Hoyer, James D.

Background In humans, maximal exercise capacity is reduced during hypoxic environmental conditions. In response to hypoxia, the hemoglobin binding affinity is decreased, promoting oxygen off‐loading at the tissue level and causing a right‐shift of the oxyhemoglobin dissociation curve (ODC). However, comparative biology demonstrates many species (e.g. Bar‐headed geese), who are adapted to hypoxia instead have high‐affinity hemoglobin (left‐shifted ODC). Therefore we studied unique human participants with hemoglobinopathies that result in high affinity hemoglobin (HAH) to investigate the role of oxygen affinity in exercise performance and tissue oxygenation during hypoxic conditions. Methods HAH participants (n=5, 1M/4F, Age= 34±11 yr, P 50 =16±1 mmHg) and matched control participants (n=12, 5M/7F, age=39±12 yr, and P 50 =26±1 mmHg) completed two incremental exercise tests to exhaustion on a cycle ergometer under two conditions: 1) room air (F i O 2 = 0.21, NORM), and 2) normobaric hypoxia gas mixture (F i O 2 = 0.15, HYP), separated by a 45 minute break. The vastus lateralis (VL) muscle was studied using frequency‐domain near infrared spectroscopy (NIRS) to measure concentrations of oxygenated ([OxyHb+Mb]), deoxygenated ([DeoxyHb+Mb]) hemoglobin and myoglobin, and tissue saturation (S t O 2 ). Data were analyzed using a three‐way (Inspirate [HYP, NORM], Hb type [Normal, HAH], exercise intensity [Rest, 40, 60, 80, 100% normoxic PPO]) univariate analysis of variance. Results HAH had an attenuated decline in exercise tolerance (peak power output at maximal exertion) in HYP compared to controls (HAH: 2.9±6.4% decline vs CTL: 11.7±5.4% decline, P<0.05). There was a significant effect of Hb type on [DeoxyHb+Mb] (P=0.048) suggesting less O 2 offloading in HAH compared to controls in the VL at rest and throughout graded exercise in both NORM and HYP. There were no significant effects of inspirate or exercise intensity on [DeoxyHb+Mb], and no significant differences in [OxyHb+Mb] between HAH and controls. There was a significant effect of Hb type on S t O 2 in NORM controls (P<0.01) However, during hypoxic exercise S t O 2 was the same in both HAH and controls except at end exercise (HAH 64.1±6.4% vs CTL: 62.1±4.2%, P<0.05). Conclusion HAH individuals have less oxygen off‐loading at rest and during exercise but higher tissue saturation during exercise in NORM compared to controls. Our results suggest that the similar levels of exercise capacity in hypoxia for HAH are due, at least in part, to maintenance of microvascular extraction similar to that during normoxic exercise. Future studies utilizing direct measurements of skeletal muscle blood flow during exercise are needed to understand integrative exercise in HAH patients. Support or Funding Information This work was funded by NIH R‐35‐HL139854 and the Mayo Foundation. Muscle oxygenation during graded exercise tests in normoxia and hypoxia. Data are presented as means ± standard error. Abbreviations: [OxyHb+Mb], concentration of oxygenated hemoglobin+myoglobin; [DeoxyHb+MB], concentration of deoxygenated hemoglobin+ myoglobin; PPO, peak power output. † denotes significant main effect of Hb types across all workloads. * denotes significant difference from CTL at same %PPO. Figure 1

