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Introduction

The Bernard-Soulier syndrome (BSS) is an extremely rare congenital disease that is characterized by macrothrombocytopenia and defective binding of platelets to the von Willebrand factor (vWF), which participates in the adherence of platelets to the vascular subendothelial layer (1). BSS disease pathogenesis lies in the expression of vWF receptor via the glycoprotein Ib-IX-V (GPIb-IX-V) complex (2). The GPIb-IX-V complex consists of four subunits, glycoprotein (GP)Ibα, GPIbβ, GPIX and GPV, which are encoded by glycoprotein Ib platelet subunit alpha (GP1BA), glycoprotein Ib platelet subunit beta (GP1BB), GP9 and GP5(3). Mutations that occur in GP1BA, GP1BB and GP9 may lead to BSS (4). The major subunit, GPIbα, holds an essential ligand-binding site for vWF within its N-terminal domain, and this features the leucine-rich repeat (LRR) (5,6). Pathogenic mutations in its GP1BA gene are classically inherited as a biallelic, autosomal recessive trait (4). A milder phenotype caused by monoallelic autosomal dominant inheritance that shares characters with BSS, including macrothrombocytopenia, has been previously reported (6-12). However, this aforementioned phenotype does not lead to severe bleeding or serious surgical or obstetric hemorrhage that is commonly seen in BSS. Therefore, the autosomal dominant variant of BSS can be easily misdiagnosed as immune thrombocytopenia (ITP) and managed with intravenous immunoglobulin and glucocorticoids, which are used as a first-line treatment for ITP (13,14).

In the present case report, sequence analysis performed on an 18-month-old girl identified an NM_000173 c.97T>A (p.Cys33Arg) (p.C33R) heterozygous missense mutation in GP1BA. This specific mutation has not been previously reported and may further expand the information regarding mutations in BSS.

Case report

An 18-month-old girl with thrombocytopenia consulted the Hematology Oncology Center, Beijing Children's Hospital, Capital Medical University (Beijing, China). A period of six months prior to admission to the department, the patient underwent a physical examination, which revealed a baseline platelet count of 31x109/l. Since this examination, the patient had no bleeding symptoms, including easy bruising, epistaxis, gingival bleeding and other symptoms as a result of internal bleeding. A peripheral blood smear indicated giant platelets and a reduction in platelet count. vWF antigen (vWF Ag) levels was 94.6% (42.0-140.8%), aggregation response of platelets after adenosine diphosphate (ADP) was 55% (55-90%), collagen was 62.1% (55-90%), arachidonic acid was 82.6% (55-90%) and ristocetin was including low concentration and [76.1% (55-90%); low concentation] and [2.6% (<3%); standard concentration], all of which were within the normal range. CBC, peripheral blood smear and platelet aggregation were performed on the patients parents, and the patients father shared a similar phenotype, including enlarged platelet size and normal aggregation rate of platelet as follows: Aggregation response of platelets after adenosine diphosphate (ADP) was 56.4% (55-90%), collagen was 72.3% (55-90%), arachidonic acid was 85.7% (55-90%) and ristocetin, including low concentration and standard concentration, was 66.7% (55-90%) and 1.3% (<3%) respectively. Considering the probability of inherited thrombocytopenia and abnormal morphology of platelets, DNA samples for genetic analysis were collected from her parents. Written consent from the patient's parents was obtained at the time of sample collection. The present study was performed with approval from the Ethics Committee of Beijing Children's Hospital (Beijing, China; reference no. 2020-Z-113).

Approximately 2 ml peripheral blood of the patient, parents and one sibling were collected in new EDTA containing tubes (as anticoagulant) for genetic analysis. Exome sequencing of germline DNA samples was performed using a QIAamp DNA Mini kit (Qiagen, China Co., Ltd.), according to the manufacturer's protocol. Selected targeted genes that were associated with thrombocytopenia were analyzed using a gene capture strategy with a GenCap custom enrichment kit (MyGenostics, Inc.), following the manufacturer's protocol. The final enriched sequencing libraries were sequenced for paired-end reads of 150 bp using Illumina NovaSeq 6000 platform (Illumina, Inc.). The identified variants were annotated using ANNOtate VARiation (ANNOVAR; http://annovar.openbioinformatics.org/en/latest/).

The sequencing analysis identified a heterozygous nucleotide substitution in the GP1BA gene (NM_000173: exon2:c.97T>G). An amino acid of cysteine 33 was substituted with arginine in the N-terminal domain of GPIbα protein on the platelet membrane. A validation of GP1BA sequencing was performed on the patient's parents and one sibling. The pedigree analysis (Fig. 1) indicated an autosomal-dominant mode of inheritance. The individuals father carried a similar heterozygous p.C33R variant, while her mother carried a wild-type sequence (Fig. 2). The structural effect of the sequence variant was predicted to be deleterious via in-silico analysis (Fig. 3), using the following software with default parameters: Mutation Taster (http://www.mutationtaster.org/), Sorting Intolerant From Tolerant (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and Genomic Evolutionary Rate Profiling (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html). According to the ACMG guidelines (15), the variant was classified as ‘likely pathogenic’, which confirmed BSS.

Figure 1 Proband's (V-1) family pedigree indicates mild bleeding and thrombocytopenia. Black star indicates the analysis of family members by next-generation sequencing.

Figure 2 Sanger sequencing showing the nucleotide of GP1BA at position 97 in the reference sequence as T. The proband and her father were heterozygous for the variant C, and her mother was wild-type. The red arrows indicate the site of the new mutation, and the blue arrow indicates the wild-type sequence. GP1BA, glycoprotein Ib platelet subunit alpha.

