22q11.2 deletion syndrome (22q11.2DS) presents with various clinical symptoms and several congenital anomalies, including congenital heart diseases, palatal abnormalities, immune deficiencies, craniofacial features, and developmental delay. Bernard-Soulier syndrome (BSS) is an extremely rare autosomal recessive genetic disease, characterized by giant platelet cells, thrombocytopenia and prolonged bleeding time. BSS is caused by a problem with glycoprotein Ib-IX-V, a platelet receptor complex that attaches to von Willebrand factor (vWF). The gene encoding glycoprotein Ib-beta (GPIbb) protein exists on chromosome 22q11.2. Because the majority of 22q11DS patients have a heterozygous deletion of the GPIbb gene, they are not usually diagnosed with BSS, which is expressed as an autosomal recessive disorder. We report a case of Bernard-Soulier syndrome diagnosed in a patient with 22q11.2 deletion syndrome who showed a severe bleeding tendency. Abnormal platelet aggregation tests and gene mutations confirmed by gene sequencing confirmed the presence of BSS. The Institutional Review Board of the Asan Medical Center approved this case report (IRB approval no: 2023-1042).
A 5-year-old Korean boy presented with headache and increased head circumference (>99%). His symptoms gradually worsened over 1 week. He had no history of trauma, and physical examination revealed no head in-juries. At 1 month of age, he underwent heart surgery for an atrial septal defect and a ventricular septal defect. At the time, his platelet count was around 80-100×103/mL. He underwent surgery to repair a cleft palate at age 4. Thrombocytopenia had been present since birth, and platelet transfusion was performed for surgeries. Addi-tionally, he had a history of frequent infections, speech impairment, and facial dysmorphism (hypertelorism, narrow palpebral fissure, bulbous nose, hypernasal voice). Conventional karyotyping confirmed normal chromosomes, but fluorescent in situ hybridization (FISH) confirmed del(22)(q11.2q11.2)(TUPLE1-), and he was diagnosed with 22q11.2 deletion syndrome.
At this presentation, peripheral blood tests showed thrombocytopenia (77×103/mL) and anemia (Hb 8.5 g/dL), and white blood cells were normal. Mean platelet volume could not be measured, but large platelets were often observed on microscopy. The bleeding time, prothrombin time, partial thromboplastin time, thrombin time, and fibrinogen levels were all normal. Subgaleal hemorrhage was confirmed on a brain computed tomography (Fig. 1), and there were no findings of skull fracture or bleeding at other sites. Additional tests were conducted for the possibility of a bleeding disorder. Factor VIII level, Von Willebrand factor antigen, and ristocetin cofactor activity were normal. In the platelet aggregation test, using a platelet function analyzer-100, normal plasma platelet aggregation was observed when exposed to ADP, collagen and epinephrine, but a hypoaggregation response was shown to ristocetin (maximal aggregation of 16.4%). These results suggested the diagnosis of Bernard-Soulier syndrome.
Multiplex ligation-dependent probe amplification (MLPA) was performed to analyze the patient’s DNA sequence. On one side of the patient’s chromosome, a 1.9 Mb deletion containing the low-copy repeats (LCR) A and LCR B regions of the chromosome 22q11.2 region was identified, including the GPIbb gene. This refers to the child’s previously known 22q11.2 deletion syndrome. On the other side of the chromosome, the mutation c.[124C>T](p.[Arg42Cys]) of the GPIbb gene was confirmed (Fig. 2). Family genetic testing confirmed that the mother was a carrier with the same mutation.
Finally, he was diagnosed with BSS accompanied by 22q11.2DS, and received multiple platelet transfusions. After 2 weeks of hospitalization, the headache and head circumference improved, and he was discharged without additional bleeding. Brain computed tomography also showed improvements in the subgaleal hemorrhage (Fig. 3). Later, when he was 10 years old, he was admitted to the hospital with a hematoma in his left buttock caused by a fall. Platelet was transfused, but the bleeding did not stop, so embolization was done instead. He also has scoliosis requiring surgery, but he is under observation without surgery due to the high risk of bleeding.
22q11.2 deletion syndrome, previously called DiGeorge or velocardiofacial syndrome, is now known to show highly variable phenotypes . 22q11.2 DS is rare but is one of the most common chromosomal microdeletion disorders. The estimated live-birth prevalence has a wide range from 1.7 to 4.7 per 10,000 live births [1,2]. 22q11.2DS is an autosomal dominant genetic disease inherited with a 50% probability, but 90% of cases are known to occur de novo.
