CARRIER DETECTION IN DMD FAMILIES WITH POINT MUTATIONS, USING PCR-SSCP AND DIRECT SEQUENCING

Non-isotopic single-strand conformation polymorphism (SSCP) and direct sequencing was used for carrier diagnosis in four families of DMD/BMD patients with previously characterized point mutations, leading to th e identification of eight carriers and four noncarriers. When the muta tion caused a;distinctly altered migration pattern of the single stran ds, in principle, the SSCP-technique allowed determination of carrier status in the extended family of the probands without direct sequencin g. However, because SSCP measures a function of not only the mutation, but of the entire sequence of the PCR product, it can lead to false n egative and/or false positive diagnoses due to intronic and exonic seq uence heterogeneity in the family. As we discovered this pitfall in on e of the reported families, we concluded that for carrier testing the SSCP approach must be performed in essential conjunction with an indep endent assessment of the mutation site by direct sequencing.