Application of High-Throughput Sequencing in the Diagnosis of Inherited Immune-Thrombocytopenia from Children Chronic/Refractory ITP

BACKGROUND: Inherited thrombocytopenias are a group of hereditary diseases with reduced platelet counts and associated bruising and bleeding as the main clinical manifestations. These entities may be hard to distinguish from ITP, particularly difficult immune thrombocytopenia complicates primary immunodeficiency and immunological treatments are effective in increasing the platelet count. Then when the course of thrombocytopenia is prolonged and other abnormalities, eg. infection, either are non-existent or subtle, distinguishing such diseases from ITP may be clinically almost impossible. However, in order to carry out proper disease management, accurate diagnosis is very necessary and urgently needed, especially in childhood.

OBJECTIVES: To evaluate: 1) the detection rate of inherited immune thrombocytopenia by high-throughput, next-generation sequencing (NGS) from children with apparent chronic and refractory ITP, and 2) the value of NGS in screening and diagnosis of inherited "immune" thrombocytopenia.

METHODS: We retrospectively collected 245 cases of chronic and refractory ITP in children with transient response to Intravenous Immunoglobulin (IVIG) and/ or glucocorticoid and/ or other immunosuppressive therapy, all of whom underwent genetic testing from April 2016 to April 2019. Their clinical data were systematically recorded and analyzed. We introduced a high-throughput, NGS platform into the routing diagnosis of those patients and analyzed the gene-sequencing results. We compared the differences between patients with positive gene mutations and those who carried suspected gene mutations. All subjects and their legal guardians gave written informed consent to the investigation.

RESULTS: Sixteen patients were excluded as their final diagnosis was malignancy, aplastic anemia (AA), or myelodysplastic syndrome (MDS). Among the remaining 229 cases, 32 patients (14%) received a genetic diagnosis. Twenty-five patients (11%) had pathogenic mutations in 12 genes including CASP10(2), WAS(12), LRBA(1), CARD(1), ITGA2B(2), ITGB(1), CD36(1), NFKB2(1), NBEAL2(1), UNC13D(1), KMT2D(1), TNFRS13B(1) known to be included in lymphoproliferation or autoimmunity, whereas 7 patients (3%) carried a suspected pathogenic variant in 6 genes including: GATA(1), MYH-9(1), PTPN-11(1), RUNX(1), SLX4(2), TUBB1(1) that had not been reported in the context of autoimmune diseases. Among the 25 patients with known mutations, 16 patients (7%) could be definitely diagnosed as inherited immune thrombocytopenia and formed the Diagnosed Group (DG) according to their phenotype, inheritance and pathogenicity of the mutated gene, while 9 cases in this category and 7 patients who carried probable pathogenic variants constitute the Suspected Diagnosed Group (SDG). We compared their clinical and laboratory phenotype with the biggest difference identified in age of onset (median: 4.08 months in DG vs 54.00 months in SDG, P =0.002). Other variables analyzed included duration of time with misdiagnosis (median: 13.5 months vs 26.0 months, P=0.430), baseline platelet count (median: 6×10⁹/L vs 5×10⁹/L, P=.0.282), level of IL-4 (median: 0 vs 0, P=0.232), level of IL-6 (median: 13.32 vs 7.48 P=1.000), bleeding severity (without any bleeding: 1 vs 0; merely petechia/ecchymosis: 3 vs 6; bleeding in skin and one another location: 6 vs 4; bleeding in more than 2 location: 2 vs 2. P=0.542) and rate of identification of autoimmune antibodies between the two groups (P=0.662).

CONCLUSIONS: Definite or suspected genetic etiologies consistent with inherited immune thrombocytopenia were identified in approximately one-seventh of cases of apparent chronic ITP. These cases would have been classified as "routine" cases of childhood ITP based on response to standard first-line ITP treatments and the absence of overt other findings. Eventually, their chronicity would have increased suspicion of an underlying etiology and the correct diagnosis made. The definite diagnosis group and the suspected group were identical clinically and in laboratory testing in every way except for age of onset suggesting that the suspected group was also likely inherited immune thrombocytopenia. Wide-ranging genetic screening (NGS) should be offered in children chronic/refractory ITP. The genetic findings have prognostic significance and may guide the choice of a targeted treatment in the future.