Abstract BACKGROUND: Acquired hemoglobin disorders including α thalassemia (hemoglobin H (HbH) disease) can complicate neoplastic myeloid disorders, especially myelodysplastic syndrome (MDS). Acquired α thalassemia in MDS has recently been linked to mutations in the trans-acting ATRX at Xq13, a gene previously associated with inherited ATR-X (α thalassemia-mental retardation-X linked) syndrome. In the 3 instances where acquired ATRX point mutations predicted to affect splicing have been encountered, mRNA has been unavailable for assessment or unexpressed; in the 3 cases where ATRX splicing changes were described in MDS, the responsible genomic DNA mutation could not be located. Here we report a novel genomic point mutation in an ATRX splice donor motif and define the mutation’s splicing and expression consequences. METHODS AND RESULTS: A 59 year old anemic Swiss man with chronic myelomonocytic leukemia (CMML) and multiple autoimmune complications including granulomatous uveitis, diabetes insipidus, and pleuropericarditis, developed anemia (Hb 10 g/dL) with MCH of 16.7 pg and MCV of 54 fL. We detected 50% HbH-containing cells by supravital staining and 7.5% HbH by weak cation chromatography, confirmed as HbH by isoelectric focusing. Peripheral blood granulocytes, mononuclear cells and bone marrow mononuclear cells were isolated using dual density Ficoll-Hypaque, and DNA and RNA extracted for mutation screening. ATRX coding region (35 exons) and splice site DNA from mononuclear cells was amplified and subjected to denaturing high performance liquid chromatography (DHPLC). A heteroduplex peak was encountered in the amplicon centered on exon 4. Sequencing demonstrated thymidine to cytidine point mutation with high proportional clonality in the canonical GT splice donor motif 3′ to ATRX exon 4 (IVS 4+2 T&gt;C), also detectable in patient granulocytes. Electrophoresis of raw PCR product from the 5′ end of ATRX cDNA demonstrated multiple abnormal bands. Subcloning and sequencing of cDNA demonstrated 2 splicing consequences of this mutation: exon 4 skipping (i.e., exon 3 was spliced directly to exon 5), and a second isoform with intact exons 3, 4, and 5 but retaining 43 base pairs of intron 4 (including the T&gt;C point mutation in the splice donor site), activating a previously unrecognized cryptic splice site. Since exon 4 is 53 base pairs in length, both aberrant splicing consequences result in a frameshift and generation of a premature stop codon in the 5′ end of exon 5. Exon 6 is normally alternatively spliced, and this variation was retained, so there were two subforms of each aberrant spliceoform. Expression of ATRX mRNA in the patient’s monocytes, measured by RT-PCR probing the 3′ helicase domain, was 11.1% of healthy controls; in the granulocytes, expression was 15.3% of normal. CONCLUSION: Pre-mRNA splicing abnormalities such as that described here are increasingly recognized in association with human disease and particularly neoplasia, and may represent a potential therapeutic target. Point mutations in general are sought in myeloid disorders as they can provide proof of clonality in ambiguous cases and allow tracking of clonality with therapy and disease evolution. Germline 5′ ATRX splicing abnormalities are only rarely associated with inherited ATR-X syndrome, presumably because of the devastating consequences on gene expression and fetal development, but may be more common in acquired thalassemia. In future analyses of acquired HbH disease, when seeking ATRX mutations, splicing abnormalities should be routinely considered.

Abstract Hemoglobin Lufkin is a rare and mildly unstable hemoglobin variant with increased oxygen affinity. Since 1977, two cases of hemoglobin Lufkin trait and one hemoglobin Lufkin/hemoglobin S have been described. This report is the first case of hemoglobin Lufkin/beta-zero thalassemia in a 10 year-old Caucasian male of Irish/Italian/German background. The patient presented with jaundice, splenomegaly and thalassemia major facies. On blood smear examination, RBC morphology showed hypochromia, microcytosis, many target cells, some spherocytes and basophilic stippling of RBC’s. On both alkaline hemoglobin (Hb) electrophoresis and isoelectric focusing (IEF) there was an absence of Hb A with a predominant band slightly anodal to the Hb A position. Hb A2 (4.8%)and Hb F (5.3%) were elevated as measured by high performance liquid chromatography. The heat unstable Hb test was abnormal. DNA sequencing of the beta globin gene confirmed a GCC to GAC mutation at codon 29 (gly to asp) consistent with Hb Lufkin. DNA sequence analysis also revealed a beta-zero thalassemia mutation, IVS-1-1, (G to A). The mother’s sample also showed the same beta-thalassemia mutation. Neither hemoglobin Lufkin nor beta-thalassemia were identified in the father; further studies are being done.

Abstract Background: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal stem cell disorder characterized by intravascular hemolysis, thrombosis, bone marrow failure and reduced survival. In this current single institution study of patients with PNH, we investigate the risk factors predictive of survival and thrombosis. Methods: Data were abstracted from medical records of a consecutive cohort of patients with PNH seen at the Mayo Clinic. Diagnosis was confirmed by the presence of intravascular Hemolysis and either a positive Ham’s test, and/or by the presence of a GPI-deficient hematopoietic clone. Follow-up data were obtained by contacting the patients and by reviewing the social security death index. Results: i. Patients and treatment: The study cohort included 68 patients with PNH (median age 32 years, range 10–77; 33 females) with a median follow up of 84.5 months (range, 1–538 months). Presenting features included infection (n=9), thrombosis (n=9), neutropenia (absolute neutrophil count (ANC) &lt; 1000) (n=32) severe intravascular hemolysis (n=16) and splenomegaly (n=11). Prior to the diagnosis of PNH, 6 patients (8.8%) carried a diagnosis of aplastic anemia (AA) and 2 (2.9%) myelodysplastic syndrome (MDS). Complications documented after diagnosis included major thrombosis (n=11), major hemorrhage (n=4), and development of AA (n=7), MDS (n=4), and non-hematologic cancer (n=4) but none experienced leukemic transformation. Treatment included prednisone (n=30), other immunosuppressive agents (n=6), androgens (n=10), and bone marrow transplantation (n=4). Review of treatment with systemic anticoagulation disclosed only 2 patients that were receiving prophylactic warfarin therapy and both patients remain free of thrombosis after 1 and 2 years of therapy. ii. Thrombotic events and risk factors: Overall, 20 patients (29.4 %) experienced one or more episodes of major thrombosis. Among several clinical and laboratory parameters evaluated at presentation, only the detection of urine hemoglobin and infection were found to be significantly associated with the occurrence of thrombosis (p=0.05) iii. Risk factors for survival To date, 18 patients (27%) have died and median survival for the study cohort is projected at 21.1 years. Multivariate analysis identified thrombosis (p=0.003) and an ANC of &gt; 1000 (p= 0.05) at presentation as independent risk factors for inferior survival. In addition, the occurrence of major hemorrhage was significantly associated with poor survival (p= 0.04). Conclusion The current study provides natural history information in a relatively large cohort of patients with PNH and identifies certain disease features that could be further explored for their potential as risk factors for both thrombosis and survival.

