==== Front Clin Case Rep Clin Case Rep 10.1002/(ISSN)2050-0904 CCR3 Clinical Case Reports 2050-0904 John Wiley and Sons Inc. Hoboken 10.1002/ccr3.7273 CCR37273 CCR3-2022-11-2414.R1 Case Report Case Report More than just mild thrombocytopenia: Clinical clues in the diagnosis of germline predisposition to malignancy from a rare ETV6 variant Fang et al. Fang Shannon https://orcid.org/0000-0001-5991-0269 1 Botero Juliana Perez 2 3 Hackney Lisa R. 4 lisa.hackney@uhhospitals.org 1 Case Western Reserve University School of Medicine Cleveland Ohio USA 2 Versiti Diagnostic Laboratories Milwaukee Wisconsin USA 3 Division of Hematology and Oncology Medical College of Wisconsin Milwaukee Wisconsin USA 4 Pediatric Hematology and Oncology UH Rainbow Babies and Children's Hospital Cleveland Ohio USA * Correspondence Lisa R. Hackney, Pediatric Hematology and Oncology, UH Rainbow Babies and Children's Hospital, Cleveland, OH, USA. Email: lisa.hackney@uhhospitals.org 02 7 2023 7 2023 11 7 10.1002/ccr3.v11.7 e727315 3 2023 30 11 2022 12 4 2023 © 2023 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. https://creativecommons.org/licenses/by/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Key Clinical Message In the evaluation of patients with longstanding mild thrombocytopenia, emphasis on family history, genetic testing, and collaborative clinical and laboratory‐based family studies can ensure proper diagnosis and monitoring for malignancies. Abstract We report the diagnostic approach to mild and non‐specific thrombocytopenia with unclear genetic findings in two sisters. Genetic sequencing revealed a rare variant in ETS Variant Transcription Factor 6, which is associated with inherited thrombocytopenia with predisposition to hematologic malignancy. Familial studies provided sufficient evidence for a likely pathogenic classification. Familial testing reveals a rare ETV6 variant likely causing inherited thrombocytopenia in this family. genetics hematology oncology pediatrics and adolescent medicine thrombocytopenia University Hospitals Rainbow Babies & Children's Department of Pediatric Hematology Oncology source-schema-version-number2.0 cover-dateJuly 2023 details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.3.0 mode:remove_FC converted:02.07.2023 Fang S , Botero JP , Hackney LR . More than just mild thrombocytopenia: Clinical clues in the diagnosis of germline predisposition to malignancy from a rare ETV6 variant. Clin Case Rep. 2023;11 :e7273. doi:10.1002/ccr3.7273 ==== Body pmc1 INTRODUCTION Thrombocytopenia can be the first indication of an underlying condition in children and includes a wide range of differential diagnoses. Inherited thrombocytopenias are a heterogenous group of disorders with low platelet counts, with or without platelet dysfunction. With increasing access to genetic testing, the number of genes associated with this phenotype and number of patients diagnosed continue to grow. 1 Over 50 genes have been described to be associated with inherited thrombocytopenia and an estimated 2.7% are from disease‐causing variants in ETS Variant Transcription Factor 6 (ETV6). 1 , 2 ETV6 is a transcription repressor in the ETS family of transcription factors, and it is implicated in normal hematopoiesis, thrombopoiesis, and platelet function. 3 , 4 Distinguishing ETV6‐related thrombocytopenia (ETV6‐RT) from other etiologies is important, given its associated risk of malignancy, 2 , 3 , 4 and requires genetic testing for diagnostic confirmation. This report describes the diagnostic approach and clinical challenges in a family with inherited thrombocytopenia that was found to carry a rare disease‐causing ETV6 variant. 2 CASE DESCRIPTION 2.1 Patient A An 11‐year‐old female patient was referred to the pediatric hematology clinic for isolated thrombocytopenia, which was identified after presenting to the emergency department with bilateral toe bruising. Her fingers and toes would become purple for a prolonged period of time, but bleeding symptoms were absent. Family history was positive for bruising in the mother and anemia in the maternal grandmother. The patient was taking atomoxetine, risperidone, hydroxyzine, and medroxyprogesterone acetate. Physical examination showed cyanosis in the feet after exposure to cold, slow capillary refill, and macular papular red erythematous rashes above the ankles bilaterally. Complete blood count (CBC), screening coagulation studies, serum immunoglobulins, and autoimmune studies were obtained (Table 1). Mild thrombocytopenia with normal sized platelets and normal granulation on peripheral smear was noted. The clinical presentation was most consistent