Our patient had congenital heart disease (ventricular septal defect, atrial septal defect), cleft palate, frequent infections, facial dysmorphism (hypertelorism, narrow palpebral fissure, bulbous nose, hypernasal voice), developmental delay, and scoliosis as clinical symptoms. His parents had normal chromosomes, so he was a de novo case.
22q11.2DS patients often show thrombocytopenia, and the hematological changes seen in 22q11.2DS may be due to the genetic disorder itself or may be secondary findings accompanying clinical findings such as immunodeficiency [3,4]. Some 22q11DS patients have a decrease in platelet count and an increased platelet size and volume . Most of them are asymptomatic and do not show a severe bleeding tendency. Our patient also had thrombocytopenia of 100×103/mL from birth and was misdiagnosed with idiopathic thrombocytopenic purpura (ITP) and received intravenous immunoglobulin (IVIg) treatment. Although his platelet size was not quantitatively measured, microscopy revealed the presence of large platelets. In the absence of severe thrombocytopenia, subgaleal hemorrhage without trauma suggested the possibility a bleeding disorder. Additional tests confirmed the reduced response to ristocetin, an agonist to stimulate vWF-platelet glycoprotein Ib interactions, that is not corrected by the addition of normal plasma in a platelet aggregation study. This characteristic is consistent with findings observed in BSS. Previously, cases of BSS in which platelet aggregation was reduced by ristocetin were reported in patients with 22q11DS [6,7]. However, it was also reported that platelet aggregation could be reduced due to heterozygous loss of platelet GPIbb protein by chromosome 22q11.2 deletion .
Subsequently, flow cytometry has been used as a confirmatory test for BSS. Marked reduction of glycoprotein can be confirmed through flow cytometry analysis using platelet glycoprotein specific monoclonal antibodies. However, because BSS is extremely rare, flow cytometry is often difficult to perform. Instead, many causative genes have been identified, so when clinical suspicion is present, the diagnosis is often confirmed through genetic testing for platelet glycoprotein. Therefore, we also used genetic analysis using Multiplex Ligation-dependent Probe Amplification (MLPA) to diagnose BSS. To detect mutations including gene deletions/duplications, Southern Blot and FISH techniques have been widely used. However, these methods are time consuming and have limitations in finding small intragenic rearrangements. However, MLPA is much faster and cheaper, and has high resolution that can detect even single exon deletion/dupli-cations, so MLPA has recently been preferred for detection of gene mutations.
As a result, in one chromosome, a large deletion of 22q11.2, including the GPIbb gene region, was identified. In the other chromosome, a missense mutation c.[124C>T](p.[Arg42Cys]) in the GPIbb gene was identified. This is a novel gene mutation that has not been reported before. So, we confirmed that there were deletions and mutations in both alleles of the GPIbb gene. We assumed that the mutated GPI transcripts were improperly translated, preventing platelet glycoprotein Ib from binding to vWF and causing subgaleal hemorrhage. To establish the pathogenicity of the novel mutation in the GPIbb gene that has been found, it will be necessary to conduct molecular confirmation using flow cytometry and do further study. Nevertheless, based on the patient’s clinical symptoms and the observed hypoaggregation response to ristocetin in the platelet aggregation test, it may be inferred that the identified missense mutation c.[124C>T](p.[Arg42Cys]) is very probable to represent a pathogenic variation of BBS.
There are a few previous case reports of combinations of haploinsufficiency of the GPIbb gene by chromosome 22 microdeletion and allelic mutation of chromosome 22 [6,9]. However, these reports only molecularly confirmed the 22q11.2 deletion and did not confirm the mutation of the GPIbb gene. Our report is the only case report that confirmed the GPIbb gene mutation and obtained family genetic test results.
In conclusion, we report a patient with 22q11.2 deletion syndrome who also had a GPIbb gene mutation, which is associated with Bernard-Soulier syndrome. This case highlights an association of BSS with 22q11.2DS. Although bleeding events in 22q11.2DS patients are rare, physicians should consider the possibility of BSS and perform platelet aggregation tests or DNA sequencing if bleeding symptoms are observed. Proper preparation for hemostasis should be undertaken if surgical procedures are planned for these patients.
The authors have no conflict of interest to declare.