Rapid Combined Genotyping Assay for Four Achondroplasia and Hypochondroplasia Mutations by Real-Time PCR with Multiple Detection Probes

Achondroplasia (ACH) and hypochondroplasia (HYCH) are the most prevalent genetic short-stature syndromes. Whereas the diagnosis of ACH can be established on clinical and radiologic grounds alone in the majority of cases, HYCH is more difficult to confirm. Molecular genetic analysis of both skeletal dysplasias can be especially helpful for the purpose of prenatal diagnosis, in early childhood to differentiate definitively between the largely overlapping phenotypes, and in atypical presentations. The two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide. These mutations can be identified by a variety of molecular methods, including PCR with restriction enzyme digestion or direct DNA sequencing. We have developed a single-step, real-time PCR assay in which two detection probes are applied in combination with a single anchor probe at each mutation position. Because the two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide, this approach guarantees optimal differentiation during probe dissociation analysis after amplification. This assay, which is performed on the Light- Cycler thermocycler, enables the rapid and reliable detection of the two most common FGFR3 mutations associated with ACH (1138G → A and 1138G → C; G380R) and HYCH (1620C → A and 1620 C → G; N540K) in a single test.