Hoyer, James D.

Lepore hemoglobins (Hbs) such as Hb Lepore-Boston-Washington [δ(1-87) β(116-146)], Hb Lepore-Baltimore [δ(1-50) β(86-146)], Hb Lepore-Hollandia [δ(1-22) β(50-146)] are δβ gene fusion/deletion variants (ε- Gγ- Aγ-[δβ] v )(Figure 1). They are controlled by the weak δ-globin gene promoter and synthesized at approximately 10-15% of hemoglobin fractions. This results in a similar phenotype as β +-thalassemia (microcytosis and hypochromia ± mild anemia). Anti-Lepore hemoglobins such as Hb P-Nilotic (β(1-22) δ(50-146) are βδ gene fusion/duplication variants (ε- Gγ- Aγ-δ-[βδ] v-β) associated with normal CBC values. Herein we describe a novel βδβ double fusion variant (ε- Gγ- Aγ-δ-[βδβ] v). Due to high homology between the δ and β genes, this double fusion event encodes an identical protein to Hb Lepore-Hollandia, albeit linked to the β-promoter and therefore expressed at ~40% of hemoglobin fractions, which would be expected to enhance any intrinsic properties of the variant. This novel hemoglobin variant is named Hb Lepore Rochester-MN. Two cases have been identified (Table 1). Case 1 was a 10-month-old Caucasian/Native American infant referred for an abnormal newborn screen and a reported family history of Hb G-Coushatta (β22, GAA>GCA, Glu>Ala; HGVS: c.68A>C, p.E23A). Case 2 was a 3-month-old infant referred for an abnormal newborn screen. CBCs were normal for age including MCV. Cation-exchange high performance liquid chromatography (HPLC) showed a shouldered peak in the Hb A 2 window typical for Hb Lepore (elution time: 3.41-3.46 mins), although the variant percentage was higher than expected, mimicking Hb G-Coushatta (expected elution time: 3.33 mins). Capillary electrophoresis identified a peak in the D zone (Z(D) zone), which is expected for Hb Lepore or Hb G-Coushatta. Intact mass spectrometry was consistent with Hb Lepore-Hollandia at 15836 amu, differing from Hb G-Coushatta (Glu>Ala, 15809 amu). Multiplex ligand probe amplification (MLPA) was normal because the highly variable fusion regions are unsuitable for probe placement. Sanger sequencing of the β-globin gene identified multiple single base substitutions which were challenging to interpret but matched the sequence expected for a complex double crossover event (NM_000518.4:c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). The first crossover is located between c.-29 and c.9, the second crossover is located between c.69 and c.92+17. Long-read sequencing was performed (Sequel, Pacific Biosciences) which detected the multiple missense mutations in cis conformation, as was corroborated by the mass spectrometry data. In summary, Hb Lepore Rochester-MN represents a novel double fusion [βδβ] variant class that will pose a diagnostic challenge for laboratories. Although it is likely a result of sequential crossover events (deletion and duplication) between Lepore and anti-Lepore variants, MLPA analysis is normal due to technical constraints and high homology. DNA sequencing (both Sanger and long read methods) yielded complex results that require sophisticated analysis and correlation with protein data including intact mass spectrometry. HPLC and CE data including variant percentage are very similar to Hb G-Coushatta. In addition, although resulting in an identical protein, the variant should not be mistaken for Hb Lepore-Hollandia, which is produced at lower levels and causes a thalassemic phenotype. Hb Lepore Rochester-MN is controlled by the β-promoter and therefore is associated with approximately 40% variant and apparently normal CBC values in the two infants.

Abstract Background Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-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 β-thalassemia. Notably, ϵγδβ-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 ψβ loci with intact LCR, δ-, and β-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 ψβ (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 ϵγδβ-thalassemia.

