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. This article is protected by copyright. All rights reserved.

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.

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Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.