Identification of a novel variant of epsilon‐gamma‐delta‐beta thalassemia highlights limitations of next generation sequencing

To the Editor: Next generation sequencing (NGS) techniques enable the characterization of an individual's complete genetic information within a single laboratory approach 1, 2. Recently, they emerged as popular tools in the diagnosis of rare or clinically unrecognizable genetic disorders 1-3. In this study, we present an Austrian kindred with apparently inherited microcytic anemia, which could not be diagnosed by conventional laboratory diagnostic approaches. Therefore, we aimed to elucidate its genetic background using NGS. The index patient was a 47-year-old Austrian male, presenting with anemia (hemoglobin 11.2 g/dL, normal range [NR] 13.0–17.5). Erythrocytes were microcytic with MCV 74.3 fL (NR 80.0–98.0), MCH 23.9 pg (NR 28.0–33.0), and MCHC 32.2 g/dL (NR 33.0–36.0), reticulocytes were mildly increased (0.127 × 1012/L, NR 0.027–0.095). Importantly, a detailed family history revealed normal blood counts in the patients' parents but pronounced microcytic anemia, resembling the one of the index patient, in two of his three children (aged 19–31 years; Fig. 1A,B). Further work-up of the index patient revealed a history of lung cancer treated successfully by operation, cirrhosis of the liver with splenomegaly and diabetes mellitus Type 2. Laboratory evaluation demonstrated signs of hemolysis with decreased haptoglobin (<0.07 g/dL, NR 0.30–2.00) and slightly increased bilirubin (1.34 mg/dL, NR 0.10–1.20), lactate dehydrogenase (LDH) was within normal limits (226 U/L, NR 120–240). Conventional hemoglobin electrophoresis revealed no signs of thalassemia with HbA 96.7%, HbF 0.3%, and HbA2 3.0% (HbF staining was negative). Alpha-thalassemia hot-spot variants, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, immune-mediated hemolysis, spherocytosis and lead intoxication could also be excluded. Epsilon-gamma-delta-beta thalassemia, Austrian I. (A) Pedigree analysis, showing the occurrence of this genetic condition in an Austrian family. The index patient of this study is marked with an arrow. (B) Peripheral blood smears showing anisocytosis, basophilic stippling and polychromasia of red cells, as well as the presence of target cells in the index patient (II:2, 7282) and his two affected children (III:1, 7523; III:2, 7524). His healthy daughter lacked these abnormalities (III:3, 7525). (C) MLPA delineated a deletion affecting the beta globin gene cluster. Ratio chart of peak areas of amplified probes from the index patient normalized to healthy controls and arranged by genomic location. Threshold ratios for deletion and duplication were set at <0·7 (red line) and >1·3 (blue line), respectively. Although clinically suspicious of a hemoglobinopathy, a proper diagnosis could not be established by these conventional laboratory approaches. Therefore, we extracted genomic DNA from leukocyte pellets and performed whole exome sequencing in all four individuals as previously described 4, 5. This approach revealed 88 different variants that were unique to the index patient and his two affected children, however, only two of those were located in genes which have been associated with erythrocytes previously. The first affected SLC4A1 (p.Ser350Gly), a gene that has been linked to the pathogenesis of hereditary spherocytosis, hereditary stomatocytosis, and Southeast Asian ovalocytosis 6, 7. Importantly, however, all of these disorders are incompatible with the hematologic features observed in our patients. The second variant affected HMOX2 (p.Met113Leu; rs150288371), which has been shown to play an essential role in hem catabolism 8. However, in-vivo knockout models did not show any effects on red blood cell morphology 9. Furthermore, as both variations were stated as "benign polymorphisms" by computational prediction tools, we excluded them as potential culprit mutations. Hemoglobinopathies might rarely be caused by deletions, a genomic variation that is frequently overlooked by NGS, particularly in case when larger regions are affected 2. Therefore, we decided to use multiplex ligation-dependent probe amplification (MLPA) in a next step, which delineated a monoallelic hemoglobin deletion in the index patient and his two affected children, thereby establishing the diagnosis of epsilon-gamma-delta-beta thalassemia (Fig. 1C). Further characterization of the breakpoints by high-resolution array comparative genomic hybridization (CGH) revealed a 339255 bp deletion on the short arm of chromosome 11, affecting the entire cluster of beta globin genes. This deletion has not been described previously 10 and is therefore named Austrian I. In conclusion, we identified a novel variant of epsilon-gamma-delta-beta thalassemia by combining a detailed clinical characterization with pedigree analysis and a broad range of conventional laboratory and molecular techniques. Most importantly, however, this case serves as a prime example for the limitations of NGS and demonstrates that its results should only be interpreted in sight of the entire clinical picture. The authors are thankful to all members of the family analyzed within this study. The study is dedicated to Karl Wagner, Professor emeritus of Hematology and Oncology. Armin Zebisch1*, Eduard Schulz1, Michela Grosso2,3, Barbara Lombardo2,3, Giovanni Acierno2,3, Heinz Sill1, and Achille Iolascon2,3 1Division of Hematology, Medical University of Graz, Graz, Austria 2Department of Molecular Medicine and Medical Biotechnology, University Federico II of Naples, Naples, Italy 3CEINGE–Advanced Biotechnologies, Naples, Italy Contract grant sponsor: Land Steiermark (to A.Z.); Contract grant number: GZ: Abt.08 – 16.H-76/2012-4; Contract grant sponsor: Italian Ministero dell'Universitàe della Ricerca (to A.I.); Contract grant number: MUR-PS 35–126; Contract grant sponsor: Ind. Regione Campania; Contract grant number: DGRC2362/07.