Immune thrombocytopenic purpura (ITP) is one of the most common hemorrhagic disorders in childhood, often caused by an acute self-limiting event. However, 30% of these children develop chronic ITP. Identification of the underlying causes in ITP is an important challenge. Inherited thrombocytopenia (IT) is a rare, underdiagnosed disease, included among the chronic platelet disorders. Next-Generation-Sequencing (NGS) could be an efficient way of discovering potential IT-associated mutations in children with chronic ITP.

The purpose of this retrospective study was to investigate children with chronic ITP using a targeted NGS, in order to identify IT-associated mutations.

Between June 2017 and April 2020, mutational screening by a targeted NGS was performed on 19 children, either with a familial history of IT [4 unrelated patients (pts)], or with chronic ITP (15 pts), after all other causes of thrombocytopenia were excluded. Nineteen relatives were also investigated. This study was carried out in collaboration with the Laboratory of Genetics, IRCCS Burlo Garofolo in Trieste, that developed a targeted NGS method for the simultaneous analysis of 28 IT genes. The cost of the NGS tests were supported by the public healthcare service.

We retrospectively divided our cohort of 19 pts, into three subgroups: Group I included 4 unrelated pts with familial IT; Group II consisted of 6 pts with chronic ITP and a clinical history and/or laboratory features associated with familial IT; and, Group III included 9 pts with chronic ITP refractory to several treatments (Table 1). The median age at the initial diagnosis of thrombocytopenia was lower in Group I than in Groups II and III (19/12 years vs 1310/12 years and 9 years, respectively, p=0.33). The median time between the diagnosis of thrombocytopenia, and the time of the study, was shorter in Group I compared to Groups II and III (11.7 months vs 45.3 and 51.7 months, respectively, p=0.16). Median platelet count at the disease onset was lower in Group III than in Groups I and II (21 x 109/L vs 99 x 109/L and 38 x 109/L, respectively, p=0.28). The median MPV values were 12.5 fL, 9.85 fL and 8.8 fL in Groups II, III, and I respectively. Bleeding symptoms requiring treatment were present at diagnosis in 1/6 (16%) and in 5/9 (55%) children of Groups II and III, respectively. Genetic variants, usually detected in IT, were found in heterozygosity in all children in Groups I and II, and in 7/9 (78%) in Group III. Two out of 4, 2/6 and 2/9 children in Groups I, II, and III, respectively, presented ≥2 variants. Among the 4 children of Group I, ANKDR26 variant was found in 2 pts, together with GP1BA and NBEAL2 (pt#1) and TUBB1 (pt#2). ANKDR26 variant was also recorded as a single mutation in their relatives. Two different variants involving GP1BA (c.98T>A and c.515C<T) were detected in the remaining 2 children of Group I and in their relatives. Pt #4 with GP1BA c.515C>T mutation with mild macrothrombocytopenia had relatives with a previous diagnosis of monoallelic Bernard-Souliers syndrome.

As shown in table 1, ABCG8, ACTN1, ETV6, GP1BA, MYH9, SLFN14, or WAS variants, found in combination in 2 pts (pt#5, pt#8), were also detected in the children in Group II, as well as, at least one of the relatives (for a total of 7 cases). ABCG5, ABCG8, ETV6, FLNA, GP1BA, NBEAL2, or SLFN14 were found as variants in patients of Group III. The peripheral blood smear evaluation confirmed the diagnosis of grey platelet syndrome with two NBEAL2 mutations in pt #16. SLFN14, as a single variant, was associated with macrothrombocytopenia in one pt (#10). Three pts (#5, #6, #17), with variants of ABCG8 had hypercholesterolemia. In the cohort of pts with chronic ITP, 4 (#12, #13, #14, #15) had relatives with thrombocytopenia, and 2 (#11, #13) had a familial history of hematological malignancies. Segregation analysis in families, and functional studies to evaluate the pathogenic role of the variants reported, are still in progress.

The clinical significance of IT-associated mutations in chronic ITP is uncertain, and yet to be clarified. However, our experience has shown that an in-depth clinical history, and accurate peripheral blood smear examinations, are important to better characterize chronic ITP in children. A targeted NGS method for the simultaneous analysis of different IT genes, has demonstrated to be an effective approach to explore in-depth the IT-associated mutations in children with chronic ITP, refractory to treatment.

Disclosures

Giona:Novartis: Research Funding; Takeda: Speakers Bureau; Sanofi Genzyme: Research Funding, Speakers Bureau.

Author notes

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Asterisk with author names denotes non-ASH members.