with Raynaud's, and it was thought that the mildly decreased platelets could have been the result of viral suppression or side effects from risperidone. A repeat CBC 1 month later was recommended. TABLE 1 Laboratory values in members of a family with the ETV6 c.1085A>G variant. Patient A Patient B Patient C Father Reference Platelets 133 105 137 138 150–400 × 109/L WBC 7.7 5.5 7.4 11.2 4.5–13.5 × 109/L Hemoglobin 15.3 11.1 11.2 15.6 12.0–16.0 g/dL Hematocrit 42.6 31.1 32.9 45.3 36.0%–46.0% MCV 88 89 88 97 78–102 fL ANC 4.69 2.36 2.68 6.75 1.20–7.70 × 109/L Absolute lymphocyte 2.33 3.14 3.58 2.54 1.80–5.00 × 109/L Note: Abnormal values are in bold. All units are in accordance with the reference values. Abbreviations: ANA, antinuclear antibody; ANC, absolute neutrophil count; MCV, mean corpuscular volume; WBC, white blood cell. 2.2 Patient B A 3‐year‐old female patient with a past medical history of chronic urticaria and reactive airway disease was referred to hematology by allergy and immunology due to low platelet count. She had no history of infection, fever, or sick exposures. She reported easy bruising when active and presented with bilateral leg bruising. CBC results were similar to patient A (Table 1). Family history revealed Patient B is the half‐sister of Patient A, both of whom share a father. The father and three of his children have a history of low platelet counts. No other family members were symptomatic or needed treatment for their thrombocytopenia. While the differential diagnosis for isolated mild thrombocytopenia is broad, family history pointed towards an inherited etiology. Von Willebrand factor (VWF) plasma studies were normal. Platelet transmission electron microscopy (PTEM) showed ultrastructurally normal platelets, with some platelets having large and decreased alpha granules. Bone marrow biopsy/aspirate had normal maturing trilineage hematopoiesis and showed no evidence of leukemia. There were reduced megakaryocytes and few hypolobated forms. 2.3 Genetic testing A next‐generation sequencing (NGS) based panel of genes associated with inherited thrombocytopenia was performed at Versiti Diagnostic Laboratories in a blood sample of patient B. The germline heterozygous ETV6 c.1085A>G (p.Asp362Gly) missense variant was identified (Figure 1). This variant occurs in a well‐conserved nucleotide and is located in the ETS domain, the functional domain in ETV6 that binds DNA. The variant had not been reported in the literature or in the general population. Due to limited evidence, using the criteria developed by the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP), the variant was classified as a variant of uncertain significance (VUS) (PM2, PP3, PP4). However, it was suspected to be implicated in the low platelet counts seen in this family. FIGURE 1 Sanger sequencing confirming the heterozygous ETV6 c.1085A>G variant analyzed by Mutation Surveyor. The upper panel shows reference sequence; the middle panel shows patient sequence; the lower panel shows the variant as a peak. Familial testing to provide additional evidence for variant classification using segregation was undertaken (Figure 2). The variant segregated with the thrombocytopenic trait in all individuals tested. Those who were ETV6 wild type had normal platelet counts. Subtle elevations in red blood cell size were noted and considered part of the collective additional evidence. Familial studies provided sufficient evidence to reach a likely pathogenic classification for the familial variant (PM2, PP1_mod, PP4, PP3). FIGURE 2 Pedigree showing the segregation of the ETV6 c.1085A>G variant within the extended family of the proband (patient B). WT, wild type gene confirmed by genetic testing. Arrow indicates proband. 3 DISCUSSION ETV6‐RT, as illustrated by the study of this family, presents with mild and variable clinical manifestations that can easily be overlooked, especially when historical platelet counts and a complete family history are not readily available. Typical platelet counts in patients with ETV6‐RT are in the mild to moderate thrombocytopenia range, usually >75 × 109/L. 4 , 5 Platelet size is normal, and platelet morphology or peripheral smear findings do not show distinguishing features that set it apart from other inherited thrombocytopenias. 3 , 5 Mean corpuscular volume can be mildly elevated in patients with ETV6‐RT, 4 , 6 and while not specific to this disorder, is a useful clinical clue when present. Bleeding is often variable; impaired platelet aggregation with adenosine diphosphate and arachidonic acid, as well as abnormal alpha granule morphology on PTEM, have been described. 