εγ-Thalassemia, a New Hemoglobinopathy Category.

BACKGROUND: Large ß-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or ß-, δß-, γδß-, and ϵγδß-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for ß-thalassemia. Notably, ϵγδß-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψß loci with intact LCR, δ-, and ß-regions in 2 women and newborn twins. METHODS: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. RESULTS: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψß (HBBP1) loci. CONCLUSIONS: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδß-thalassemia.

Isolated anemia in patients with large granular lymphocytic leukemia (LGLL).

Patients with large granular lymphocytic leukemia (LGLL) frequently present with neutropenia. When present, anemia is usually accompanied by neutropenia and/or thrombocytopenia and isolated anemia is uncommon. We evaluated a cohort of 244 LGLL patients spanning 15 years and herein report the clinicopathologic features of 34 (14%) with isolated anemia. The patients with isolated anemia showed a significantly male predominance (p = 0.001), a lower level of hemoglobulin (p < 0.0001) and higher MCV (p = 0.017) and were less likely to have rheumatoid arthritis (p = 0.023) compared to the remaining 210 patients. Of the 34 LGLL patients with isolated anemia, 13 (38%) presented with pure red cell aplasia (PRCA), markedly decreased reticulocyte count and erythroid precursors, and more transfusion-dependence when compared to non-PRCA patients. There was no other significant clinicopathologic difference between PRCA and non-PRCA patients. 32 patients were followed for a median duration of 51 months (6-199). 24 patients were treated (11/11 PRCA and 13/21 non-PRCA patients, p < 0.02). The overall response rate to first-line therapy was 83% [8/11 (72.7%) for PRCA, 12/13 (92.3%) for non-PRCA], including 14 showing complete response and 6 showing partial response with a median response duration of 48 months (12-129). Half of non-PRCA patients who were observed experienced progressive anemia. During follow-up, no patients developed neutropenia; however, 5/27 (18.5%) patients developed thrombocytopenia. No significant difference in overall survival was noted between PRCA and non-PRCA patients. In summary, this study demonstrates the unique features of LGLL with isolated anemia and underscores the importance of recognizing LGLL as a potential cause of isolated anemia, which may benefit from disease-specific treatment. LGLL patients with PRCA were more likely to require treatment but demonstrated similar clinicopathologic features, therapeutic responses, and overall survival compared to isolated anemia without PRCA, suggesting PRCA and non-PRCA of T-LGLL belong to a common disease spectrum.

Improved Characterization of Complex ß-Globin Gene Cluster Structural Variants Using Long-Read Sequencing.

Complex insertion-deletion (indel) events in the globin genes manifest in widely variable clinical phenotypes. Many are incompletely characterized because of a historic lack of efficient methods. A more complete assessment enables improved prediction of clinical impact, which guides emerging therapeutic choices. Current methods have limited capacity for breakpoint assignment and accurate assessment of mutation extent, especially in cases containing duplications or multiple deletions and insertions. Technology, such as long-read sequencing, holds promise for significant impact in the characterization of indel events because of read lengths that span large regions, resulting in improved resolution. Four known complex ß-globin gene cluster indel types were assessed using single-molecule, real-time sequencing technology and showed high correlation with previous reports, including the Caribbean locus control deletion (g.5,305,478_5,310,336del), a large ß-gene duplication containing the Hb S mutation (g.4,640,335_5,290,171dup with g.5,248,232T>A, c.20A>T; variant allele fraction, 64%), and two nested variants (double deletions with intervening inversion): the Indian Gγ(Aγδß)0-thalassemia (g.5,246,804-5,254,275del, g.5,254,276_5,269,600inv, and g.5,269,601_5,270,442del) and the Turkish/Macedonian (δß)0 thalassemia (g.5,235,064_5,236,652del, g.5,236,653_5,244,280inv, and g.5,244,281_5,255,766del). Our data confirm long-read sequencing as an efficient and accurate method to identify these clinically significant complex events. Limitations include high-complexity sample preparation requirements, which hinder routine use in clinical laboratories. Continued improvements in sample and data workflow processes are needed to accommodate volumes in a tertiary clinical laboratory.

High-oxygen-affinity hemoglobinopathy-associated erythrocytosis: Clinical outcomes and impact of therapy in 41 cases.

