A unique combination of inhibitory and partially activating mutations in β3 of a patient with variant-type Glanzmann thrombasthenia

To the Editor

In 1999 we published the case of a male patient from the Bordeaux area of France with variant-type Glanzmann thrombasthenia (GT) linked to a heterozygous c.685T > C transition in exon 5 of ITGB3 giving rise to a L196P (L222P, including the leader sequence) substitution [1], [2]. This mutation was detected following an initial screening of the exons and splice sites of the ITGB3 and ITGA2B genes of the propositus (pat 1 here) by single-strand conformation polymorphism analysis of PCR products. All living close family members, namely his mother, sister and son were also heterozygous carriers of the mutation. While pat 1 showed the typical signs of GT with an absent aggregation to physiologic agonists, the other three family members all gave a normal response to ADP although retaining a delayed and poor response to collagen [2]. This latter finding remains unexplained. Whereas his mother, son and sister have no bleeding symptoms, pat 1 has a lifelong history of mucocutaneous bleeding especially epistaxis. A sister died from nasal bleeding when she was a child. Whereas each of the three family members had somewhat decreased but >50% expression of αIIbβ3 in their platelets, pat 1 only possessed of the order of 8% of the normal platelet content of αIIbβ3 [1]. Nevertheless, unlike for classic type I GT, his platelets contained detectable amounts of stored and presumably endocytosed Fg. An independent report on a second French family identified the presence of a homozygous ITGB3 c.685C > T nucleotide substitution and L196P substitution in a patient with about 10% of the expression of αIIbβ3 as revealed using a radiolabeled monoclonal antibody in binding studies [3], [4]. Again some α-granule stored Fg was present. All of the evidence therefore pointed to the presence of a second, undetected mutation in pat 1. Direct sequencing of all exons and splice sites of ITGA2B (30 exons) and ITGB3 (15 exons) by the French National Sequencing Center (Génoscope, Evry, France) while confirming the heterozygous ITGB3 exon 5 c.685C > T substitution for pat 1, has additionally shown the presence of a heterozygous ITGB3 exon 11 c.1871G > A transition leading to a C598Y (C624Y) substitution. A newly adapted high-resolution DNA melting curve (HRM) analysis procedure in the presence of a saturating DNA-binding dye [5] and direct sequencing confirmed that whereas the heterozygous ITGB3 exon 5 substitution was indeed present in all of the family members, the propositus was the only family member to possess the exon 11 mutation (Figure 1).

Figure 1. Mutation screening by HRM analysis and direct sequencing of PCR-amplified products. Results are shown for ITGB3 exons 5 and 11 for family members of pat 1. In panels A and B, we illustrate normalized and temperature-shifted melting curves of mutated and control PCR amplicons (Roche Light cycler 480 ResoLight Dye; Roche Diagnostics, Meylan, France) (c.685C > T, Leu196Pro; and c.1871G > A, Cys598Tyr). Control patterns (pink) mutated patterns (red) are clearly distinguished. Whereas the ITGB3 exon 5 substitution was present in all of the family members (see superimposed lines), the propositus was the only family member to possess the exon 11 mutation. In panels C and D are shown the sequencing profiles of respective heterozygous mutated PCR products. Methodological details will be supplied on request.

A heterozygous ITGB3 c.1871G > A transition leading to a C598Y substitution has been previously reported for a third unrelated French patient (pat 2 of this study), where it was found together with an ITGB3 exon 5 c.724C > T nonsense mutation giving an R216X (R242X) stop codon [6]. Historically, this patient was one of the first GT patients to be characterized and possessed of the order of 10% residual αIIbβ3 able to bind fibrinogen (Fg) when platelets were incubated with ADP [7–11]. The effects of the L196P and C598Y substitutions on β3 function have both been previously examined by site-directed mutagenesis followed by transfection in CHO cells [4], [12]. The presence of 196P within the I-domain of β3 is inhibitory, and blocks the Fg-binding capacity of both αIIbβ3 and αvβ3. In fact, this is one of the few mutations where a dual effect on both β3 integrins has been clearly established. In contrast, the C598Y mutation, like many of the β3 cysteine mutations in the cysteine-rich EGF domains (C598 forms a disulfide with C588 in EGF-4) is partially activating, allowing the spontaneous binding of the activation-dependent monoclonal antibody PAC-1 to transfected CHO or BHK cells but not the binding of soluble Fg [12], [13]. It should be noted that whereas the residual αIIbβ3 of pat 1 would be expected to be a mixture of the integrin containing either β3P196 or β3Y598, that of patient 2 is likely to contain only β3Y598 (the second mutation is a stop codon giving rise to a severely truncated β3 not present in her platelets). Thus while both inhibitory and partially activating β3 subunits prevent normal αIIbβ3 maturation in megakaryocytes, possibly by delaying transport through the Golgi apparatus [14], residual integrin surface expression does occur. Of note is the fact that platelets of each patient contain substantial amounts of α-granule stored Fg, implying that a functional β3 I- domain or intact cysteine-rich EGF domains are not required for Fg uptake. It is highly probable that the inability of platelets of pat 2 to aggregate is due to the low density of αIIbβ3 on the platelet surface. Also of interest is that patient 2 has not been clinically protected by the presence of residual partially active integrin as she has a life-long history of mucocutaneous bleeding intermingled with several severe episodes requiring transfusions (pat 41 in [15]). Whether or not the C598Y mutation affects αvβ3 expression or function is not known.

In conclusion, our study helps define the pathophysiology of two rare ITGB3 mutations giving rise to GT, and describe the first patient with a qualitative αIIbβ3 deficiency with both inhibitory and partially activating amino acid substitutions on the β3 subunit.

Acknowledgements

This study was financed by contract N° AP07/08.42 with the Génoscope d'Evry and from INSERM (ANR-08-GENO-028-03). Informed consent was obtained and the study performed under the national ethical guidelines of the CRPP.