Hein, Molly S.

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.

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.

Background: Genetic variants resulting in quantitative defects in the production of α-spectrin are associated with autosomal recessive hereditary spherocytosis (HS). Because erythroid cells produce α-spectrin in a 3-4:1 ratio in relation to β-spectrin, one defective allele is typically well-tolerated and a single null allele combined with a second partially defective allele may display a sub-clinical phenotype. Protein expression can be reduced by nonsense, frameshift, missense or splicing variants but reported large deletion events are extraordinarily rare (n = 1). Splicing variants include the low expression alleles αLELY - Low Expression LYon (SPTA1:c.6531-12C>T, p.?) and αLEPRA-Low Expression PRAgue (SPTA1:c.4339-99C>T, p.?) which reduce protein expression by 50% and 16%, respectively. Here, we present a family study with a whole gene SPTA1 deletion that characterizes the patient phenotype when a full gene mutation is co-inherited with the αLEPRA and αLELY low expression alleles. Methods: The 6 month-old male proband had a history of congenital Coombs negative hemolytic anemia requiring transfusion from 3 months of age and in whom a red cell membrane disorder was clinically suspected. Pretransfusion CBC and peripheral blood smear results were unknown. Transfused CBC indices were as follows: RBC 4 x 1012/L, HGB 11 g/dL, MCV 76 fL, MCH 27 pg, MCHC 36 g/dL and a reticulocyte count of 1.8%. The proband's mother, of Cuban descent, and father, of European descent, had no history of anemia or HS. Targeted high throughput sequencing of genes related to hereditary hemolytic anemia was performed for the detection of single nucleotide variants (SNVs) and small insertion/deletion variants (indels). Relative quantitative real time PCR (RT-PCR) was utilized to confirm large structural variants. Results: Sequencing identified a large heterozygous deletion that spanned the entire SPTA1 gene (NC_000001.11:g.(?_158656337)_(158581024_?) in the proband and his mother. In addition, the proband had inherited hemizygous αLEPRA from his father. The mother had hemizgous αLELY. The father showed heterozygous αLEPRA and no other SPTA1 variants (Table 1). Conclusion: To our knowledge, only Iolascon et al in 2011 has reported a SPTA1 gene large deletion (SpectrinExeter, exon 11-exon 12 deletion, loss of 83 amino acids) which resulted in a structurally abnormal protein and ellipto-poikilocytosis phenotype when in trans to αLELY. Our current case is the first reported SPTA1 full gene deletion and our family study allows comparison of the effects of coinheritance of the low expression alleles αLEPRA and αLELY. When in combination with complete absence of the SPTA1 gene in trans configuration, αLELY manifests in subclinical effects and αLEPRA causes a recessive HS phenotype, which resulted in severe anemia. This contrast further demonstrates that, unlike αLEPRA, αLELY does not decrease the α-spectrin concentration sufficiently to contribute to the baseline phenotype regardless of the severity of the second allele mutation for quantitative disorders such as HS. Disclosures No relevant conflicts of interest to declare.

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 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.

ObjectiveOur aim was to identify cytokines and chemokines in patients with adult dermatomyositis (DM) and juvenile dermatomyositis (JDM) that predict changes in disease activity.MethodsMultiplexed immunoassays (Meso Scale Discovery) enabled simultaneous measurement of interferon (IFN)‐regulated chemokines and other pro‐ and anti‐inflammatory cytokines specific to differentiation of specific T‐cell and innate pathways. Cytokine scores were computed for IFNCK (IP‐10, MCP‐1), Th1 (IFNɣ, TNFα, and IL2), Th2 (IL4, IL10, IL12, and IL 13), Th17 (IL6, IL17, IL1β), macrophage (MIP‐1α, MIP‐1β, IL8), and regulatory (IL10, TNFα) factors. Spearman correlation and mixed models were used to examine whether cytokines at a previous visit predict change in disease activity at the next visit.ResultsThe study included 36 patients (16 DM and 20 JDM) with at least two visits (87 patient intervals between two visits). Mean age (SD) at inclusion was 56.9 (18.4) years for DM and 10.8 (6.6) years in JDM, 67% of patients were female, 89% Caucasian. The mean (SD) physician global, muscle and extra‐muscular disease activity Visual Analog Scale scores at inclusion were 41 (26), 36 (30), and 34 (21) mm, respectively. The change in IFN score from one visit to the next was associated with the change in physician global (P = 0.010) and extramuscular (P < 0.001) disease activity scores. Preliminary results revealed significant correlations of previous IFNCK score and IL‐6 with subsequent disease activity measures, but after adjustment for multiple visits per patient, these associations did not reach statistical significance.ConclusionThere is a potential relationship between IFNCK and other cytokine scores seen in adult and juvenile DM with future disease states.