**2. Technical aspects of molecular diagnosis**

**1. Introduction**

202 Cystic Fibrosis in the Light of New Research

detected within a single family.

The autosomal recessive mode of inheritance of Cystic Fibrosis (CF) was suggested by Andersen and Hodges in 1945 when they described this disease as a pathological entity.

Since the discovery of the *CFTR* gene (and the predicted protein cystic fibrosis transmembrane conductance regulator) in 1989 [1], close to 2, 000 variations have been identified on this locus. *CFTR* gene studies represent one of the most frequent genetic analyses routinely performed worldwide, either to confirm the clinical diagnosis of CF or CFTR-related disorders (CFTR-

The most common severe mutation, p.Phe508del, is found in approximately 70% of CF alleles of European descent (therefore it is present in 49% of homozygous and 42% of compound heterozygous CF patients), with significant variations depending on ethnicities [2]. There is a clear decreasing northwest to southwest gradient in p.Phe508del frequency across Europe. Only four other mutations represent more than 1% of CF cases: p.Gly542\*, p.Gly551Asp, p.Asn1303Lys, and p.Trp1282\*. All other mutations are rare and many are private, only

**Figure 1.** Two models for the classification of CFTR variants: (1) Functional: five classes of defective CFTR protein [3, 4,

5]; and (2) Clinical: four classes based on phenotypic expression in patients [6].

RDs), or to offer carrier testing, prenatal or pre-implantation genetic diagnosis.

The *CFTR* genetic testing strategy depends on the clinical and familial context and is classically performed step by step, as recommended [7]. As shown in Figure 2, tests are carried out in various situations, including confirmation of a clinical diagnosis, prenatal diagnosis (familial context or foetal suspicion of CF) and carrier screening.

A wide range of techniques is still used to identify *CFTR* gene sequence variations (presented in Table 1) and there is no gold standard or preferred method for routine testing. However, laboratories should be aware of the limitations of their chosen method (e.g. some mutations are not identified). Moreover, assay performance should always be verified before diagnostic use, even though commercial kits are CE-marked *in vitro* diagnostic devices (IVDD). In the perspective of accreditation (expected in France in 2020 for hospital diagnostic laboratories), all methods used in molecular biology should be validated before diagnostic use, notably by comparison with a reference method (Sanger sequencing for Single Nucleotide Variations and quantitative PCR for Copy Number Variations detection). This technical validation should be undertaken for NGS in the step 1 and 2 diagnosis.

Undetected *CFTR* mutations may lie within the introns or regulatory regions, which are not routinely explored but that will be soon analysed by NGS. Comparison between NGS and reference methods for the identification of *CFTR* variants on these unexplored regions will be a challenge.

**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.
