Analysis of clinical characteristics and treatment efficacy in two pediatric cases of ANKRD26-related thrombocytopenia

Abstract ANKRD26-related thrombocytopenia (ANKRD26-RT or THC2, MIM 188 000), an autosomal dominant thrombocytopenia, is unresponsive to immunosuppressive therapy and susceptible to hematological malignancies. A large number of pediatric patients are diagnosed with immune thrombocytopenia (ITP) every year; however, thrombocytopenia of genetic origin is often missed. Extensive characterization of ANKRD26-RT will help prevent missed diagnosis and misdiagnosis. Furthermore, identification of ANKRD26-RT will help in the formulation of an accurate diagnosis and a treatment plan. In our study, we report cases of two Chinese pediatric patients with ANKRD26-RT and analyze their clinical characteristics, gene mutations, and treatment modalities. Both patients were 1-year-old and presented with mild bleeding (World Health Organization(WHO) score grade 1), different degrees of platelet reduction, normal mean platelet volume, and megakaryocyte maturation impairment not obvious. Genetic tests revealed that both patients had ANKRD26 gene mutations.Patient 1 had a mutation c.-140C>G of the 5’ untranslated region (UTR), and patient 2 had a mutation of c.-127A>T of 5’UTR. Both patients were treated with eltrombopag, and the treatment was no response, with no adverse reactions. Plain Language Summary What is the background? ANKRD26-RT is an autosomal dominant thrombocytopenia which is unresponsive to immunosuppressive therapy and susceptible to hematological malignancies. It is rare and lacks specific clinical features, making misdiagnosis easy. Some studies report that eltrombopag is safe and effective for short-term treatment of the disease; however, these reports are limited. What we did and summary of findings. We retrospectively studied the clinical manifestations and diagnosis process of ANKRD26-RT and discussed the treatment efficacy of immunosuppressants and eltrombopag for its management. We found two pediatric cases of patients with ANKRD26-RT with varying degrees of thrombocytopenia, mild bleeding, normal mean platelet volume, and megakaryocyte maturation impairment that was not obvious. Immunosuppressant treatment wasunresponsiveor temporarily responsivebut not sustained , and short-term administration of eltrombopag (25 mg/day) was safe, but it did not effectively improve the patients’ platelet counts. What is the impact? If patients clinically diagnosed with immune thrombocytopenia do not respond to immunosuppressive agents, genetic testing should be conducted to exclude hereditary thrombocytopenia, and a normal mean platelet volume should not exclude the possibility of the disease. For patients with ANKRD26-RT, eltrombopag is safe for short-term use;however, 25 mg/day treatment is unresponsive. Ourreport complements data on the diagnosis and management of ANKRD26-RT disease in children.


Introduction
2][3] The clinical features include varying increased bleeding propensity, mild to moderate thrombocytopenia with normal mean platelet volume, increased serum TPO (Thrombopoietin) level, impaired bone marrow megakaryopoiesis, and susceptibility to myeloid tumors and other hematological malignancies.In terms of treatment, immunosuppressive treatments are not responsive; however, short-term use of eltrombopag has been reported to safely and effectively improve platelet count. 4,5However, these reports are limited.Therefore, we report the clinical characteristics and treatment efficacy of eltrombopag in two Chinese pediatric patients with the aim to provide data that will assist in the diagnosis and treatment of this heterogeneous disorder.

Patients
In this study, we enrolled two patients diagnosed with ANKRD26-related thrombocytopenia and eligible for pedigree verification at the Pediatric Department of the First Affiliated Hospital of Guangxi Medical University between January 2019 and December 2020.Clinical characteristics, laboratory tests, gene tests, and therapeutic results were retrospectively analyzed.The exclusion criteria were secondary thrombocytopenia, including thrombocytopenia induced by autoimmune disease, thyroid disease, proliferative diseases of the lymphatic system, myelodysplastic (aplastic anemia and myelodysplastic syndrome), hematologic malignancies, and chronic liver disease hypersplenism, drug-induced thrombocytopenia, alloimmune thrombocytopenia, platelet depletion and secondary thrombocytopenia caused by infections, pseudo-thrombocytopenia, and microangiopathy hemolytic anemia, including acquired and hereditary thrombotic thrombocytopenic purpura.This study was approved by the Ethics Committee of Guangxi Medical University.Informed consent was obtained from both participants' legal guardians in accordance with the institutional ethics guidelines.

