Oliveira, Jennifer L.

Abstract Abstract 3209 Background: The red blood cell folate (RCF) assay has historically been recommended as a more reliable indicator of tissue folate stores compared to the serum folate (SF) assay, as it is not affected by recent ingestion of food. However, the RCF assay suffers from inherent problems with imprecision and accuracy, which are not encountered with SF measurements. Furthermore, following the advent of required folic acid supplementation of many foods by the Food and Drug Administration (FDA) in 1992, folate deficiency is increasingly rare. Very few studies have looked at the value of the RCF versus the SF. We undertook a 10 year retrospective analysis of RCF and SF results to determine the clinical utility of RCF beyond that of SF. Methods: We retrieved all RCF and SF results from the laboratory information system at Mayo Clinic (Rochester, MN) ordered on inpatients and outpatients between 1999–2009. Data for patients who had concurrent orders for SF and RCF were analyzed and chart reviews were conducted on those patients with normal SF but low RCF. Abnormal values were defined by the National Health and Nutrition Examination Surveys (NHANES)/Center for Disease Control (CDC) criteria for folate deficiency (SF< 3.0 ng/ml, RCF < 140 ng/ml). Results: A total of 152,166 SF and 15,708 RCF were performed over the decade of the study. The prevalence of folate deficiency using only SF values was 0.39% and 0.27% using only RCF values. There were 1082 patients in which SFA and RCFA were ordered concurrently (Table 1). Only 1 patient (0.09%) had both abnormal SF and RCF. Chart reviews of the 4 patients with a normal SF but low RCF were as follows: 1) a 6 year old (yo). male with known folic acid transporter deficiency treated with Leucovorin. 2) a 58 yo male with history of gout, hypertension, psoriasis, and hyperlipidemia with normal hemoglobin (Hb) and MCV. 3) a 65 yo male with chronic diarrhea and suspected alcohol abuse; slight macrocytosis (MCV=100.3 fL) but normal Hb. 4) a 51 yo male with multifactorial gait disorder and alcohol abuse. There was a previous history of vitamin B12 deficiency but B12 levels were normal at this time. The CBC was notable for macrocytosis (MCV=115.1 fL) without anemia. Only in patient 4 did the RCF value result in the institution of folic acid supplementation. Conclusions: The RCF provides no additional information beyond that provided by the SF in virtually all situations. Thus SF alone is sufficient for assessment of folate stores. However, there is no evidence to support routine ordering of either SF or RCF, as true folate deficiency in the current era of FDA mandated folic acid supplementation is exceedingly rare. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders. Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data. Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested). Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case. Table 1. Clinically significant high oxygen affinity Hb variants at Mayo Clinic n Beta Alpha >30 Malmo Tarrant 10 - 20 Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, Abruzzo Dallas 5 - 10 Ty Gard, Ypsilanti, Alberta, Coimbra, North Chicago J-Cape Town 2 - 4 Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, Regina Chesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano 1 Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South Milwaukee Linwood, Longview*, Milledgeville, Sarasota Springs, Voorhees * novel Disclosures Pardanani: Stemline: Research Funding.

