Application of high‐throughput sequencing for hereditary thrombocytopenia in southwestern China

Abstract Background The aim of this study was to design and analyze the applicability of a 21‐gene high‐throughput sequencing (HTS) panel in the molecular diagnosis of patients with hereditary thrombocytopenia (HT). Methods A custom target enrichment library was designed to capture 21 genes known to be associated with HTs. Twenty‐four patients with an HT phenotype were studied using this technology. Results One pathogenic variant on the MYH9 gene and one likely pathogenic variant on the ABCG8 gene previously known to cause HTs were identified. Additionally, 3 previously reported variants affecting WAS, ADAMTS13, and GP1BA were detected, and 9 novel variants affecting FLNA, ITGB3, NBEAL2, MYH9, VWF, and ANKRD26 genes were identified. The 12 variants were classified to be of uncertain significance. Conclusion Our results demonstrate that HTS is an accurate and reliable method of pre‐screening patients for variants in known HT‐causing genes. With the advantage of distinguishing HT from immune thrombocytopenia, HTS could play a key role in improving the clinical management of patients.


| INTRODUC TI ON
Hereditary thrombocytopenias (HT) are a group of disorders characterized by spontaneous hemorrhage in the early postnatal period and excessive blood loss after trauma or surgery. 1,2 HTs represent thrombocytopenia and/or abnormal platelet function. 3,4 Due to the lack of specificity of clinical manifestations and screening methods, it is often misdiagnosed as immune thrombocytopenia (ITP). 5,6 Two important clinical characteristics for recognizing hereditary thrombocytopenia syndromes are the age of presentation and chronicity/ duration of symptoms. 7 In recent years, although significant progress has been made in the molecular pathogenesis of the disease, such as the discovery of abnormal gene expression in most patients, the pathophysiological mechanism of the disease is still unclear, and diagnosis is still difficult. In this study, we used high-throughput target gene capture sequencing technology to establish a liquid-phase capture chip of genes related to HTs in the form of a chip containing 21 genes known to be related to the disease. This microarray was used to detect these genes in children with a potential HT diagnosis. This study could provide a simple and feasible gene detection method for HT diagnosis in children in southwestern China, analyze the relationship between gene mutations and clinical characteristics, and provide a basis for the further study of pathophysiological mechanisms for HTs.

| Patients
Twenty-four patients (15 males, 9 females; age range, 1 month to 13 years) from 24 unrelated southwestern Chinese families were enrolled in this study. All patients had a bleeding history. Most patients suffered from mild bleeding symptoms including cutaneous bruising, bleeding, and epistaxis, in addition to more severe bleeding symptoms in a few. Among the 24 patients in this study, 12 had been diagnosed with persistent or chronic ITP and had undergone ineffective treatments. The study was approved by the ethics committee of Children's Hospital of Chongqing Medical University, and informed consents were obtained.

| Platelet counts and morphology
Platelet counts and morphology were studied in peripheral blood by sheath flow DC detection using the Sysmex XE-2100.

| DNA library preparation
For exome sequencing, we fragmented 1-3 μg of genomic DNA, extracted from each sample, to an average size of 180 bp with a Bioruptor Sonicator (Diagenode). Paired-end sequencing libraries then were prepared using a DNA sample prep reagent set 1 (NEBNext). Library preparation included end repair, adapter ligation, and PCR enrichment and was carried out as recommended by Illumina protocols.

| Bioinformatics analysis
After sequencing, the raw data were saved as a FASTQ format and then followed the bioinformatics analysis: First, Illumina sequencing adapters and low-quality reads (<80 bp) were filtered by cutadapt. After quality control, the clean reads were mapped to the UCSC hg19 human reference genome using BWA. Duplicated reads were removed using picard tools, and mapping reads were used for variation detection. Second, the variants of SNP and InDel were detected by GATK HaplotypeCaller, then using GATK VariantFiltration to filter variant, the filtered standard as follows: (a) variants with mapping qualities <30; (b) the total mapping quality zero reads <4; (c) approximate read depth <5; (d) QUAL < 50.0; (e) phred-scaled p-value using Fisher's exact test to detect strand bias >10.0. After above two steps, the data would be transformed to VCF format; variants were further annotated by ANNOVAR and associated with multiple databases, such as 1000 genome, ESP6500, dbSNP, EXAC, Inhouse (MyGenostics), HGMD, and predicted by SIFT, PolyPhen-2, MutationTaster, GERP++.

| Variants selected
In this course, five steps using to select the potential pathogenic mutations in downstream analysis: (a) Mutation reads should be more than 5, mutation ration should be no less than 30%; (b) removing the mutation, the frequency of which showed more than 5% in 1000 g, ESP6500,

| Sanger sequencing
Sanger sequencing has been used to validate variants of seven patients identified by the high-throughput sequencing. The primers used have been listed in Table 1. All 24 unrelated Table 4.

| Candidate variants and variant prevalence in 12 patients
In total, DNA samples from 24 patients were analyzed by a HTspecific HTS panel. Following post-sequencing bioinformatics analysis, candidate variants previously implicated in HT genes were observed in the patients. In total, 14 variants were noted in 12 patients, with a variant in a gene previously known to cause HT. One patient was observed with two variants in two different genes, and two patients were noted with two variants occurring within the same gene. Two variants were observed in a hemizygous state, and the others were observed in a heterozygous state.
Thirteen of the variants identified were missense variants affecting a single amino acid. In addition, one splicing variant was noted in patient 18 (VWF; c.2823-19G>C) (displayed in Table 5).

| Pathogenicity prediction and variant classification
Of the 14 variants noted across all patients investigated, one variant was classified as "pathogenic" and one as "likely pathogenic" when considering the ACMG consensus guidelines. The remaining 12 variants, without a positive prediction of pathogenicity, were classified as of "uncertain significance." A pathogenic variant was identified in   believed to lead to a broad range of congenital malformations, affecting craniofacial structures, skeleton, brain, viscera, and urogenital tract. 16 Hence, we consider that the variant we identified may be pathogenic.
Patient 10 was a rare case of a variant-type GT in which the pathogenic variant was c.50T>G (p.Leu17Arg) on ITGB3. This variant was newly discovered and derived from the patient's father.
According to Nurden's report, 17 the primary feature of variant GT is a subtle reduction of the number of GPIIB/IIIa receptor molecules on the surface of platelets; however, the platelet aggregation function is defective, and some patients have reductions in their platelet counts. This patient was a nine-month-old boy with mild bleeding symptoms and a severely low platelet count.
Patient 11 had two unreported missense variants c.295C>T (p.Ar-g99Trp) and c.4169C>T (p.Ser1390Leu) on the NBEAL2 gene, which related to Gray platelet syndrome. 18,19 This patient was a 4-year-old boy who had normal or slightly reduced platelet size rather than large platelet size. However, myelofibrosis or splenomegaly was not present. Patient 21 was a 13-year-old girl with mild bleeding symptoms since early life and manifested as menorrhagia when she came to our hospital. We found that she had a known risk variant in the ADAMTS13 gene. 20 However, she did not present with hemolytic anemia or nervous system symptoms, and we noticed that her plate-

ACK N OWLED G EM ENT
We would like to thank all the patients who volunteered for this study.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest associated with this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.