Abstract Deletion 5q in Acute Myeloid Leukemia (AML): a Clinicopathological Description of 79 Cases From a Single Institution Rafael Santana-Davila, Shernan G. Holtan, Rhett P. Ketterling, Ryan A. Knudson, David P. Steensma, Dong Chen, James D. Hoyer, Curtis A. Hanson, Ayalew Tefferi. Background: Deletion 5q (del(5q)) is a lenalidomide-sensitive cytogenetic abnormality commonly seen in patients with myeloid disorders. In this study we analyze the clinicopathological characteristics of patients with AML and del(5q). Methods: Patients with del(5q) and AML were identified through a database query of our institutional cytogenetic database from May, 1992 through December, 2005. Both peripheral blood and bone marrow specimens from each patient were morphologically examined by a hematopathologist (DC, JDH and/or CAH). A morphologic diagnosis was given based on current WHO criteria. A critical review of the patient history was performed for each patient. Results: A total 79 patients were identified. Median age was 71 (range 39–87) years, 56% (n=43)were female. The most common subtype according to the FAB classification was M2 (n=23, 29%). In 35 (44%) patients AML developed a median of 9.4 (range 1–294) months since the diagnosis of a pre-existing myeloid malignancy, of which myelodysplastic syndrome accounted for 24 (9 cases of MDS NOS, 6 of RA, 6 of RAEB1, 2 of RAEB-2, and a single RARS), a myeloproliferative neoplasm in 10 (4 cases of a CMPD NOS, 4 cases of PV and a single case of ET and PMF each) and 1 case of a prior history of AML with a t(8;21). The del(5q) was documented in 7 of this 24 patients in the antecedent hematologic disorder. Neither a history of prior exposure to cytotoxic agents nor a prior myeloid disorder was found in 44% (n=35) of the patients. Del(5q) was commonly associated with a complex karyotype (n=67), however in 9 patients it was found to be the sole abnormality. The most frequent breakpoint was q13q33 (n=47) followed by q15q33 (n=12). Initial pathological description did not identify any morphological abnormalities in 6 cases, in contrast myelodysplasia involving at least two cell lines was appreciated in 58% (n=46) of the patients. The median survival was 2.8 months. The majority of the subjects (n=51, 65%) received palliative treatment, 7 patients were lost to follow up and no treatment information was available for review and the remaining 21 received induction chemotherapy with a curative intent, in this group median survival was 8.8 months with 52% (n=11) of patients achieving a complete response. Surprisingly the only factor that appeared to alter survival in the whole group was the presence of the breakpoint in chromosome 5 being distal to q15 (median 2.8 vs 4.1 months, p=0.025) Conclusions: This study constitutes the largest review of del(5q) in AML, its presence is currently associated with advanced age and a poor survival. Close to half of the patients in our series were classified as de novo AML. Although more commonly encountered as part of a complex karyotype it can also be present as a single abnormality. The different cytogenetic characteristics of the del(5q) may impact survival and this deserves further investigation. Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line). Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line).