We describe presenting features, treatment strategies, and follow-up events involving 41 patients (median age 39 years, range 1-81; 54% males) with high oxygen affinity (HOA) hemoglobinopathy-associated erythrocytosis, seen at our institution (1973-2020). Thirty-four (83%) patients carried ß-chain (13 Malmo, 4 Olympia, 3 San Diego, 2 Wood) and 7 (17%) α-chain (4 Dallas and one each Columbia-Missouri, Jackson, and Wayne) variants. Median (range) hemoglobin (Hgb)/hematocrit (Hct), serum erythropoietin and p50 were 18 g/dL/52.9% (16-21.9/48-66), 10.4 mIU (4-36.3), and 20 mmHg (12-25), respectively. Family history was documented in 24 patients and history of thrombosis in two (5%). Treatment included phlebotomy in 23 and antiplatelet therapy in 21 patients. At a median follow-up of 10 years, 23 (56%) patients reported one or more symptoms that were thought to be related to their increased Hct while thrombosis was documented in 10 (24%) patients. Neither Hgb/Hct level nor active phlebotomy showed a significant correlation with either thrombotic or nonthrombotic symptoms (p > .1 in all instances). Among 23 pregnancies recorded, 78% resulted in live births and no fetal loss was attributed to erythrocytosis. The current study does not implicate Hgb/Hct level as a major contributor of morbidity in HOA hemoglobinopathy-associated erythrocytosis and suggests limited therapeutic value for phlebotomy.

Clinical Heterogeneity of the VEXAS Syndrome: A Case Series.

The objective of this study is to describe the clinical features and outcomes of patients with the newly defined vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome. Nine men with somatic mutations in the UBA1 gene were identified; the most frequent variant was p.Met41Thr (7 of 9, 78%). The median age at VEXAS diagnosis was 74 (67, 76.5) years, and patients had a median duration of symptoms for 4 years before diagnosis. Refractory constitutional symptoms (88%), ear and nose chondritis (55%), and inflammatory arthritis (55%) were common clinical features. Vasculitis was noted in 44%. All patients had significantly elevated inflammatory markers and macrocytic anemia. Thrombocytopenia was present in 66% at diagnosis of VEXAS. Eight patients had bone marrow biopsies performed. All bone marrows were hypercellular, and there was vacuolization of the erythroid (100%) or myeloid precursors (75%). Glucocorticoids attenuated symptoms at prednisone doses ≥20 mg per day, but no other immunosuppressive agent showed consistent long-term control of disease. One patient with coexisting plasma-cell myeloma received plasma-cell-directed therapy with improvement of the inflammatory response, which is a novel finding. In conclusion, VEXAS syndrome is a clinically heterogeneous, treatment-refractory inflammatory condition caused by somatic mutation of the UBA1 gene. Patients often present with overlapping rheumatologic manifestations and persistent hematologic abnormalities. As such, internists and subspecialists, including pathologists, should be aware of this condition to avert diagnostic delay, now that the etiology of this syndrome is known.

Spectrum of hematological malignancies, clonal evolution and outcomes in 144 Mayo Clinic patients with germline predisposition syndromes.

Germline predisposition syndromes (GPS) result from constitutional aberrations in tumor suppressive and homeostatic genes, increasing risk for neoplasia in affected kindred. In this study, we present clinical and genomic data on 144 Mayo Clinic patients with GPS; 59 evaluated prospectively using an algorithm-based diagnostic approach in the setting of a dedicated GPS/ inherited bone marrow failure syndrome (IBMFS) clinic. Seventy-two (50%) patients had IBMFS (telomere biology disorders-32,Fanconi anemia-18, Diamond Blackfan Anemia - 11, congenital neutropenia-5, Schwachman-Diamond Syndrome-5 and Bloom Syndrome-1), 27 (19%) had GPS with antecedent thrombocytopenia (RUNX1-FPD-15, ANKRD26-6, ETV6-2, GATA1-1, MPL-3), 28 (19%) had GPS without antecedent thrombocytopenia (GATA2 haploinsufficiency-16, DDX41-10, CBL-1 and CEBPA-1) and 17 (12%) had general cancer predisposition syndromes (ataxia telangiectasia-7, heterozygous ATM variants-3, CHEK2-2, TP53-2, CDK2NA-1, NF1-1 and Nijmegen Breakage Syndrome-1). Homozygous and heterozygous ATM pathogenic variants were exclusively associated with lymphoproliferative disorders (LPD), while DDX41 GPS was associated with LPD and myeloid neoplasms. The use of somatic NGS-testing identified clonal evolution in GPS patients, with ASXL1, RAS pathway genes, SRSF2 and TET2 being most frequently mutated. Fifty-two (91%) of 59 prospectively identified GPS patients had a change in their management approach, including additional GPS-related screening in 42 (71%), referral for allogenic HSCT workup and screening of related donors in 16 (27%), medication initiation and selection of specific conditioning regimens in 14 (24%), and genetic counseling with specific intent of fertility preservation and preconceptual counseling in 10 (17%) patients; highlighting the importance of dedicated GPS screening, detection and management programs for patients with hematological neoplasms.

