The incidence of inherited rare bleeding, thrombotic and platelet disorders (BPD) is estimated to be 200-250 per million individuals. For at least 15% of these cases the molecular basis is unresolved (Lentaigne et al, Blood, 2016). We aim to discover the genetic basis of these unresolved BPDs, to improve diagnosis and treatment. In addition this will increase our knowledge of the molecular pathways of megakaryopoiesis, haemostasis and thrombus formation. For this purpose we have established a prospective BPD cohort, which at time of writing consist of 1,378 probands, 123 affected relatives and 41 unaffected relatives. After consenting, all these individuals have been precisely phenotyped using human phenotype ontology (HPO) terms (Westbury et al, Genome Medicine, 2015). This includes clinical parameters, laboratory results and pedigree history. Ten thousand DNA samples from the BPD cases, patients with other rare diseases and their close relatives, who were all enrolled in the NIHR BioResource were analysed by whole genome sequencing (Turro et al, Science Trans. Med, 2016). We applied phenotype similarity regression to identify statistical associations between presence of a coding variants with consequences in a gene and similarity to a latent HPO-coded phenotype (Greene et al, AJHG, 2016). We identified a strong statistical association between presence of 8 unique rare coding DNA variants with consequences in GP1BB and 8 probands with macrothrombocytopenia (SimReg posterior probability = 0.93 with inferred characteristic phenotype preferentially included the term "Increased mean platelet volume", Fisher's p = 2.10 x 10-6). We sought to validate these discovery findings through identification of further cases in the cohorts of 75 and 301 macrothrombocytopenia cases from the ThromboGenomics consortium (Simeoni et al, Blood, 2016) and the Nagoya Medical Center in Japan, respectively. Three additional variants in GP1BB were identified in 9 individuals from 8 pedigrees. Systematic review of the sequencing results of 27 BPD genes (including GP1BA, GP9) implicated in thrombocytopenia in 10 probands did not reveal any alternative variants that could plausibly explaining the phenotype.
In aggregate 59 affected macrothrombocytopenia cases were observed in 16 pedigrees with 9 unique GP1BB variants, with the Y113C variant, which was observed in 6 pedigrees thought to be a Japanese founder variant. The means of the count and volume of platelets of the probands was 104.6 x109/l (range 47-172 x109/l) and 12.6 fL, respectively. Inspection of blood smears revealed anisocytosis with a small number of giant platelets and electron micrograph images were reminiscent of those from platelets of a patient with Bernard Soulier syndrome (BSS). In 11 pedigrees measurement by cytometry showed reduced levels of the GpIb/IX/V complex on the platelets of 8 genetically independent individuals and bleeding diathesis was reported in 7 of 16 pedigrees.
Altogether, we identified 9 unique variants in the GP1BB gene, which encodes the 202 amino acid long Type 1 transmembrane protein GpIb▢, which together with GpIba, GpV and GpIX form the receptor complex for von Willebrand Factor on megakaryocytes and platelets. They result in a disruption of the canonical methionine start codon, another resulting in a premature stop at residue 46, 5 missense variants at L16P, G43W, T68M, Y113C and L132Q, a deletion removing PAL at residues 79-81, and finally a frameshift in the codon for residue A150 leading to an alternative open reading frame predicted to result in a protein of 193 instead of 202 amino acids long. All 9 variants but the G43W one, which was observed in one of the 61,000 ExAC subjects were unobserved in the ExAC database.
In summary before our study there was only one isolated report of a Gp1ba-R42C variant assumed to be causal of macrothrombocytopenia, but no segregation study was performed to corroborate this observation. Our findings in 16 pedigrees with 59 subjects with macrothrombocytopenia provide robust statistical and convincing co-segregation evidence that some variants in GP1BB if present as a single alleleexert a dominant effect on the count and volume of platelets, resulting in some pedigrees in a bleeding diathesis rejecting the dogma that BSS is mainly an autosomal recessive disorder.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.