Data collection
We collected and summarized the clinical data of the two pediatric patients, including the age of onset, sex, clinical presentation, and family history.Laboratory test results before treatment were also collected and analyzed, including the lowest platelet count, white blood cell count, hemoglobin level, mean platelet volume, platelet morphology, and bone marrow cytology.Bleeding tendency was measured using the World Health Organization (WHO) bleeding scale: grade 0, no bleeding; grade 1, petechiae; grade 2, mild blood loss (clinically significant); grade 3, gross blood loss; and grade 4, debilitating blood loss. 6

Genetic screening
Peripheral venous blood (5 mL) was collected from the two patients and their respective family members using an ethylenediaminetetraacetic acid (EDTA) anticoagulant tube that was then sent to Beijing Kangxu Medical Laboratory for genetic testing.Mutations in ANKRD26 were detected by high-throughput second-generation sequencing and confirmed by Sanger sequencing.To screen for previously reported mutations, the sequence was compared to reference sequences using HGMD pro, PubMed, Clinvar, and other databases.Single nucleotide polymorphisms (SNPs) were excluded from the ESP, 1000 genome, database and EXAC databases.

Treatment evaluation
We retrospectively analyzed the treatment of two patients with immunosuppressive agents and the thrombopoietin receptor agonist eltrombopag.Both patients were initially diagnosed with immune thrombocytopenia(ITP) and administered immunosuppressive therapy.The type and dose of immunosuppressants, the total course of treatment, platelet counts, and bleeding features at week 4 and throughout the course of treatment were recorded.After immunosuppressive therapy was unresponsive or responded temporarily but could not be maintained and genetic testing indicated mutations in the ANKRD26 gene, the two patients were treated with oral eltrombopag.The dosage and total course of treatment, platelet counts and bleeding features at weeks 4 and 8, and at the end of the course of treatment, were recorded.The observation endpoint of the total treatment course was the end of drug withdrawal or until December 2020.Definitions of response to treatment for the study were as follows: Complete response (CR): A platelet count ≥ 100 × 10 9 /L and the absence of bleeding; Response (R): platelet count ≥ 30 × 10 9 /L and a greater than 2-fold increase in platelet count from baseline and the absence of bleeding; no response (NR): platelet count < 30 × 10 9 /L or less than 2-fold increase in platelet count from baseline or the presence of bleeding. 7Adverse events were graded using CTC-NCI version 5.0. 8

Clinical characteristics
Patient 1, a 1-year-old boy, presented with easy bruising (grade 1 WHO bleeding scale).No history of hematological malignancy was reported in the family; however, the father had a history of thrombocytopenia (76-91.5 × 10 9 /L) with no bleeding symptoms.Patient 2, a 1-year-old girl, presented with recurrent hemorrhagic spots(WHO bleeding scale 1).Similar to patient 1, no history of hematological malignancy was reported in the family, but the father had a history of thrombocytopenia (34-82 × 10 9 /L) with no bleeding symptoms (Table I).

Genetic results
Both patients had mutations in ANKRD26.Patient 1 harbored a previously reported mutation c.-140C>G in the 5'UTR, 9,10 inherited from his father.Patient 2 had a mutation c.-127A>T in the 5'UTR.The mutation c.-127A>T, first reported in 2011, 11 was inherited from her father.Both fathers had thrombocytopenia with no bleeding symptoms (Table II, Figure 1).

Treatment results
Both patients were initially diagnosed with ITP and treated with immunosuppressants.Patient1 had a severely low platelet count of 2 × 10 9 /L, which was treated with intravenous immunoglobulin-(IVIG), prednisone, dexamethasone, and rituximab for a total observation period of 3 months.Dosage: (1) IVIG: a single dose of 1.0 g/kg/1-2d.;(2) Dexamethasone: 0.6 mg/kg/d for 4 days, intravenously (IV), max40mg/d; Prednisone: initial dose of 1.5 mg/kg/d, dosage adjusted according to platelet count and bleeding symptoms; (3) Rituximab: 100 mg/week for 4 weeks.After treatment with IVIG, dexamethasone, prednisone, and rituximab, the platelet count was 22.4 × 10 9 /L at week 4, and the curative effect was considered unresponsive.After the second dose of rituximab, the platelet count rose briefly to 55 × 10 9 /L, but was not maintained, and decreased to 4.9 × 10 9 /L at week 6.The platelet count fluctuated from 4 × 10 9 /L to 51 × 10 9 /L throughout the 3 months observation period.Thus, the treatment efficacy was temporary response, but was not maintained.Patient 2 had a platelet count of 40 × 10 9 /L before treatment, with recurrent hemorrhagic spots and ecchymosis.Following her family's wish to initiate treatment, she received 15 months of discontinuous treatment with prednisone, including 7 months of irregular treatment in other hospitals.Dosage: initial dose 1.5 mg/kg/d, dosage adjusted according to platelet count and bleeding symptoms.After 4 weeks of treatment, the platelet count was 60 × 10 9 /L, which fluctuated from 40 × 10 9 /L to 60 × 10 9 /L throughout the 7 months of treatment.After 2 months of complete prednisone cessation, the platelet count dropped to 34 × 10 9 /L,  After the failure of immunosuppressive therapy and genetic testing revealed ANKRD26 gene mutations, both patients were administered oral thrombopoietin receptor agonist eltrombopag (25 mg/d).The total course of treatment for patient 1 was 2 months, with a platelet count of 29.8 × 10 9 /L before treatment, 18 × 10 9 /L and 11.3 × 10 9 /L at 4 and 8 weeks of treatment, respectively, showing no increase compared to the pre-treatment period.The total curative effect was considered no responsive, with no adverse events.After eltrombopag was discontinued, the platelet count was no longer monitored, and no bleeding symptoms were observed during follow-up.The patient 2 family decided to stop the administration of eltrombopag after 1 month of treatment, and the platelet count observed subsequently only increased from 40 × 10 9 /L to 55 × 10 9 /L after treatment, showing an unresponsive curative effect.No adverse events were observed during the treatment period, except for a small rash that resolved on its own(CTC-NCI grade 1).After discontinuation of the drug, the platelet count was monitored irregularly and fluctuated from 32 × 10 9 to 50 × 10 9 /L.No bleeding symptoms were observed (Table IV).