Abstract Background: Large deletions involving the beta globin complex are relatively rare. They can be categorized generally into five groups by deletion size and/or location: 1) beta zero thalassemia (BZT); 2) delta beta thalassemia (DBT); 3) hereditary persistence of fetal hemoglobin (HPFH); 4) gamma delta beta thalassemia (GDBT); and 5) epsilon gamma delta beta thalassemia (EGDBT). These deletions are not well understood but often have significant clinical impact, either when present alone or in combination with other hemoglobin mutations. In this study, we analyze phenotypic and molecular data on a large number of cases with deletions in the beta globin gene complex to better classify these five groups of deletions as they occur in isolation. Methods: A query of the routine clinical testing patient files from the Mayo Clinic Metabolic Hematology and Molecular Genetics Laboratories from 2010 to 2015 identified 179 patients with a deletion confirmed by a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. Twenty-four probes sets were placed from the 5' locus control region (LCR) to the 3' hypersensitivity region, spanning the beta globin gene complex. Using a Luminex LX200 flow cytometer, a gene dosage ratio was calculated for each probe set using the median fluorescent intensity value collected. The size and location of the deletion and patient phenotype were compared. Results: Of the 179 total cases, the following large deletions were identified: beta gene (HBB) (n = 47), delta (HBD) through HBB (n = 105), A-gamma (HBG2) through HBB (n = 20), and locus control region (LCR) through HBB (n = 7). One case had a deletion involving the LCR epsilon with the rest of the complex left intact. A subset (n = 60) of cases had compound hemoglobin mutations that altered the phenotype. The BZT cases had relatively high Hb A2 levels and variable Hb F levels consistent with promotor region loss. The main differences between DBT and HPFH included Hb F and Hb A2 levels. GDBT cases presented with median Hb F levels higher than that observed in DBTs, normal Hb A2, and microcytic anemia. EGDBT cases had variable features according to age of the patient and Hb F level; severe microcytic anemia was observed in neonates, milder microcytic anemia in young children, and microcytosis without anemia in an adult case. The phenotypic features of 119 patients with isolated large deletions are compiled in table 1. Conclusion: In general, all five categories of large deletions in an isolated heterozygous state can present with microcytic anemia and are typically benign with the exception of transient severe microcytic anemia in neonatal EGDBT cases. Although phenotypes associated with large deletions involving the beta globin gene complex are frequently distinctive, significant phenotypic overlap can be seen in a subset of cases. These cases require molecular analysis due to their clinical importance when in combination with another beta globin gene complex mutation for an adequate diagnosis and treatment approach. Table 1. Deletion type Age n HbF (%) HbA2 (%) Hb (g/dL) MCV (fL) RBC (10^12/L) RDW (%) MCH (pg/cell) BZT 20 6.3 (0.6-94.4) 6.8 (3.4-11.6) 11.1 (8.3-14.5) 65.4 (60.8-77.2) 5.4 (4.2-6.2) 19.2 (16.6-21.2) 20.9 (18.3-25.7) DBT 56 10.6 (2.7-22.4) 2.7 (2.5-3.1) 11.7 (8.6-14.4) 68.9 (61.3-83.5) 5.3 (4.1-7.3) 21.4 (18.2-26.8) 21.6 (19.9-39.2) HPFH 23 25.9 (17.6-39.7) 2.0 (1.5-2.4) 11.6 (8.1-16.7) 78.4 (60.2-101.9) 4.4 (3.0-6.3) 17.5 (14.1-22.3) 25.4 (17.6-29.7) GDBT 14 13.3 (8.2-19.0) 2.6 (1.8-2.7) 11.0 (8.6-14.1) 72.5 (57.9-82.1) 5.1 (3.5-6.2) 20.6 (17.4-23.5) 22 (17.9-25.1) EGDBT* 28 Y 1 0.3 3 13.3 59.4 6.9 15.4 19.2 1-4 Y 3 0.9 (0-1.6) 3.2 (2.9-3.5) 9.5 (8.8-13.3) 57.8 (57.6-59.4) 5.2 (4.9-6.9) 16.6 (15.4-17.4) 18.5 (18.1-19.2) <6 month 2 21.4 (14.8-27.9) 2.6 (2.2-2.9) 6.3 (6.0-6.6) 61.3 (59.9-62.6) 3.4 (3.3-3.3) 21.5 (21.2-21.7) 18.4 (18.1-18.7) medians, (min, max); *stratified by age Disclosures No relevant conflicts of interest to declare.

