A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology

Abstract

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing ebased method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling.

The true panel of cystic fibrosis mutations in the Sicilian population

“…The high allele heterogeneity is explained by the multiple invasions during the centuries especially from Northern populations. Different commercial kits for first level analysis [30] and NGS-assay screen for a large panel of mutations [31] but none of them test overall CFTR mutations found here. Respectively 12 and 6 of the mutations found in the present work would not have been detected with the strategies described elsewhere [30,31].…”

Genotype-Phenotype Correlations of the Course of Cystic Fibrosis in Russian Children. The First Description of Eleven New Mutations

“…В последние годы для молекулярно-генетической диагностики кистозного фиброза стали широко использоваться методы исследования, созданные на основе технологии секвенирования нового поколения. При этом применяются различные модификации этой технологии, позволяющие как анализировать набор определенных мутаций, так и проводить анализ всего гена, включая его некодирующие области [13]. В случае необнаружения биаллельных мутаций вышеперечисленными методами применяются дополнительные методы исследования, такие как количественная полимеразная цепная реакция (ПЦР) в режиме реального времени и мультиплексная лигазная ПЦР (MLPA), позволяющие выявлять протяженные структурные изменения гена CFTR, носящие патогенный характер [14].…”

Evaluating sequence data quality from the Swift Accel-Amplicon CFTR Panel

“…In the past three decades, the detection of CFTR mutations has evolved through various molecular methods, including reverse dot blot, restriction fragment length polymorphism (RFLP), and Sanger sequencing 8,9 . The advent of next generation sequencing (NGS) leads to a higher clinical sensitivity by screening more targeted CFTR mutations and sequencing of the exonic gene regions, as well as a higher throughput by multiplexing many samples into one sequencing run 10,11 . While NGS excels at generating large amount of data, it is time-consuming and less cost-effective for sequencing few targets and low volume of samples.…”