Interpreting sulfhemoglobin and methemoglobin in patients with cyanosis: An overview of patients with M-hemoglobin variants.

INTRODUCTION: Methemoglobin (MetHb) and sulfhemoglobin (SHb) measurements are useful in the evaluation of cyanosis. When one or both values are elevated, additional analysis is important to establish the etiology of the disorder. Methemoglobinemia occurs from acquired or hereditary causes with diverse treatment considerations, while true sulfhemoglobinemia is only acquired and treatment is restricted to toxin removal. Some toxic exposures can result in a dual increase in MetHb and SHb. Hereditary conditions, such as M-Hemoglobin variants (M-Hbs), can result in increased MetHb and/or SHb values but are clinically compensated and do not require treatment if they are cyanotic but otherwise clinically well. METHODS: Herein, we report 53 hemoglobin variant cases that have associated MetHb and SHb levels measured by an adapted Evelyn-Malloy laboratory assay method. RESULTS: Our data indicate M-Hbs cause variable patterns of MetHb and SHb elevation in a fairly reproducible pattern for the particular variant. In particular, α globin chain M-Hbs can mimic acquired sulfhemoglobinemia due to an isolated increased SHb value. CONCLUSION: If the patient appears clinically well other than cyanosis, M-Hbs should be considered early in the evaluation process to differentiate from acquired conditions to avoid unnecessary testing and treatment regimens and prompt genetic counseling.

Genetic and Clinical Studies of Patients With Increased Multinucleated Megakaryocytes in Bone Marrow as an Isolated Finding: A Diagnostic Pitfall for Myelodysplastic Syndrome.

The presence of increased multinucleated megakaryocytes (aka osteoclast-like) is considered a dysplastic feature in myelodysplastic syndrome; however, its clinical significance in isolation is uncertain. Herein, we report the clinicopathologic and genetic features of 18 such cases of 40,539 bone marrow biopsies spanning 10 years. All 18 patients had ≥25% multinucleated megakaryocytes in otherwise normal bone marrow biopsies, which were evaluated for plasma cell neoplasms (n=9), lymphoma (n=4), or anemia/neutropenia (n=5). None of the 17 patients tested showed acquired cytogenetic abnormalities. Sixteen patients underwent targeted gene panel next-generation sequencing: 9 patients had no pathogenic mutations; 3 harbored a single pathogenic mutation with variant allele frequencies of 7.5%, 7.6%, and 10.7%, likely representing clonal hematopoiesis of indeterminate potential; 1 had 2 pathogenic mutations, 1 of which had a variant allele frequency >20%. Fourteen of 18 patients had a follow-up period >6 months (median: 36.5 mo, range: 7 to 110 mo) and no patients developed a new-onset cytopenia, a progressive cytopenia, or a myeloid neoplasm. The patient with 2 mutations had persistent anemia, worrisome for an emerging MDS. However, given the absence of thrombocytopenia, increased multinucleated megakaryocytes in this patient could be an unrelated incidental finding. Our study indicates that increased multinucleated megakaryocytes as an isolated finding is a rare phenomenon, and this sole morphologic finding is not diagnostic of myelodysplastic syndrome. Diagnostic approaches in the presence of increased multinucleated megakaryocytes are proposed based on different clinical and pathologic scenarios.

Pathologic Spectrum and Molecular Landscape of Myeloid Disorders Harboring SF3B1 Mutations.

