome editing tools, in particular zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system, a wide range of therapeutic options have been-and will be-developed at an unprecedented speed, which will change the clinical routine of various disciplines in a revolutionary way. This review summarizes the fundamentals of genome editing and the current state of research. It particularly focuses on the advances made in employing engineered nucleases in hematopoietic stem cells for the treatment of primary immunodeficiencies and hemoglobinopathies, provides a perspective of combining gene editing with the chimeric antigen receptor T cell technology, and concludes by presenting targeted epigenome editing as a novel potential treatment option.

Humoral Immune Reconstitution Kinetics after Allogeneic Hematopoietic Stem Cell Transplantation in Children: A Maturation Block of IgM Memory B Cells May Lead to Impaired Antibody Immune Reconstitution.

Although T cell immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been well studied, long-term B cell immune reconstitution remains less characterized. We evaluated humoral immune reconstitution among 71 pediatric allo-HSCT recipients. Although tetanus toxoid antibody levels were normal at 1 year after allo-HSCT, antipolysaccharide carbohydrate antibodies remained persistently low for up to 5 years. While naive B cell counts normalized by 6 months, IgM memory B cell deficiency persisted for up to 2 years (P = .01); switched memory B cell deficiency normalized by 1 year after allo-HSCT. CD4+ T cell immune reconstitution correlated with that of switched memory B cells as early as 6 months after allo-HSCT (r = .55, P = .002) but did not correlate with IgM memory B cells at any time point after allo-HSCT. Taken together, this suggests that allo-HSCT recipients have impaired antibody immune reconstitution, mainly due to IgM memory B cell maturation block, compared with more prompt T cell-dependent switched memory cell immune reconstitution. We further explored other factors that might affect humoral immune reconstitution. The use of total body irradiation was associated with lower naive B cells counts at 6 months after HSCT (P = .04) and lower IgM (P = .008) and switched (P = .003) memory B cells up to 2 years. Allo-HSCT recipients with extensive chronic graft-versus-host disease had lower IgM memory B cell counts (P = .03) up to 2 years after allo-HSCT. The use of cord blood was associated with better naive (P = .01), IgM (P = .0005), and switched memory (P = .006) B cells immune reconstitution. These findings may inform future prophylaxis and treatment strategies regarding risk of overwhelming infection, graft-versus-host disease, and post-allogeneic HSCT revaccination.

Fat Embolism Syndrome Secondary to Bone Marrow Necrosis in Patients with Hemoglobinopathies.

Bone marrow necrosis with subsequent embolization of the fat and necrotic tissues into the systemic circulation causing fat embolism syndrome and multiorgan failure is a rare complication of patients with hemoglobinopathies. The exact etiology of this condition is not known. Because it occurs more often in patients with compound heterozygous conditions than in sickle cell disease, some patients are unaware of their predisposition. The initial symptoms are nonspecific, such as back and/or abdominal pain, fever, and fatigue, which may rapidly progress to respiratory failure and severe neurologic compromise. Common laboratory tests reveal anemia without reticulocytosis, thrombocytopenia, leukoerythroblastic picture with immature white cells and nucleated red blood cells, increased lactate dehydrogenase, high ferritin, and, sometimes increased creatinine. The diagnosis can be delayed because of an apparent lack of awareness about bone marrow necrosis with fat embolism syndrome, its rarity, and its similarities with other conditions such as thrombotic thrombocytopenic purpura. Although a bone marrow biopsy is diagnostic, waiting for it delays definitive treatment, which appears to be essential for the recovery of end-organ damage, such as neurologic and pulmonary damage. In our experience, either multiple units of red blood cell transfusion or, preferably, red cell exchange initiated promptly, is lifesaving.