Discussion
ANKRD26-RT is a rare autosomal dominant disorder with no significant geographic or sex differences in its onset. 11We studied two Chinese pediatric cases, a boy and a girl, aged 1, and diagnosed with ANKRD26-RT and low platelet count of <50 × 10 9 /L, Table III.Therapeutic effect of immunosuppressor.the boy, had a platelet count as low as <10 × 10 9 /L.0][11][12] A clinical feature of this disease that has previously attracted attention is the increased risk of hematological malignancies, especially myeloid tumors.In this study, both patients and their family members had no history of hematologic malignancies; however, further followup is required because both patients were still very young.Apart from a normal white blood cell count, hemoglobin level, and platelet morphology, both patients had a normal mean platelet volume, which can potentially be a nonspecific distinguisher from some hereditary thrombocytopenia with abnormal platelet volume.Previous reports have shown that the platelet count is temporarily normalized during viral infection. 10,11However, this was not observed in patient 1, who had a viral infection at the beginning of the disease.However, his platelet count severely decreased, and whether this was related to the specific infectious pathogen remains unclear.It has been reported that bone marrow usually showed increased megakaryocytes and dysmegakaryopoiesis, and dystrophic forms consisted mainly of small, underdeveloped megakaryocytes and typical micromegakaryocyte.absence of features suggesting syndromic association. 14Finally, mutant molecular confirmation was performed to establish the diagnosis.Nevertheless, the mutations are sometimes difficult to identify, especially if the disease is mainly characterized by mild to moderate platelet reduction with no or slight bleeding, resulting in clinicians missing the inheritance source and correlation with the family history of thrombocytopenia.The main features that prompted our molecular examination were isolated thrombocytopenia, a normal platelet volume, and no response to immunosuppressive therapy or temporary response, but not sustained.We believe that these indicators suggest the need for genetic screening.ANKRD26 is the ancestor of a family of primate-specific genes termed POTE, 15 With regard to human bone marrow cells, ANKRD26 is expressed in megakaryocytes, and, to a lesser extent, in erythroid cells. 1,16Thrombocytopenia 2 (THC2) disorder, first described in two large families in Italy and North America, has a pathogenic gene locus located on the short arm of chromosome 10 (10p11.1-P12). 2,3After Pippucci et al. 1 revealed that THC2 was not caused by MASTL or ACBD5 defects, but was rather associated with ANKRD26 mutations, subsequent studies have reported that the specific pathogenesis is mainly due to the 5'UTR mutation of the ANKRD26 gene.This mutation has been shown to lead to the loss of RUNX1and FLI1 binding at the 5'UTR, resulting in the continued overexpression of ANKRD26, leading to abnormal activation of the ERK/MAPK pathway and impaired megakaryopoiesis. 170][21][22][23][24][25][26] In addition, a missense mutation c. 473A>G in the ANKRD26 exon region has been reported to segregate with thrombocytopenia 27 ; however, it has been proposed that this may not be the real pathogenic mutation, as this disease is caused by failing to silence ANKRD26 transcription rather than loss of protein function. 25

