Allelic discrimination of cis-trans relationships by digital polymerase chain reaction: GJB2 (p.V27I/p.E114G) and CFTR (p.R117H/5T).

Purpose: : To distinguish the cis-trans relationship of two sequence changes and to arrive at an accurate molecular diagnosis for autosomal recessive disorders, methods such as Sanger sequencing cannot differentiate whether sequence changes are in cis or trans. In addition, most techniques theoretically appropriate for allelic discrimination depend on the specific identified sequence changes for assay design, need extensive optimization, or may not be suitable. We developed a method that does not fully depend on the specific nucleotide changes. It enables efficient assay design and practical implementation of allelic discrimination.

Methods: : Digital polymerase chain reaction (PCR) was used to separate and amplify alleles. Sanger sequencing was subsequently used to identify sequence changes.

Results: : We developed a cost-effective digital PCR method for allelic discrimination of short amplicons and demonstrated it with p.Val27Ile and p.Glu114Gly in GJB2 as an example. We also successfully developed a long-range digital PCR approach to determine the cis-trans relationship of p.Arg117His and 5T in the CFTR gene.

Conclusion: : Digital PCR for allelic discrimination can be clinically implemented to determine the allelic configuration of relatively common sequence changes which frequently appear together and have clinical ramifications, such as the combination of p.Val27Ile and p.Glu114Gly in the GJB2 gene and p.Arg117His and 5T in CFTR.