6 Patients with ETV6‐RT can exhibit megakaryocytes that are small and hypolobulated, 4 , 6 , 7 as in the case of patient B. The inheritance pattern is autosomal dominant, and the penetrance of the thrombocytopenic phenotype is high. The family's history showed affected family members in multiple generations, with males and females affected equally, supporting this inheritance pattern. When the clinical evaluation of a patient and their family is suggestive of an inherited thrombocytopenia without pathognomonic clinical features, genetic testing becomes an essential tool in confirming the diagnosis and providing appropriate genetic counseling. At least three nonsyndromic autosomal dominant inherited thrombocytopenias with normal platelet size are associated with hematologic malignancy and involve variants in RUNX1, ANKRD26, and ETV6. 8 , 9 , 10 Genetic testing is the only strategy able to confidently distinguish them from each other, which is an important step in the clinical approach because the expected rate of evolution to malignancy and type of associated neoplasm varies between them. ETV6‐RT has an overall 30% risk for hematologic malignancies—including B‐cell acute lymphoblastic leukemia, acute myeloid leukemia, and myelodysplastic syndrome. 4 Results of genetic testing can be difficult to interpret, especially in cases where the identified variant is rare or novel. In absence of prior reports in the literature or functional evidence to support its pathogenicity, other evidences—including thorough knowledge of the functional domains of the protein affected, population data, and in silico prediction tools—are helpful in variant classification. However, due to insufficient evidence, frequently the classification is VUS, which is not clinically actionable. 11 As illustrated in this family, segregation can provide key additional evidence for variant classification and resolution of the VUS. Inherited thrombocytopenias with mild decrease in platelet counts and no syndromic associations, such as ETV6‐RT, are challenging to diagnose due to their non‐specific clinical presentation and low frequency compared to acquired platelet disorders. Recognizing the clinical clues of inherited thrombocytopenia is an important diagnostic skill to prevent misdiagnosis and ensure proper counseling regarding risk of malignancy. Unexplained, persistent thrombocytopenia should prompt consideration of inherited syndromes, particularly with a positive family history, and appropriate genetic counseling and testing. Special attention to family history, genetic testing, and family studies using multidisciplinary clinical and laboratory‐based teams can facilitate confirmation of an inherited thrombocytopenia diagnosis, even when limited variant‐specific data are available. AUTHOR CONTRIBUTIONS Shannon Fang: Conceptualization; visualization; writing – original draft; writing – review and editing. Juliana Perez Botero: Conceptualization; data curation; formal analysis; investigation; writing – original draft; writing – review and editing. Lisa Hackney: Conceptualization; data curation; formal analysis; funding acquisition; investigation; writing – original draft; writing – review and editing. FUNDING INFORMATION Funding was received from University Hospitals Rainbow Babies & Children's Department of Pediatric Hematology Oncology. CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest to declare. CONSENT Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy. Patient consent was obtained. ACKNOWLEDGMENTS The funding of this study was supported by University Hospitals Rainbow Babies & Children's Department of Pediatric Hematology Oncology. The authors would like to acknowledge Jamie McCreery, MS, CGC, Valerie Trapp‐Stamborski, PhD, Mia J Sullivan, Sean Tracey, and Teresa Lijewski from Versiti Diagnostic Laboratories for their contributions in data analysis and interpretation, variant classification, clinical correlation, and coordination of family studies. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed in this study. ==== Refs REFERENCES 1 Noris P , Pecci A . Hereditary thrombocytopenias: a growing list of disorders. Hematology Am Soc Hematol Educ Program. 2017;2017 (1 ):385‐399. doi:10.1182/asheducation-2017.1.385 29222283 2 Melazzini F , Palombo F , Balduini A , et al. Clinical and pathogenic features of ETV6‐related thrombocytopenia with predisposition to acute lymphoblastic leukemia. Haematologica. 2016;101 (11 ):1333‐1342. doi:10.3324/haematol.2016.147496 27365488 3 Di Paola J , Fisher MH . ETV6‐related thrombocytopenia and platelet dysfunction. 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