function
In this study, the final diagnosis in our two patients was established after mutations in ANKRD26were detected by genetic screening.Two mutations inherited from the parents were identified, namely ANKRD26 c.-140C>G in patient 1 and ANKRD26 c.-127A>T in patient 2. Mutations c.-140C>G and c.-127A>T have been previously reported to lead to thrombocytopenia, and the two fathers carrying the heterozygous mutation in this study had thrombocytopenia, which is consistent with theANKRD26-RT diagnosis.
Immunosuppressant therapy has been reported to be a no response or temporary increase but is ultimately unable to maintain platelet stability. 10,11,13In this study, our two patients were  Two pediatric cases of ANKRD26-RT 5 initially diagnosed with ITP and treated with immunosuppressants for 3 and 15 months, respectively.Patient 2 had a platelet count of 40 × 10 9 /L before treatment, with recurrent hemorrhagic spots and ecchymosis.The decision to treat relies largely on the frequency and severity of bleeding and the impact on quality of life 7 .Bleeding risk appears to increase when platelets are <20 × 10 9 /L, while patient 2 had a platelet count of >30 × 10 9 /L before treatment, with mild bleeding and no specific needs such as surgery.
Considering the degree of bleeding, we can choose to observe rather than treat it.However, she had recurrent hemorrhagic spots and ecchymosis, which affected the child's quality of life, and the family was anxious about this and strongly desired treatment.
Considering the preference of the patient's family, we chose to treat patient 2. A review of the entire course of treatment showed that immunosuppressive treatment was no response at week 4 and throughout the course of treatment in patient 2. For patient 1, the treatment was temporary response, but was not sustained.As immunosuppressants have certain toxic side effects, an accurate diagnosis would help choose the correct treatment plan which and avoid the excessive use of immunosuppressants.
Patients with hereditary thrombocytopenia usually require transfusion of platelets to improve platelet counts before surgery or other invasive procedures as well as in cases of significant spontaneous bleeding.The use of platelet receptor agonists helps to avoid the risks associated with platelet transfusion, such as exposure to acute reactions, transmission of infectious diseases, alloimmunity, and blood shortage.A previous study has reported that the platelet count in an adult patient with ANKRD26-RT can be successfully improved by short-term preoperative administration of eltrombopag at a dose of 50-75 mg/d instead of platelet transfusion. 4A phase II clinical trial has revealed that eltrombopag can safely and effectively increase the platelet count in patient with ANKRD26-RT, even though it was still observed that the degree of platelet response in patient with ANKRD26-RT was generally lower than that in other types of hereditary thrombocytopenia, such as MYH9 related and mBSS related thrombocytopenia.Most participants in the trial had a mild response (platelets were at least 2 times higher than baseline), and for most of those who did not have this response, the platelet count later increased after increasing the dose to 75 mg/d for 3 weeks. 5In our study, the two patients were administered oral eltrombopag at a dose of 25 mg/day for 8 and 4 weeks, respectively, but the treatment was no response.To evaluate the association of the degree of treatment response with eltrombopag dose, further clinical studies are needed.Patients with ANKRD26-RT are at risk of myeloid tumors, and the TPO/MPL pathway can be activated by thrombopoietin receptor agonists.Thus, the association risk of probable disease progression to myeloid tumors remains a major concern.No adverse events were observed in our two patients during the course of treatment, except for a small rash(CTC-NCI grade 1) that faded on its own in patient 2, with no other blood changes except for platelet count.This is consistent with the previous reports that the short-term use of eltrombopag is safe.However, the overall course of treatment in this study was not long and the number of cases was small.The safety of long-term eltrombopag use requires further investigation.
In conclusion, we report two Chinese pediatric patients with ANKRD26-RT who initially presented with mild bleeding accompanied by different degrees of platelet reduction, normal mean platelet volume, and megakaryocyte maturation impairment not obvious.The normal mean platelet volume can be distinguished by ANKRD26-RT from other hereditary thrombocytopenia with abnormal platelet volume; however, no other laboratory tests revealed differential markers.If patients clinically diagnosed with ITP do not respond to immunosuppressive agents, genetic testing is necessary to exclude hereditary thrombocytopenia.The short-term use of eltrombopag at a dose of 25 mg/day failed to effectively improve the platelet count for our two patients.However, the association of the degree of treatment response with dose was not characterized in the study.For patients with ANKRD26-RT, eltrombopag is safe for short-term use; however, its long-term effects and safety remain unclear.Our report complements data on the diagnosis and management of ANKRD26-RT disease in children.

Figure 1
Figure 1.chromatograms from direct sequencing of the ANKRD26 gene.The red arrow indicates the mutation position.(a) represents the mutation c.-140C>G of ANKRD26 in 5'UTR in patient 1.(b) represents the mutation c.-127A>T of ANKRD26 in 5'UTR in patients 2.
of the total course Temporary response, but not sustained No response PLT: Platelet, IVIG: Intravenous immunoglobulin.

Figure 3 .
Figure 3. Representative mutations in 5'UTR of the ANKRD26 gene associated with ANKRD26-RT.Position c-172 indicates the transcription start site (TSS).Green and blue areas represent RUNX1 and FLI1 binding sites.The red position represents the location of the mutation in our patient.

Table I .
Patients' clinical characteristics.

Table IV .
Therapeutic effect of eltrombopag.