Hemoglobinopathy evaluation in routine carrier testing: Results to inform guidelines and protocols Craig Adamski, MS, CGC1, Catherine Terhaar MS, CGC1, Jeana DaRe, PhD, FACMG1, Priscilla Fernandes, PhD, FACMG1, Christina Settler MS, CGC1, Lindsay Dohany, MS, CGC1

1Progenity, Inc., Ann Arbor, MI, United States

Introduction FIGURE 1. Distribution of positive evaluation results by race/ethnicity. FIGURE 2. Interpretation of abnormal results on hemoglobin evaluation

Hemoglobinopathies, as a group, represent the most commonly inherited genetic n=732 diseases worldwide. It is estimated that 5-20% of individuals in the United States <1% 600 are carriers for hemoglobinopathies.¹ However, there are limited publications 11% u African American/Black (n=883) 500 n=499 demonstrating the general population results experienced in routine carrier testing 39% u Other or Mixed (n=388) n=36 Delta trait in the US. 400 n=64 trait u Hispanic (n=377) ACOG Guidelines for hemoglobinopathies recommend a 16% 300 n=89 Other with red blood indices for all women as well as hemoglobin electrophoresis for u Caucasian/White (n=357) n=233 n=230 n=120 A2 Prime women of certain high-risk ethnicities or with low mean corpuscular hemoglobin 200 u Asian (n=250) (MCH) or (MCV). DNA genotyping is recommended for 100 n=112 women with an abnormal MCV or MCH and normal hemoglobin electrophoresis.² 17% 17% u Not Provided (n=23) n=163 Limited data is available on the outcomes of these recommendations given the NUMBER OF POSITIVE RESULTS 0 technological advancements over the past few decades. Possible alpha Sickle cell Elevated Possible beta Hemoglobin Other thalassemia trait trait hemoglobin F thalassemia trait C trait Aim RESULT INTERPRETATION The goals of this study were to estimate the positivity rate for hemoglobinopathies in the general population and evaluate the benefit of concurrent genotyping. TABLE 1. TABLE 2. Rare hemoglobin identified with hemoglobin evaluation Individuals suspected to have hemoglobinopathy disease based on hemoglobin evaluation results

Method A2 Prime G-Philadelphia J-Medellin P-Nilotic Disease Number of affected individuals suspected Our database was queried for abnormal hemoglobin evaluations from 42,166 Anamosa Grady J-Toronto Q-India Sickle- disease 9 consecutive samples of mixed ethnicities. Hemoglobin evaluation consisted of Athens GA G-San Jose Korle Bu Riyadh Hemoglobin S–beta-thalassemia disease 6 red blood indices and high-performance liquid chromatography followed by, Belleville K-Woolwich San Bruno Hasharon 4 if necessary, hemoglobin electrophoresis. A subset (n= 2,863) also had genotyping Chicago Hopkins II Lepore Baltimore Seal Rock Hemoglobin E disease 2 for common variants known to cause alpha-thalassemia, beta-thalassemia, and Constant Spring Izmir Lima Sherwood Forest Hemoglobin E/ disease 2 hemoglobin S, C, D, E and O.* Abnormal reports were evaluated individually, Fannin-Lubbock J-Baltimore N-Baltimore Tarrant and test metrics and interpretations were tabulated. Fontainbleau J-Bangkok NYU Twin Peak Sickle-hemoglobin E disease 1 Osu-Christiansborg Wayne Hemoglobin C disease 1 *HBA1/HBA2: -alpha3.7, -alpha4.2, -alpha20.5, --SEA, --MED, --FIL, and --THAI deletions; HBB: 138C>T, -78A>G/-28A>G, -79A>G/-29A>G, G-Coushatta J-Broussais 118C>T, 126_129delCTTT, 135delC, 25_26delAA, 27dupG, 315+1G>A, 316-2A>C, 316-2A>G, 47G>A, 51delC, 52A>T, 59A>G, 75T>A, Westmead Compound heterozygous Hb K-Woolwich and Hb Little Rock 1 92+1G>A, 92+5G>C, 92+6T>C, 93-1G>A, 93-21G>A, 20A>T, 19G>A, 364G>C, 79G>A, and 364G>A. Double heterozygous of hemoglobin Westmead/E trait 1

Results Concurrent genotyping was completed in some cases and identified silent carriers ofalpha- Of the 42,166 samples, 2,278 (5.4%) had abnormal hemoglobin evaluation results. Conclusions thalassemia (-α/αα) that would otherwise have been missed. Of patients who had a hemoglobin shows the distribution of these results by race/ethnicity. Of these evaluation with no indication of alpha thalassemia trait, and went on to have abnormal Figure 1 Our study supports an approximate 5% carrier positivity rate in the general population for 2,278 abnormal results, the most common interpretation (32%) was ‘possible genotyping, 13% were positive for a single pathogenic variant in HBA1 or HBA2. These hemoglobinopathies. The most common result was ‘possible alpha-thalassemia trait’ based alpha-thalassemia trait’, followed by ‘’ (22%), ‘possible beta-thalassemia patients are at risk to have a child with hemoglobin H disease if their partners are heterozygous on abnormal hemoglobin evaluation. trait’ (10%), ‘elevated hemoglobin F’ (10%), and ‘hemoglobin C trait’ (5%) (Figure 2). α0 – thalassemia carriers. There were over 35 rare hemoglobin identified Table( 1). Hemoglobin evaluation also Although ACOG recommends genotyping for patients with abnormal hemoglobin evaluation, This study supports hemoglobin evaluation and concurrent genotyping for hemoglobinopathies. identified several individuals with results indicating that they may be affected rather alpha-thalassemia carrier status was not confirmed in 59% of samples with abnormal MCV/MCH. This complete approach allows patients to have clear, definitive answers about carrier than just a carrier of hemoglobinopathy (Table 2). These results are suggestive of a high false positive rate for alpha-thalassemia based on status in a timely manner. This approach also decreases false positive test results and hemoglobin evaluation alone, without genotyping. Of the 2,863 samples that had genotyping, 146 (5.1%) had abnormal molecular results. identifies more silent alpha-thalassemia carriers than current recommendations. Genotyping confirmed 100% (5/5) of samples with abnormal hemoglobin evaluation indicating ‘consistent with alpha-thalassemia trait’ and 41% (17/41) indicating References Acknowledgements: Thank you to Genetic Counselor Assistants Spencer Fuller and Ashley Cosenza; as well as Leanne Cartwright, MS, CGC for contributing to data collection for this project. : Craig R. Adamski, MS, CGC Senior Medical Science Liaison, ‘possible alpha-thalassemia trait’. Thirteen percent (14/105) of samples with abnormal 1. Hemoglobinopathies Clinical Manifestations, Diagnosis, and Treatment. Kohne, E Dtsch Arztebl Int. 2011 Aug; 108(31-32): 532–540. Contact information Progenity, Inc. [email protected] molecular results, and normal hemoglobin evaluation for alpha-thalassemia, were silent 2. Carrier screening for genetic conditions. Committee Opinion No. 691. American College of Obstetricians and Gynecologists. Obste Gynecol 2017: 129: e41-55 alpha-thalassemia carriers. 5230 S. State Road, Ann Arbor, MI 48108 © 2019 Progenity, Inc. All rights reserved. Progenity® is a registered service mark of Progenity, Inc.