*2.1.3. Advantages and limits*

**Figure 2.** CF molecular diagnosis: a step-by-step strategy (modified from [7]). *NBS*: newborn screening; *SNV*: Single Nucleotide Variation; *CNV*: Copy Number Variation; *MLPA*: Multiplex Ligation-dependent Probe Amplification.

These approaches consist of genotyping a panel of frequent CF mutations using commercial kits (Table 1) that classically cover more than 80% of CF known mutations in European populations. Additional search for mutations specific to certain regions or ethnicities (fre‐ quency higher than 1% of CF alleles in the targeted population) completes the analyses.

Data on disease and carrier frequencies or mutation frequencies in various populations are available in the WHO report [8] and should be accurately known and used by laboratories.

For many patients carrying CF-causing mutations included in commercial panels, *CFTR* molecular analysis generally stops at this step. There is no need for additional studies, except the confirmation of mutations by a second method, as recommended by international guide‐

The high heterogeneity of *CFTR* mutations in CF and CFTR-RD populations makes the complete molecular screening of the 27 exons and parts of the regulatory regions (5'UTR,

Therefore, the analysis of the *CFTR* locus can be performed as follows:

**2.1. Current techniques to detect** *CFTR* **variants**

*2.1.1. Screening techniques (Step 1)*

204 Cystic Fibrosis in the Light of New Research

lines for genetics diagnosis.

*2.1.2. Scanning techniques (Step 2)*

3'UTR and partial intronic regions) essential.

Classical screening or scanning methods used to detect mutations (in step 1 and 2) have high specificity and sensitivity (Table 1). Older techniques such as RFLP (restriction fragment length polymorphism), DGGE (denaturing gradient gel electrophoresis) or DHPLC (denaturing high performance liquid chromatography) were widely used in the past 20 years but were difficult to set up and showed inconstant performance. They have been progressively replaced by easyto-use commercial kits, more appropriate for larger sample series. These kits allow PCR multiplexing and thus enable the detection of the most common CF mutations in one reaction. Sanger sequencing and quantitative PCR are the most effective to identify unknown mutations. However, all these classical methods are labour-intensive and time-consuming.


**Table 1.** Overview, advantages and limits of screening and scanning techniques used in molecular genetics diagnosis (adapted from [7])
