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Ledger's uncle Haydn had claimed that the late actor may have become a dad at 17 after an affair with a married 25-year-old woman in Perth, Australia.
But the woman, who has since remarried, said there is no proof that her daughter has any connection to the Brokeback Mountain star.
She told a Western Australian newspaper: "I'm not answering anything. I think it's very, very rude that I'm being posed all of this. I really do. I can't talk about anything.
"You have no proof, there's no background, there's no grounds, there's no nothing."
Her husband said: "I'm the step-dad, but there's nothing to hide... and if I asked [the mother], I'm sure that she wouldn't mind [having a DNA test]."
He added: "It would probably be a great thing if she was [Heath's], but no."
Ledger died of a prescription drug overdose in January. He had two-year-old daughter Matilda with ex-fiancée Michelle Williams.
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