P2Y2 receptor polymorphisms and haplotypes in cystic fibrosis and their impact on Ca2+ influx

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Activation of P2Y2 receptors in airway epithelia by ATP and UTP stimulates a Ca2+-regulated Cl- channel, which regulates Cl- secretion in cystic fibrosis (CF). We hypothesized that genetic alterations in the P2Y2 receptor may act as disease modifiers in CF and thus analyzed the coding region of this gene for polymorphisms in 146 CF patients and 64 healthy controls. We also assessed the impact of the genetic variants on Ca2+-influx of P2Y2-null cells transfected with several P2Y2 receptor haplotypes. We identified three frequent nonsynonymous P2Y2 receptor polymorphisms: Leu46Pro; Arg312Ser and Arg334Cys, of which only Arg312Ser was significantly more common in CF: Arg = 0.80, Ser = 0.20 (CF) vs. Arg = 0.72, Ser = 0.28 (controls), P < 0.05; for Leu46Pro, Leu = 0.92, Pro = 0.08 (CF) vs. Leu = 0.96, Pro = 0.04 (controls), P = 0.65 and for Arg334Cys, Arg = 0.79, Cys = 0.21 (CF) vs. Arg = 0.84, Cys = 0.16 (controls), P = 0.79. The most frequent haplotype was Leu46Leu/Arg312Arg/Arg334Arg (28% in CF, 31% in controls) but 6% of CF patients and none of the controls had Leu46Leu/Ser312Ser/Arg334Cys or Leu46Leu/Arg312Arg/Cys334Cys. To assess function of the receptor haplotypes, we stably transfected 1321N1 (P2Y-null) cells to similar levels of mRNA expression with Leu46Leu/Arg312Arg/Arg334Arg (wild-type), Leu46Leu/Ser312Ser/Arg334Arg and Leu46Leu/Arg312Arg/Cys334Cys and measured ATP-stimulated transient Ca2+-influx. Cells expressing the homozygous Cys334 variant had significantly increased Ca2+-influx compared to wild-type (P<0.01). The increase in Ca2+-influx was more pronounced in cells carrying the homozygous Ser312 variant than in cells with the other two genotypes (P<0.01). These data indicate that P2Y2 receptor gene haplotypes influence intracellular Ca2+-release. Such genetic variants might therefore represent modifiers of Cl- secretion or of response to P2Y2 agonist therapy in CF.