Abstract The εγδβ thalassemias (EGDBT) are infrequently occurring deletions of the β globin gene cluster on chromosome 11. EGDBTs can be an unsuspected cause of severe neonatal anemia that resolves during the first year of life into a normal Hb A2 thalassemic phenotype, presumed to be due to an imbalance of β-like chains during development. First described in 1972, they are categorized into two groups, both with loss of the five DNase I hypersensitivity sites upstream of the β gene (HBB) called the locus control region (LCR). In group 1 deletions, all or most of the β globin gene cluster including HBB is deleted; in group 2 deletions, removal of the LCR silences an intact HBB. Large deletions that do not involve LCR but remove the embryonic (HBE) and fetal genes (HBG2/HBG1) [LCR(ε Gγ Aγ) 0δβ] have not been reported. It is not known whether a deletion with intact LCR as well as δ and β genes would similarly manifest as neonatal anemia that resolves into a normocytic phenotype with normal hemoglobin fraction percentages after infancy. Four individuals with an LCR(ε Gγ Aγ) 0δβ deletion have been evaluated. These include monochorionic, diamniotic twin girls whose newborn screen (NBS) returned significant for Hb A percentage greater than Hb F for both twins. NBS was repeated two weeks later with the same result. The newborns were induced late pre-term (36 weeks and 6 days) after an uncomplicated pregnancy, including no features of twin to twin transfusion. Both infants had no anemia and bilirubin levels remained in the low to low-intermediate risk zones. Neither twin received transfusions or phototherapy during admission. At a follow up visit at 20 months of age a hemoglobin electrophoresis was performed which showed borderline Hb A2 and no Hb F in both twins (Table 1). Ferritin levels were low-normal in Twin A (14 µg/L) and low in Twin B (9 µg/L). Both infants were found to have a heterozygous large deletion affecting HBE, HBG2, HBG1, and HBBP1 loci by Multiplex Ligation-dependent Probe Amplification (MLPA), a method that uses multiple DNA probe pairs to determine the size of the DNA segment deleted. MLPA deletion/duplication testing on the α genes was negative. No β globin variants were identified by DNA sequencing. An evaluation was performed on the 25 year-old healthy mother and showed similar findings including the heterozygous large deletion. Mother and infants continue to be asymptomatic with normal red cell indices for age (Table 1). The fourth was an unrelated 40-year-old female with mild anemia screened by hemoglobin electrophoresis for obstetric evaluation who was found to have borderline Hb A2 (see table 1). MLPA showed a heterozygous deletion involving the same loci. Again, no β globin variants were identified by DNA sequencing. All four cases showed the same MLPA pattern except one non-specific probe commonly altered in common gamma gene conversion events. Long range sequencing performed on one case confirmed a single contiguous 32,599 bp deletion that matched the MLPA data (g.5262276-5294875). The detected deletion has not been reported in literature and differs from EGDBT mutations in that the LCR is intact. As the LCR controls expression of the entire gene cluster, this mutation is expected to display different phenotypic features than classic EGDBT. HBD and HBB are not deleted and therefore adequate transcription of Hb A2 and Hb A are expected with no imbalance of α/β chains after the neonatal period. It is postulated this finding explains the decreased Hb F levels in the NBS test result. The Hb A2 increase is likely a product of this deletion and does not indicate β-thalassemia. The heterozygous absence of HBE and HBG2/HBG1 may result in upregulation of HBD and HBB expression through loss of FKLF and FKLF-2 binding. In addition, HBB-EKLF binding would have less competition as the gamma promoter elements have been heterozygously deleted. In summary, in contrast to classic EGDBTs which cause transient severe neonatal anemia and resolve to normal Hb A2 thalassemic indices, this novel LCR(ε Gγ Aγ) 0δβ deletion was associated with normal CBC values with an absence of severe neonatal anemia, inverted Hb F/Hb A percentages at birth and borderline Hb A2 levels. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background Approximately 100 high oxygen affinity (HOA) hemoglobin (Hgb) variants have been reported to date; review of the Mayo Clinic laboratory database (1974-2018) identified 80 distinct variants including 12 novel variants (60 β, 20 α). One-third of HOA Hgb variants result in secondary erythrocytosis, provoking concern regarding increased risk for thrombosis. Current management guidelines lack supporting evidence regarding the utilization of phlebotomy in such cases. We describe presenting features, treatment strategies and follow-up events involving 41 consecutive cases seen at our institution. Methods Study patients were recruited from our institutional laboratory and clinical Hgb variant database. Initial evaluation for Hgb variants was conducted by capillary electrophoresis and high-performance liquid chromatography. Additional testing which included mass spectrometry and isoelectric focusing was pursued as necessary. Since the majority of variants were difficult to identify by protein studies, DNA sequencing of HBB, or HBA1, HBA2 genes was performed for confirmation. Symptoms and thrombosis, both at presentation and during follow up, were recorded. Therapeutic interventions were based on physician discretion and mostly included phlebotomy and/or aspirin therapy; a careful response assessment was performed to determine the impact of each therapy on symptoms and/or thrombosis. Results A total of 41 patients with HOA Hgb variant-associated erythrocytosis (median age 39 years, range 1-81; 54% males) were seen at our institution between January 1973 and February 2020. The majority of the patients carried β-chain variants (n=34; 83%), common variants being Hgb Malmo (n=13), Olympia (n=4), San Diego (n=3), and Wood (n=2). Among the 7 patients with α-chain variants, Hgb Dallas was the most frequent (n=4). Presenting median values (range) for Hgb/Hct, serum erythropoietin and p50 were 18 g/dl/52.9%(16-21.9g/dl/48-66%) 10.4 mIU (4-36.3 mIU), and 20 mmHg (12-25 mmHg), respectively. Family history was documented in 34 patients, of which 24 (71%) reported one or more affected family members. Family history of thrombosis was documented in 7 patients (21%). CV risk factors were present in half of the patients; by contrast, history of thrombosis prior to or at diagnosis was documented in only two patients (5%). Of 23 pregnancies reported in 12 women, live birth rate was 78% (n=18); none of the fetal losses were attributed to erythrocytosis. Active therapies at the time of initial referral consisted of phlebotomy (n=12), aspirin (n=11) and systemic anticoagulation (n=1). At a median follow-up of 10 years (range; 0.04-44), 23 patients had reported one or more symptoms, attributed to hyper-viscosity, such as headaches, fatigue, and lightheadedness. Neither Hct level at diagnosis (p=0.32) nor phlebotomy (p=0.16; 75% patients on vs 52% not on phlebotomy) or aspirin therapy (p=0.75; 55% patients on vs 60% not on aspirin) appeared to influence the occurrence of symptoms. Phlebotomy relieved symptoms in 7 (42%) symptomatic patients; however, 7 (30%) of 23 patients on phlebotomy reported one or more adverse symptoms that were attributed to phlebotomy-induced iron deficiency. Ten patients (24%) experienced thrombosis prior to or following diagnosis: 6 arterial and 4 venous. Median age at thrombotic event was 51 years and median hematocrit 52%; active therapies at the time of event included phlebotomy in 5 patients, aspirin in 4, and systemic anticoagulation in 2. Hct level at diagnosis (p=0.10) or the time of event (p=0.67) did not correlate with occurrence of thrombosis. Additionally, the incidence of thrombosis was no different among patients receiving or not receiving phlebotomy (5/23(22%) vs 5/18(28%), respectively; p=0.66). The presence of CV risk factors was predictive of arterial events (p=0.002). Two of the 4 venous events developed in the context of concomitant thrombophilia. Eight patients (20%), median age 28 years, without CV risk factors, were observed without therapy for a median of 9.5 years (range; 0.4-21) and have not experienced any thrombosis to date. Conclusions We found no association between Hct level and either thrombotic or non-thrombotic symptoms in HOA hemoglobinopathy-associated erythrocytosis; furthermore, implementation of aggressive phlebotomy did not provide a clear benefit with respect to thrombosis risk reduction. Figure 1 Figure 1. Disclosures Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

AbstractWe 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 β‐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.

AbstractIntroductionMethemoglobin (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.MethodsHerein, we report 53 hemoglobin variant cases that have associated MetHb and SHb levels measured by an adapted Evelyn‐Malloy laboratory assay method.ResultsOur 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.ConclusionIf 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.

Abstract 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.