Received on October 31, 2008
Accepted on March 27, 2009

Molecular Diagnostics and Genetics

A Novel Approach to CFTR Mutation Testing by Pyrosequencing-Based Assay Panels Adapted to Ethnicities

¹ Department of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, Mainz, Germany
² CF Center of the Children's Hospital, Johannes Gutenberg University Mainz, Mainz, Germany
³ Department of Pulmonology, Thorax Clinic of the University Clinic of Heidelberg, Heidelberg, Germany
⁴ Institute of Human Genetics, Medical School, Hannover, Germany

^* To whom correspondence should be addressed. E-mail: rossmann{at}zentrallabor.klinik.uni-mainz.de.

BACKGROUND: Cystic fibrosis (CF) is a common autosomal recessive genetic disorder caused by a variety of sequence alterations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)]. Because the relative prevalence of mutations strongly depends on the ethnic background, first-level testing of CF as defined by recent consensus recommendations ought to be adaptable to the ethnicity of patients.

METHODS: We therefore developed and implemented a diagnostic approach to first-level testing for CF based on published mutation frequencies and Pyrosequencing (PSQ) technology that we complemented with standard procedures of mutation detection at the second level.

RESULTS: The current test system of PSQ assays for 46 target CF mutations [including CFTRdele2,3 (21 kb) and 5T/7T/9T] permits recombinations of single assays to optimize sensitivities for certain ethnicities. Increasing the sensitivities of first-level tests with other ethnic groups facilitates easy expansion of the original mutation panel, provided that the mutation frequencies are known. The test was validated with our local, ethnically mixed, but mainly German population (155 patients). The mutation-detection rate for the 92 patients whose CF was confirmed by the sweat test was 89.0% for the patients of German descent (73 of the 92 patients) and 73.7% for the patients of any other origin (19 of the 92 patients). Ethnicity-adapted testing panels for our foreign CF patients will increase the sensitivities for the respective groups by approximately 5%.

CONCLUSIONS: PSQ-based genotyping is a reliable, convenient, highly flexible, and inexpensive alternative to conventional methods for first-level testing of CFTR, facilitating flexible adaptation of the analyzed mutation panel to any local ethnic group.