Abstract Background: The control of hemoglobin F (Hb F) expression has proven an elusive puzzle with several postulated but not mutually exclusive hypotheses. A silencing mechanism hypothesis, which predicts the existence of a regulatory element that suppresses HbF expression, has been supported by recent investigations into a 3.5 kb region upstream from the delta globin gene (HBD). To test this hypothesis, we have investigated our historical case files in a tertiary care high-throughput clinical laboratory and compared the patient phenotype (Hgb, MCV, MCH, RDW, Hb F %, and flow cytometry Hb F distribution) with the status of this 3.5 kb region. Methods: A query of clinical testing patient files from Mayo Clinic Metabolic Hematology Laboratory yielded 179 patients confirmed by Multiplex Ligation-dependent Probe Amplification (MLPA) to contain large deletions located within the epsilon through beta globin genes. Of these, 27 unrelated patients with breakpoints between the pseudobeta (HBBP1) and HBD genes were identified. Four additional MLPA probe pairs were placed in this region and the patient phenotypes were compared. Results: The interior two (of the four added) probes between HBBP1 and HBD stratified all 27 cases. The other two (flanking) probes were never discriminatory, thus refining the area of interest to 2.4 kb. Sixteen cases showed breakpoints within hg19 g.5260154-5259135 (5' region) and eleven cases showed breakpoints within hg19 g.5259136-5257692 (3' region) upstream of HBD. Patients who displayed a phenotype of HPFH (n = 9) contained breakpoints in the 5' region whereas those with a delta beta thalassemia (DBT) phenotype (n = 11) were associated with breakpoints in the 3' region. A subset (n = 7) with breakpoints in the 5' region had indefinite phenotypic features, but of these, all but one showed homocellular Hb F distribution. Conclusion: Our molecular and phenotypic correlation of 27 patients with large deletional breakpoints between HBBP1 and HBD supports the hypothesis of a silencing element located upstream of HBD and further narrows the area that segregates many HPFH and DBT patient phenotypes from 3.5 kb to 2.4 kb. Several potential silencing effectors with binding sites in this region include HDAC1, GATA1 and H3K27me3; interestingly, the BCL11A binding site may be outside of the regulatory area. Table 1. Phenotypic data of patients with deletions between HBBP1 and HBD. Case Age/Sex Hb F Hb A2 Hb X Hgb RBC MCV MCH MCHC RDW Hb F Flow (Y) (%) (%) (%) (g/dL) ( 10^12/L) (fL) (pg/cell) (hg/cell) (%) 3' Region 1 47 M 25.5 2.1 16.7 6.3 81.4 26.7 32.8 15.7 H 2 5 M 28.3 2.4 12.5 4.9 74.6 25.4 34 15.9 H 3 24 F 30.3 1.7 11.6 4.23 92.7 27.4 29.6 15.2 H 4 9 F 36.3 2.2 S = 62 13.1 5.2 69.9 NA NA 14.8 5 24 F 37.3 2.1 C = 61 12.9 5.1 72.9 25.5 35 14.3 6 3 M 39.7 2.1 12.4 4.4 88.7 28.1 31.7 16.3 7 27 F 40.2 3.4 S = 56 12.2 4.4 79.7 27.8 34.9 15 8 16 M 43.7 2.2 S = 54 15.9 6.8 71.5 23.5 32.9 14.1 9 7m M 100.0* 0.0 12.8 4.8 76 26.5 34.9 16.8 3' Region 10 28 F 20.9 2 10.11 4.4 70.9 22.7 32.1 19 H 11 64 M 21.4 2.3 10.51 4.1 81.1 25.8 31.8 18.1 12 2.5 M 24 2.1 10.21 5.3 59.4 19.3 32.5 20.9 H 13 58 F 24 2.1 9.51 4.4 72.2 21.5 29.8 17.7 H 14 14 F 25.4 1.5 10.81 4.9 69.4 22 31.8 17.6 H 15 52 F 26.9 1.8 8.42 3.6 72 23.5 32.7 19.4 T 16 34 M 38.7 1.7 S = 60 11.4 4.7 71.6 NA NA 21.6 5' Region 17 24 F 2.7 2.9 12.3 6.1 62.2 20 32.2 18.2 18 32 F 6.1 2.7 10.2 5.1 61.3 19.9 32.5 19.3 19 27 F 7.2 2.9 11 5.1 72.9 21.7 29.8 19.9 20 29 F 7.8 2.7 10.2 4.7 72.2 21.7 30 20.5 21 15 M 7.8 2.5 12.7 6.2 64.7 20.4 31.5 21.2 22 42 M 7.8 3.1 10.6 4.9 75.1 21.5 28.6 23.7 23 60 M 10.1 2.5 11 4.5 74.8 24.7 33 23.4 24 41 M 10.7 2.8 8.6 4.1 73.3 21.2 29 26.8 25 30 F 11.5 2.7 12.1 5.5 67.4 21.9 32.5 21.8 26 25 F 13.2 2.8 12.1 5.3 68.4 22.8 33.3 21.6 27 3 F 18.3 2.6 11.9 5.79 63.6 20.6 32.3 22.7 T Normal values <1 2-3.3 0 H = homocellular; T= heterocellular; I = indeterminate; *homozygous deletion -- = no deletion; + = deletion present (all alpha deletions were single -3.7 kb 'rightward' deletions) 1 = Suspected Fe deficiency; 2 = recent transfusion Disclosures No relevant conflicts of interest to declare.

