Novel variants in FERMT3 and RASGRP2—Genetic linkage in Glanzmann-like bleeding disorders

2.1 Clinical symptoms and phenotype

Index patient 1.II.5 is the fifth child of asymptomatic Libyan parents who reported not to be consanguineous (Figure 1A). The eldest brother died at the age of 7 days due to neonatal sepsis, whereas the younger three siblings are healthy. The 4-year-old female patient with failure to thrive had a marked history of frequent mucocutaneous bleeding tendency, predominantly epistaxis, gum bleedings, and hematomas. After surviving neonatal sepsis, she suffered from recurrent nonpussing bacterial infections and chronic anemia, aggravated by repetitive acute blood loss, which regularly demanded antibiotic treatment and red blood cell transfusions. Severe bleeding episodes could hardly be overcome by tranexamic acid or desmopressin, but always terminated after platelet transfusion. She successfully underwent matched unrelated-donor hematopoietic stem cell transplantation at the age of 9 years following myeloablative conditioning with thiotepa (8 mg/kg), treosulfan (42 g/m²), fludarabine (150 mg/m²), and antithymocyte globulin (60 mg/kg). Prophylaxis of graft-versus-host disease was provided with methotrexate and cyclosporine A. Neutrophil engraftment was recorded at day 18 leading to discharge of the patient at day 34. Under complete donor chimerism, neither bacterial infections nor bleeding episodes occurred, and spleen size declined significantly.

The 15-year-old female index patient 2.II.2 is the second child of asymptomatic, consanguineous parents (first-degree cousins) originating from Turkey (Figure 1B). Since early childhood, she suffered from a pronounced mucocutaneous bleeding tendency, in particular epistaxis, gum bleedings, hematomas, and prolonged menstrual hemorrhage. An adenoidectomy and a surgical abscess incision were complicated by diffuse postinterventional bleeding. The patient repetitively needs oral or even intravenous iron substitution due to secondary iron deficiency anemia. Perimenstrual treatment with tranexamic acid or desmopressin did not lead to significant improvement of the bleeding tendency. Recommended prophylaxis with an oral contraceptive to reduce menstrual bleeding episodes has been refused by her parents so far. The elder sister, 2.II.1, reported prolonged menstrual bleedings without additional bleeding symptoms.

2.2 Laboratory findings and functional assays

Accessible main laboratory findings of the investigated index patients and family members are summarized in Table 1. In all the investigated individuals, the platelet count and mean platelet volume were normal. The whole blood count of patient 1.II.5 showed marked leukocytosis with an elevated neutrophil count. PFA-100^® (platelet function analyzer) closure times and light transmission aggregometry were significantly pathologic in both index patients and, to a milder extent, in other members of family 2, while von Willebrand and plasmatic coagulation factors were normal. Anemia was consistent with blood loss and secondary iron deficiency. Slightly reduced factor XI activity was recorded for two individuals.

For both index patients, flow cytometric analysis of platelets proved normal expression of integrins α_IIb (CD41a) and β₃ (CD61), which form the fibrinogen receptor (Table 1), of the von Willebrand receptor GPIb/V/IX, and of the other investigated platelet receptors (data not shown).17 However, conformational change, and thus, activation of α_IIbβ₃ detected by PAC-1 antibody binding was diminished in both index patients upon low- (2 µM) and optimal-dose (5 µM) adenosine diphosphate (ADP) as well as thrombin receptor activating peptide 6 (TRAP6) treatment, though to a lesser extent (Figure 1C; data not shown). In contrast, P-selectin (CD62P) neo-expression upon TRAP6 was not impaired in patient 1.II.5 (data not shown), whereas CD62P neo-expression upon ADP treatment was clearly, and upon TRAP6 treatment, slightly reduced in patient 2.II.2 (Figure 1C).

2.3 Genetic analysis and variant assessment

Since the clinical phenotype and the functional assays suggested Kindlin-3 deficiency in patient 1.II.5, we sequenced the coding region of FERMT3 (NM_178443.2) and found a novel homozygous variant, c.1185delT (p.D397Tfs*29), leading to a premature stop codon and intracellular lack of the truncated protein (Figure 1D). Both parents 1.II.1 and 1.II.2 were heterozygous for the detected variant (Figures 1A and 1D). No variant was discovered in RASGRP2 (NM_153819.1) (data not shown).

In patient 2.II.2, we identified two homozygous variants in FERMT3 and RASGRP2 by targeted next generation sequencing, as previously described.18 Both variants were confirmed by Sanger sequencing (Figure 1E). The novel missense variant c.119C > T (p.S40L) in FERMT3, classified as a variant of unknown significance after applying in silico prediction tools (MutationTaster2: polymorphism; sorting intolerant from tolerant [SIFT]: tolerated; Polyphen-2: benign), cosegregated with the clinical phenotype in family 2 (Figures 1B and 1E). The variant c.742G > C (p.G248R) in RASGRP2 is a novel variant affecting the same amino acid position as that in a previously reported distinct missense variant (p.G248W).14 After applying in silico prediction tools (MutationTaster2: disease causing; SIFT: deleterious; Polyphen-2: probably damaging) and proving cosegregation in family 2, we classified this variant as likely pathogenic (Figures 1B and 1E).

We then verified the functional impact of the detected variants on intracellular platelet signaling in the two index patients by Rap1 pull-down assays, as previously reported.13 Normal Rap1 activation upon ADP and thrombin treatment confirmed functional signaling in patient 1.II.5. In contrast, ADP treatment showed absent Rap1 activation in patient 2.II.2, and hence, proved specifically impaired CalDAG-GEFI-mediated signaling (Figure 1F).13 With these results, we considered the RASGRP2 variant as causative in 2.II.2.