Two related patients (sisters) with a congenital bleeding diathesis associated with a severe defect of the platelet ADP receptor, P2Y12, have been described (

Cattaneo et al, ATVB 2000;20:883
). Platelets from these patients are severely defective in ADP-dependent aggregation and in Gi-coupled ADP-mediated responses, but are normal in Gq-coupled ADP-mediated responses. Molecular analysis of the P2Y12 gene of each sister revealed an identical single base pair (bp) deletion (378delC) occurring just beyond the coding sequence for the third transmembrane domain in P2Y12. The mutation results in a frame shift (Thr126 frame shift X34) and premature truncation of the protein (
Conley et al, Blood 2001; 98:43b
). In the present study, we focused on a 13-year old son (GL) of one of the affected sisters. GL has not suffered spontaneous bleedings; his bleeding time is moderately prolonged (13 min). His platelets display a phenotype that is compatible with partial defect of the platelet P2Y12 receptor: abnormal aggregation and ATP secretion induced by several agonists, moderate deficiency of platelet-binding sites for [33P]2MeS-ADP, and partial impairment of inhibition of adenylate cyclase by ADP. PCR analysis of GL’s P2Y12 gene revealed only normal DNA sequence. Therefore, we hypothesized that the abnormal platelet phenotype of GL is due to haploinsufficiency of his P2Y12 gene, and, accordingly, his mother and aunt suffer from P2Y12 deficiency owing to haploinsufficiency and the previously described single base mutation in their remaining P2Y12 allele. In order to test our hypothesis, we analyzed genomic DNA from the family by Southern blotting and real time quantitative PCR. The Southern blot results suggested that GL had one P2Y12 allele only, which he inherited from his father. Our P2Y12 gene quantification strategy was based on SYBR Green detection and normalization using two reference genes with a normal copy number, ZNF 80 (3q13.31) and GPR15 (3q12.1). P2Y12 gene copy numbers, calculated by use of a comparative Ct method, revealed that GL, his mother, and aunt each contained a single intact P2Y12 allele. Thus, GL, his mother, and aunt suffer from haploinsuffiency in their P2Y12 alleles. However, GL’s remaining allele is normal, while his mother and aunt each have a remaining mutant allele. This explains the differences in clinical phenotypes between GL and his mother and aunt. The quantitative PCR result, supported by Southern blotting results, supports our hypothesis and illustrates the platelet phenotype associated with P2Y12 haploinsufficiency.

Topics:
bleeding diathesis, blood platelets, genes, haploinsufficiency, polymerase chain reaction, southern blot assay, dna, adenylate cyclase, agonists, bleeding time procedure

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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