OBJECTIVES: SF3B1 mutations are the most common mutations in myelodysplastic syndromes (MDS). The International Working Group for the Prognosis of MDS (IWG-PM) recently proposed SF3B1-mutant MDS (SF3B1-mut-MDS) as a distinct disease subtype. We evaluated the spectrum and molecular landscape of SF3B1-mutated myeloid disorders and assessed the prognostication in MDS harboring SF3B1 mutations (MDS-SF3B1). METHODS: Cases were selected by retrospective review. Clinical course and laboratory and clinical findings were collected by chart review. SF3B1-mut-MDS was classified following IWG-PM criteria. RESULTS: SF3B1 mutations were identified in 75 of 955 patients, encompassing a full spectrum of myeloid disorders. In MDS-SF3B1, Revised International Prognostic Scoring System (IPSS-R) score greater than 3 and transcription factor (TF) comutations were adverse prognostic markers by both univariate and multivariate analyses. We confirmed the favorable outcome of IWG-PM-defined SF3B1-mut-MDS. Interestingly, it did not show sharp prognostic differentiation within MDS-SF3B1. CONCLUSIONS: SF3B1 mutations occur in the full spectrum of myeloid disorders. We independently validated the favorable prognostication of IWG-PM-defined SF3B1-mut-MDS. However it may not provide sharp prognostication within MDS-SF3B1 where IPSS-R and TF comutations were prognostic-informative. Larger cohort studies are warranted to verify these findings and refine MDS-SF3B1 prognostication.

Hemolytic disease and reticulocytopenia of the newborn attributable to maternal immunoglobulin G anti-M reacting optimally at cold temperatures.

BACKGROUND: Hemolytic disease of the fetus and newborn (HDFN) attributable to anti-M is rare, although case reports implicate anti-M in varying severities of HDFN, including fetal hydrops and intrauterine death. CASE DESCRIPTION: We describe the case of a newborn with HDFN associated with an atypical immunoglobulin (Ig) G anti-M that reacted best at cold temperatures. The maternal antibody detected in pregnancy was not reactive at 37°C, and a direct antiglobulin test (DAT) on red blood cells (RBCs) from the newborn was negative, suggesting an anti-M that should not have been clinically relevant. However, the infant developed hyperbilirubinemia (bilirubin level, 17.6 mg/dL), hemolytic anemia (hemoglobin nadir, 5.5 g/dL), and reticulocytopenia. Laboratory testing demonstrated the presence of an IgG anti-M in maternal and neonatal samples reacting best at 4°C. This passively acquired IgG anti-M provoked hemolytic anemia in the infant and likely suppressed erythropoiesis, resulting in reticulocytopenia with prolonged anemia. He was treated for IgG anti-M HDFN with 10 intravenous Ig infusions and 10 days of oral prednisone followed by a taper. He required seven transfusions with M- RBCs. His hemoglobin level normalized at 3 months of age. Follow-up at 2 years revealed no hematologic or neuro-developmental concerns. CONCLUSION: To our knowledge, this is the second report of HDFN attributable to an IgG anti-M reacting preferentially at cold temperature with no 37°C reactivity. Clinically relevant IgG anti-M may elude standard testing. Early recognition and testing for cold-reacting IgG anti-M should be considered for newborns with hemolysis, a negative DAT, and prolonged anemia.

Does Transfusion of Red Blood Cells Impact Germline Genetic Test Results?

PURPOSE: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. METHODS: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. RESULTS: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3-27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13-95%). CONCLUSION: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.

Hemoglobin S/Hemoglobin Quebec-Chori Presenting as Sickle Cell Disease: A Case Report.

BACKGROUND: Sickle cell disease is a homozygous hemoglobinopathy with vaso-occlusive complications secondary to abnormal sickling of red blood cells under stressful conditions such as hypoxia. Children with sickle cell trait have a heterozygous genetic state, typically without symptoms. OBSERVATION: An 8-year-old boy diagnosed with sickle cell trait was found to have multiple complications consistent with sickle cell disease, including pain crises, osteomyelitis, and priapism. Over a 6-year period, he underwent routine laboratory evaluations without a definitive diagnosis. The diagnosis of a compound heterozygous state of hemoglobin S/hemoglobin Quebec-Chori was eventually made on the basis of mass spectrometry and confirmed with hemoglobin subunit beta gene sequencing. CONCLUSION: Expanding diagnostic evaluation in patients with abnormal clinical presentations is vital to making the correct diagnosis and hence earlier institution of appropriate management of rare hemoglobinopathies.

Further Characterization of Hb Bronovo [α103(G10)His→Leu; HBA2: c.311A>T] and First Report of the Homozygous State.

