A single multiplex PCR and Single Nucleotide Extension Assay for the detection of common Thanatophoric dysplasia I and II mutations.

Mutation analysis is used to provide confirmation of a clinical and radiological diagnosis of Thanatophoric dysplasia types I and II (TDI & II). We developed a single multiplexed PCR and a single nucleotide extension (SNE) assay to identify 14 common mutations causing 99% of TD I and TD II, including the challenging three adjacent mutations in the stop codon of exon 18 of the FGFR3 gene. The assay design also provides a solution for resolving SNE PCR product sizing using performance optimized polymer-7 (POP-7). The assay was validated using thirty-seven previously characterized, de-identified patient samples representing the nine wild-types and 10/14 mutant genotypes. Four artificial templates were synthesized to mimic four TD I mutations not represented in the available patient samples. Fragment size and fluorophore channel for each SNE product from 10 samples and the four artificial templates were used to define bins and panels for analysis with GeneMarker v3.0 and GeneMapper v6.0 software. Allele calls (bin placement within the panels) were verified using the remaining 27 previously characterized samples. This TD I and II PCR and SNE assay is a robust multiplexed assay, streamlined, to identify 14 mutations in one single reaction. Turnaround time required for this assay performance and analysis is decreased in comparison to traditional Sanger or next generation sequencing.

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