The evidence of enlarged platelets with reduced expression of glycoprotein Ib/IX might indicate recessive inherited thrombocytopenias other than Bernard-Soulier Syndrome

Introduction: The assessment of platelet morphology represents a cornerstone in the diagnostic work-up of inherited platelet disorders (IPD). We have established an immunofluorescence-based method for phenotyping platelets on the peripheral blood smear in combination with light microscopy. The technique has been validated for 9 IPD, for which typical changes of platelet structure can be detected. To verify the efficacy of our approach as a screening tool for IPD, we contiuosly perform a blinded investigation of blood films obtained from suspected patients, who had previously undergone molecular testing.

Method: Blood smears were stained using 13 primary antibodies against different platelet structures (alpha and dense granule markers, surface glycoproteins, cytoskeletal components) and 2 fluorescence-labelled secondary antibodies. Exclusively based on the morphologic evaluation, we formulated a possible diagnosis. After uncovering patients’ information, we compared the anticipated defect with molecular outcome.

Results: Thirty subjects were enrolled to this study over the last 12 months. Based on the finding of platelet macrocytosis and reduced expression of the surface platelet glycoprotein (GP) Ib/IX, we predicted a diagnosis of Bernard Soulier Syndrome (BSS) in 4 individuals. After unblinding genetic results, we could confirm the suspicion only in one case, in whom a pathogenic, heterozygous deletion in GP1BB gene (c.236_244del [p.Pro79_Leu81del]) consistent with a dominant-inherited BSS was found. In one subject, two novel variants in compound heterozygosis in the gene encoding for the enzyme uridine diphosphate (UDP)-galactose-4-epimerase (GALE) were identified: c.382G>A [p.Val128Met] and c.590T>C [p.Ile197Thr]. In the other two patients, belonging to the same pedigree, we found a novel homozygous variant in the gene encoding for UDP-N-acetylglucosamine 2-epimerase (GNE): c.1516G>A [p.Gly506Ser]. The patient with GALE mutations presented with severe thrombocytopenia, moderate bleeding symptoms and a syndromic constellation including mental retardation, mitral valve prolapse and hip malformation. The subjects with GNE mutations had severe thrombocytopenia without clinically-relevant bleeding and no apparent syndromic picture. GALE and GNE encode enzymes involved in glycosylation reactions, which play a role at different levels of platelet production and clearance. In both disorders, the reduced externalization of GPIbIX is a consequence of impaired protein glycosylation [1] [2] [3] [4] [5].

Conclusion: The archetypal morphologic presentation of BSS (platelet macrocytosis with reduced expression of GPIb/IX) can even indicate recessive forms of inherited thrombocytopenia due to biallelic mutations in GALE or GNE gene ([Fig. 1]). The presence of syndromic manifestations and impaired platelet aggregation not limited to ristocetin but even upon stimulation with other agonists (e.g. collagen and CRP) can orient the differential diagnosis toward these disorders.

Fig. 1  Platelet size by light-microscopy and platelet expression of glycoprotein (GP) Ib/IX by immunofluorescence-microscopy in reported patients with monoallelic Bernard-Soulier syndrome (mBSS), GALE-related thrombocytopenia (GALE-RT), or GNE-related thrombocytopenia (GNE-RT) compared to healthy control. Scale bars correspond to 5 µm.

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