Hb Bronovo [α103(G10)His→Leu, HBA2: c.311A>T] is an α-globin variant that interferes with and decreases binding efficiency to α hemoglobin (Hb) stabilizing protein (AHSP), a chaperone molecule. The histidine residue at position 103 is integral to the AHSP hydrogen bond formation where disruption results in an increased quantity of cytotoxic free α-globin chains, thereby creating a similar pathophysiology as ß-thalassemia (ß-thal). We report a family with Hb Bronovo, including a homozygous proband, which resulted from maternal uniparental disomy (UPD). Although not detected by routine studies in previous reports, the variant protein is visible by intact mass spectrometry (MS).

Hemoglobin Sunshine Seth: A Case Report of Low-Oxygen-Affinity Hemoglobinopathy.

Pulse oximetry is routinely used in the newborn nursery for clinical monitoring and to detect critical congenital heart disease. The differential diagnoses for reduced peripheral oxygen saturation in an infant include congenital heart disease, respiratory distress syndrome, transient tachypnea of the newborn, persistent pulmonary hypertension of the newborn, meconium aspiration syndrome, pneumonia, pneumothorax, and sepsis. The diagnostic evaluation for neonatal hypoxemia can be invasive and expensive. When this evaluation is unrevealing, other interventions may be tried without clear benefit to the patient, including, but not limited to, supplemental oxygen. Therefore, it is important to consider alternative, albeit rare, diagnoses, including hemoglobinopathies with abnormal oxygen binding properties. Mutations in the structure of alpha- and beta-globin chains can alter the affinity of hemoglobin for oxygen, and changes in oxygen affinity may result in changes in the oxygen saturation detected by pulse oximetry. These changes may or may not be of clinical significance. This case report describes Hemoglobin Sunshine Seth, a rare low-oxygen-affinity hemoglobin variant presenting as reduced peripheral oxygen saturation in an otherwise well-appearing infant male.

Hb F-Wentzville [Gγ24(B6)Gly→Glu; HBG2: c.74G>A, p.Gly25Glu]: An Unstable Gγ-Globin Variant Associated with Neonatal Hemolytic Anemia.

A novel unstable Gγ-globin variant, Hb F-Wentzville [Gγ24(B6)Gly→Glu; HBG2: c.74G>A, (p.Gly25Glu)], was identified in a young infant who required a single transfusion of erythrocytes for hemolytic anemia. This is the first reported γ-globin variant affecting the highly conserved glycine residue at helical position B6. In the tertiary structure of hemoglobin (Hb), glycine at B6 is in close proximity to another invariant glycine residue at E8. Prior studies have shown that replacement of the B6 or E8 glycine residues with bulkier amino acids disrupts packing between the B and E helices, resulting in Hb instability. Thus, Hb F-Wentzville is analogous to the following unstable ß-globin B6 variants: Hb Savannah (HBB: c.74G>T, p.Gly24Val), Hb Riverdale-Bronx (HBB: c.73G>C, p.Gly24Arg), and Hb Moscva (HBB: c.74G>A, p.Gly24Asp).

The significance of genetic mutations and their prognostic impact on patients with incidental finding of isolated del(20q) in bone marrow without morphologic evidence of a myeloid neoplasm.

Patients with a sole del(20q) chromosomal abnormality and without morphologic features of a myeloid neoplasm (MN) have shown variable clinical outcomes. To explore the potential risk stratification markers in this group of patients, we evaluated their genetic mutational landscape by a 35-gene MN-focused next-generation sequencing (NGS) panel and examined the association of mutations to progression of MNs. Our study included 56 patients over a 10-year period with isolated del(20q), of whom 23 (41.1%) harbored at least one mutation. With a median follow-up of 32.6 months (range: 0.1-159.1), 9 of 23 patients with mutation(s) progressed to MNs, while all 33 patients without mutations did not progress to MN. Kaplan-Meier survival analysis demonstrated the presence of mutation(s) as a significant risk factor for progression to MN (P < 0.0001). MN progression was strongly associated with the presence of non-DNMT3A/TET2/ASXL1 epigenetic modifiers and nonspliceosome mutations (P = 0.003). There was no significant difference among patients with and without MN progression with respect to the number of mutations, variant allele frequency, percentage of del(20q), and other clinical/laboratory variables. This study illustrates the underlying genetic heterogeneity and complexity of isolated del(20q), and underscores the prognostic value of NGS mutational analysis in these cases.