The Impact on Genetic Testing of Mutational Patterns of CFTR Gene in Different Clinical Macrocategories of Cystic Fibrosis

Abstract

More than 2000 sequence variations of the cystic fibrosis transmembrane conductance regulator gene are known. The marked genetic heterogeneity, poor functional characterization of the vast majority of sequence variations, and an uncertain genotype-phenotype relationship complicate the definition of mutational search strategies. We studied the effect of the marked genetic heterogeneity detected in a case series comprising 610 patients of cystic fibrosis (CF), grouped in different clinical macrocategories, on the operative characteristics of the genetic test designed to fully characterize CF patients. The detection rate in each clinical macrocategory and at each mutational step was found to be influenced by genetic heterogeneity. The definition of a single mutational panel that is suitable for all clinical macrocategories proved impossible. Only for classic CF with pancreas insufficiency did a reduced number of mutations yield a detection rate of diagnostic value. All other clinical macrocategories required an extensive genetic search. The search for specific mutational classes appears to be useful only in specific CF clinical forms. A flowchart defining a mutational search that may be adopted for different CF clinical forms, optimized in respect to those already available, is proposed. The findings also have consequences for carrier screening strategies.

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

“…15,16 The same genetic test may have very different DRs, depending on its application in these different clinical forms. 17 In this article, CF refers to the classic form of the disease and excludes CFTRrelated disorders and congenital bilateral absence of vas deferens, which will be specifically mentioned when appropriate.…”

“…Limiting the scope to CF, but considering geographic areas with high genetic heterogeneity, an extended Sanger sequencing protocol of the 5 0 -flanking region, 27 exons and proximal intronic flanking regions, plus selected deep intronic zones for specific intron mutations in CFTR, have shown DRs of up to 97%. 5,17 An additional search for large CFTR rearrangements showed a DR increment of approximately 2%. 5,9,17 The high DRs of approaches with extended genetic characterization seem to be confirmed by nextgeneration sequencing (NGS) technology, which has been recently used for sequence and copy number variation (CNV) analyses of CFTR.…”

“…5,17 An additional search for large CFTR rearrangements showed a DR increment of approximately 2%. 5,9,17 The high DRs of approaches with extended genetic characterization seem to be confirmed by nextgeneration sequencing (NGS) technology, which has been recently used for sequence and copy number variation (CNV) analyses of CFTR. 18e27 NGS has been used mainly in validation studies and has been performed on a limited number of samples in comparisons of the performance of NGS to those of classic Sanger sequencing and multiplex ligationdependent probe amplification (MLPA).…”

“…In particular, the classic sequencing approach and MLPA have shown excellent laboratory operative characteristics for CFTR. 5,17,28,29 Despite the excellent operative characteristics of experimental approaches based on both extended sequencing and large rearrangement search, which are welcomed for diagnostic purposes, these approaches may not be suitable for other health programs, such as neonatal and carrier screening. In fact, in addition to the obvious problems of cost and time, the functional characterization of all of the possibly identified sequence variations and the subsequent assignment of their clinical significance 30 may be impossible tasks.…”

“…31,32 In this case, the DR of the selected panel of mutations in populations of various ethnographic origins and/or with various CF clinical forms invariably arose as a crucial variable. 17 NGS platforms seem to be particularly suited to the development of mutation panels with high a mutation number and, consequently, a high DR. However, at the moment, only a small number of articles have addressed specific CFTR mutation panels based on NGS approaches.…”