Figure 3 Crystal structure of the human N-terminal domain (~300 aa) of GPIbα. Cys33 of our patient was presented by colored spheres in the conserved N-terminal flank (amino acid 1-35), which had a role in binding to von Willebrand factor. GP, glycoprotein.

Discussion

In the current case report, a novel heterozygous c.97T>A substitution was reported in the GP1BA gene, which causes a Cys33Arg amino acid substitution. The mutation was annotated with ANNOVAR and the results demonstrated that it has not been recorded in the GnomAD (https://gnomad.broadinstitute.org/), 1000 Genomes (http://www.1000genomes.org/), ExAC database (16), the human gene mutation database (http://www.biobase-international.com/product/hgmd; http://www.hgmd.cf.ac.uk/ac), OMIM (https://www.omim.org/) or ClinVar databases (https://www.ncbi.nlm.nih.gov/clinvar/). To date, ~60 variants of GP1BA encoding GPIBα protein have been identified, and the majority of these are homozygous and contribute to symptoms of BSS (6,17). However, the symptoms of individuals in the pedigree of the current study seemed milder than the symptoms of individuals with classic homozygous BSS. Platelet aggregation of the present patients with heterozygous BSS (including the response to the low concentration of ristocetin) appeared normal despite giant platelets being identified in the results of the peripheral blood smear.

Mutations in BSS may be missense, nonsense, frameshift, insertions or deletions, and these mutations can cause abnormality in GPIBα protein expression (18,19). The dominant inheritance of BSS remains relatively rare as only six variants have been previously identified, including the Bolzano variant (p. Ala172Val), which is the most frequent type in Italy (12). The remaining four heterozygous mutations were described in single families (6-12).

The phenotype of monoallelic BSS, which is the same as that of the biallelic BSS in the degree of symptom presentation, may vary based on different genotypes and relevant changes in the expression and function of GPIbα (2). Mutations in the patient outlined in the current case report and her father were located in the conserved N-terminal flanking domain. The domain lies ahead of the LRR region of GP Ibα, which serves a role in the orientation of charged residues (6). All previously reported variants (p.Ala172Val, p.Asn57His, p.Tyr70Asp, p.Leu73Phe, p.Asn150Ser and p.Leu59Arg), which are located in the LRR region (amino acids 35-200), may lead to macrothrombocytopenia. This is due to the abnormal interaction of the cytoplasmic end of GPIbα protein with the actin-binding protein, which controls the normal process of megakaryocyte maturation, demarcation membrane system production and the size of the platelets (20,21). While the conserved N-terminal flanking region of GPIbα, where the novel mutation was discovered in the pedigree outlined the present study, may interact with C-terminal flanking (amino acid 201-268) and exhibit a weak influence on the binding of GPIbα to vWF that is induced by ristocetin or botrocetin, the mutations within the LRR caused a more significant change in the formation of GPIbα as well as a pronounced disturbance in ligand binding (22).

BSS is commonly misdiagnosed with ITP and is subsequently treated with futile therapies, including glucocorticoids and immunoglobulins (14). BSS is a rare bleeding disorder that is accompanied by thrombocytopenia, and symptoms are less pronounced in the monoallelic phenotype of the disease (6,8). BSS shares similar clinical characteristics with ITP, such as mucocutaneous bleeding, negative family history and normal platelet aggregation (14). However, the identification of giant platelets in a peripheral blood smear may help clinicians make an accurate diagnosis. In the current case report, the patient and her father shared the same mutation and similar mild manifestation. Her mother carried a wild-type genotype and did not exhibit macrothrombocytopenia and bleeding. For those suspected of having BSS, gene sequencing should be performed, and symptoms such as abnormal levels of bleeding should be investigated throughout the family to encourage early diagnosis and subsequently, the establishment of effective treatment.

In the present study, platelet immunophenotype and vWF-cofactor activity analysis (binding of VWF to recombinant mutated GPIb) could not be performed. Therefore, whether this novel dominant variant contributes to the reduced expression of GPIb-V-IX and whether the inactive interaction between platelets and vWF was indicated, which is commonly identified in classic BSS, was not confirmed. Identification of the effect of specific mutations on the structure and function of GPIbα protein and the pathogenesis to a milder phenotype is required in future studies.

A novel autosomal dominant inheritance mutation in GP1BA was identified in a family with mild bleeding symptoms and macrothrombocytopenia. This monoallelic mutation that occurs in the N-terminal flanking domain has, to the best of our knowledge, not been previously reported. Due to common misdiagnosis, gene sequencing is considered essential and urgent for diagnosing suspected patients who exhibit a blood smear showing giant platelets, with BSS.

Acknowledgements

The authors would like to thank Dr Lulu Mao and Mr. Xinwen Fu from MyGenostics, Inc. for technical support in NGS work and drawing the crystal structure.

Funding

Funding: The present study was supported by the National Natural Science Foundation of China (grant no. 81970111), the Beijing Natural Science Foundation of China (grant no. 7192064), the Pediatric Medical Coordinated Development Center of Beijing Municipal Administration of Hospitals (grant no. XTZD20180205) and the National Science and Technology Key Projects (grant no. 2017ZX09304029004).

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

JM was involved in the collection and critical analysis of clinical data and wrote the manuscript. RW contributed to the designing the study, supervision and critical review of the manuscript. ZC contributed to the supervision and critical analysis of laboratory data, including the interpretation of NGS results. GL performed the platelet aggregation. HG was involved in the collection and sorting of the clinical data. RW and ZC confirmed the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Beijing Children's Hospital (Beijing, China; reference no. 2020-Z-113) and written consent from the patient's parents was obtained.

Patient consent for publication

The patient's parents have provided consent for publication.

Competing interests

The authors declare that they have no competing interests.

References