Key points Haemoglobin affinity is an integral concept in exercise physiology that impacts oxygen uptake, delivery and consumption. How chronic alterations in haemoglobin affinity impact physiology is unknown. Using human haemoglobin variants, we demonstrate that the affinity of haemoglobin for oxygen is highly correlated with haemoglobin concentration. Using the Fick equation, we model how altered haemoglobin affinity and the associated haemoglobin concentration influences oxygen consumption at rest and during exercise via alterations in cardiac output and mixed‐venous . The combination of low oxygen affinity haemoglobin and reduced haemoglobin concentration seen in vivo may be unable to support oxygen uptake during moderate or heavy exercise. AbstractThe physiological implications, with regard to exercise, of altered haemoglobin affinity for oxygen are not fully understood. Data from the Mayo Clinic Laboratories database of rare human haemoglobin variants reveal a strong inverse correlation (r = −0.82) between blood haemoglobin concentration and P50, an index of oxygen affinity [Hb = −0.3135(P50) + 23.636]. In the present study, observed P50 values for high, normal and low oxygen‐affinity haemoglobin variants (13, 26 and 39 mmHg) and corresponding haemoglobin concentrations (19.5, 15.5 and 11.4 g dL−1 respectively) are used to model oxygen consumption as a fraction of delivery at rest ( = 0.25 L min−1, cardiac output = 5.70 L min−1) and during exercise ( = 2.75 L min−1, cardiac output = 18.9 l min−1). With high‐affinity haemoglobin, the model shows that normal levels of oxygen consumption can be achieved at rest and during exercise at the assumed cardiac output levels, with reduced oxygen extraction both at rest (16.8% high affinity vs. 21.7% normal) and during exercise (55.8% high affinity vs. 72.2% normal). With low‐affinity haemoglobin, which predicts low haemoglobin concentration, oxygen consumption at rest can be sustained with the assumed cardiac output, with increased oxygen extraction (31.1% low affinity vs. 21.7% normal). However, exercise at 2.75 l min−1 cannot be achieved with the assumed cardiac output, even with 100% oxygen extraction. In conclusion, the model indicates chronic alterations in P50 associate directly with Hb concentration, highlighting that human Hb variants can serve as ‘experiments of nature’ to address fundamental hypotheses on oxygen transport and exercise.

AbstractMultiple algorithms have been published for the evaluation of hereditary erythrocytosis (HE). Typical entry points begin after excluding the more common acquired conditions through investigations of clinical history and assessment of cardiac, pulmonary, or vascular system disorders. Prior exclusion of JAK2 mutations, particularly the common JAK2 V617F mutation, is indicated in adults but less so in pediatric populations. Key decision trees are based on serum erythropoietin levels and p50 results. Recent data reveal some overlap in clinical presentation and laboratory findings in erythrocytosis. Caveats to consider when using algorithmic approaches are discussed.

AbstractHSTCL is a highly aggressive malignancy with a poor prognosis. Case series and accounts have reported the use of different chemotherapy regimens with diverse patient outcomes. Most long‐term survivors had undergone high‐dose chemotherapy with autologous or allogeneic HCT. We describe two pediatric patients with HSTCL who were treated with chemotherapy followed by allogeneic HCT. Both patients are alive and in complete remission 2 and 8 years after therapy. Multiagent chemotherapy followed with allogeneic HCT seems to provide patients who have chemotherapy‐sensitive disease a long‐term disease‐free survival.

Hereditary erythrocytosis is associated with high oxygen affinity hemoglobin variants (HOAs), 2,3‐bisphosphoglycerate deficiency and abnormalities in EPOR and the oxygen‐sensing pathway proteins PHD, HIF2α, and VHL. Our laboratory has 40 years of experience with hemoglobin disorder testing and we have characterized HOAs using varied protein and molecular techniques including functional assessment by p50 analysis. In addition, we have more recently commenced adding the assessment of clinically relevant regions of the VHL , BPGM, EPOR , EGLN1 (PHD2), and EPAS1 (HIF2A) genes in a more comprehensive hereditary erythrocytosis panel of tests. Review of our experience confirms a wide spectrum of alterations associated with erythrocytosis which we have correlated with phenotypic and clinical features. Through generic hemoglobinopathy testing, we have identified 762 patients with 81 distinct HOA Hb variants (61 β, 20 α), including 12 that were first identified by our laboratory. Of the 1192 cases received for an evaluation specific for hereditary erythrocytosis, approximately 12% had reportable alterations: 85 pathogenic/likely pathogenic mutations and 58 variants of unknown significance. Many have not been previously reported. Correlation with clinical and phenotypic data supports an algorithmic approach to guide economical evaluation; although, testing is expanded if the suspected causes are negative or of uncertain significance. Clinical features are similar and range from asymptomatic to recurrent headaches, fatigue, restless legs, chest pain, exertional dyspnea and thrombotic episodes. Many patients were chronically phlebotomized with reported relief of symptoms.