Abstract Background: Deletion 5q is a lenalidomide-sensitive cytogenetic abnormality seen usually in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In the current study, we looked into the occurrence and clinicopathologic correlates of del(5q) in myeloproliferative neoplasms (MPNs) defined according to the World Health Organization (WHO) criteria. Methods: Patients with del(5q)-positive MPN were identified through database query involving over 2000 patients with myeloproliferative neoplasms (MPD) including no less than 500 patients with primary myelofibrosis (PMF), 600 patients with essential thrombocytosis (ET), 500 patients with polycythemia vera (PV), and the remaining comprised of patients with molecularly-undefined chronic eosinophilic leukemia, hypereosinophilic syndrome, systemic mastocytosis (SM), chronic neutrophilic leukemia, and “MPN, unclassifiable”. Results: A total of 23 patients with del(5q) were identified (12 at time of initial diagnosis), suggesting an overall prevalence rate of 1%. A central pathological review was conducted to confirm the diagnosis in each instance. Specific diagnoses included PMF (n=14; estimated prevalence of 3%), MPN, unclassifiable (n=4), PV (n=2), ET (n=1), post ET-myelofibrosis (n=1) and SM (n=1). Del(5q) was a sole abnormality in 5 patients, accompanied by one other abnormality in 6 patients, and part of a complex karyotype in 12 patients. The most common breakpoint was q13q33 (n=10) followed by q15q33 (n=6). Based on sample size adequacy, we focused on del(5q)-positive PMF patients and compared their bone marrow histological and clinical features with their del(5q)-negative counterparts seen at the Mayo Clinic during the same period of time (n=311). There was no difference in gender or age distributions. Histopathological review did not show the typical tight clusters of megakaryocytes seen in classic PMF. The megakaryocytes were not the predominate feature of the biopsy, in general were less lobulated and smaller in size, and had a higher grade of fibrosis (med = 3+) than classic PMF. Furthermore, del(5q)-positive PMF patients presented with significantly lower hemoglobin level (p=0.0009) and platelet count (p=0.0003). The percentage of patients presenting with a hemoglobin level of &lt; 10 g/dL or a platelet count of &lt; 100 × 109/L were 70% and 92% in del(5q)-positive patients as opposed to 43% and 18% in del(5q)-negative patients. However, the presence of del(5q) did not significantly affect either overall survival (Figure) or leukemic transformation in PMF (14% vs 9%). Conclusions: Del(5q) is generally rare in MPNs but might occur in as much as 3% of patients with PMF. The presence of del(5q) does not appear to affect overall survival in PMF despite its clustering with adverse prognostic features for the disease including anemia and thrombocytopenia. Finally, megakaryocyte morphology and distribution in del(5q)-associated PMF are characteristically different from classic PMF. Figure Figure

Key points Theoretical models suggest there is no benefit of high affinity haemoglobin to preserve maximal oxygen uptake in acute hypoxia but the comparative biology literature has many examples of species that are evolutionarily adapted to hypoxia and have high affinity haemoglobin. We studied humans with high affinity haemoglobin and compensatory polycythaemia. These subjects performed maximal exercise tests in normoxia and hypoxia to determine how their altered haemoglobin affinity impacts hypoxic exercise tolerance. The high affinity haemoglobin participants demonstrated an attenuated decline in maximal aerobic capacity in acute hypoxia. Those with high affinity haemoglobin had no worsening of pulmonary gas exchange during hypoxic exercise but had greater lactate and lower pH than controls for all exercise bouts. High affinity haemoglobin and compensatory polycythaemia mitigated the decline in exercise performance in acute hypoxia through a higher arterial oxygen content and an unchanged pulmonary gas exchange. AbstractThe longstanding dogma is that humans exhibit an acute reduction in haemoglobin (Hb) binding affinity for oxygen that facilitates adaptation to moderate hypoxia. However, many animals have adapted to high altitude through enhanced Hb binding affinity for oxygen. The objective of the study was to determine whether high affinity haemoglobin (HAH) affects maximal and submaximal exercise capacity. To accomplish this, we recruited individuals (n = 11, n = 8 females) with HAH (P50 = 16 ± 1 mmHg), had them perform normoxic and acute hypoxic (15% inspired oxygen) maximal exercise tests, and then compared their results to matched controls (P50 = 26 ± 1, n = 14, n = 8 females). Cardiorespiratory and arterial blood gases were collected throughout both exercise tests. Despite no difference in end‐exercise arterial oxygen tension in hypoxia (59 ± 6 vs. 59 ± 9 mmHg for controls and HAH, respectively), the HAH subjects’ oxyhaemoglobin saturation () was ∼7% higher. Those with HAH had an attenuated decline in maximal oxygen uptake () (4 ± 5% vs. 12 ± %, p < 0.001) in hypoxia and the change in between trials was related to the change in (r = −0.75, p < 0.0001). Compared to normoxia, the controls’ alveolar‐to‐arterial oxygen gradient significantly increased during hypoxic exercise, whereas pulmonary gas exchange in HAH subjects was unchanged between the two exercise trials. However, arterial lactate was significantly higher and arterial pH significantly lower in the HAH subjects for both exercise trials. We conclude that HAH attenuates the decline in maximal aerobic capacity and preserves pulmonary gas exchange during acute hypoxic exercise. Our data support the comparative biology literature indicating that HAH is a positive adaptation to acute hypoxia.