A long PCR-based molecular protocol for detecting normal and expanded ZNF9 alleles in myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) lacks the expansion on chromosome 19q13 present in DM1 and is characterized by a mutation on 3q21. It has been shown that the DM2 mutation is a huge [CCTG]n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. The longest normal allele observed has a approximately 30 CCTG repeat, whereas the range of expansion is extremely variable, starting from 75 up to 11,000 CCTGs. Direct analysis by Southern blot, after restriction enzyme digestion of genomic DNA, was the first method chosen for studying the DM2 mutation. However, the expansion size and the elevated grade of somatic instability have limited the sensitivity of the test to approximately 80% of known carriers. We developed a long PCR-formatted protocol, which involves a single genomic in vitro amplification, followed by agarose gel electrophoresis and oligospecific hybridization. We were able to detect normal alleles and expanded ZNF9 alleles, starting from low amounts of genomic DNA (>/= 1 ng) in virtually all the DM2 patients analyzed, obtaining a molecular detection rate of 100%. This method is quick, sensitive, and reproducible, and it reduces the cost of diagnostic laboratory processing for DM2 diagnosis.