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 the identification of eight carriers and four non-carriers. When the mutation caused a distinctly altered migration pattern of the single strands, in principle, the SSCP-technique allowed determination of carrier status in the extended family of the probands without direct sequencing. 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 negative and/or false positive diagnoses due to intronic and exonic sequence heterogeneity in the family. As we discovered this pitfall in one of the reported families, we concluded that for carrier testing the SSCP approach must be performed in essential conjunction with an independent assessment of the mutation site by direct sequencing.