New era of cystic fibrosis: Full mutational analysis and personalized therapy

Table 1 Cystic fibrosis transmembrane conductance regulator mutation classes and cystic fibrosis personalized therapy.

Mutation class	Functional effect	Structural defect	Mutation-specific intervention	Personalized therapy
I	Defective protein synthesis (complete lack of protein production)	Nonsense mutations	Restore synthesis by suppressors of premature stop codons in-frame (read-through drugs)	Suppressor in phase 3 trials: Ataluren
		Frameshift mutations
		Severe splicing mutations
		Deletions or insertions (a common mechanism is the onset of premature stop codons)
II	Defective protein processing and/or trafficking (severe decrease of protein in the apical membrane due to processing and/or maturation defects)	Missense mutations Small deletions or insertions	Restore processing and trafficking by correctors (chemical, molecular, pharmacological chaperones) and combined approaches (corrector + potentiator)	Combined therapy to patients: Orkambi (the corrector Lumacaftor + the potentiator Ivacaftor)
III	Defective channel regulation and/or gating (impaired channel opening)	Missense mutations	Restore channel regulation and gating by potentiators	Potentiator to patients: Ivacaftor
		Small deletions or insertions
IV	Defective Cl- conductance (reduced Cl- transport through the channel)	Missense mutations	Restore the Cl- conductance by potentiators	Under evaluation
		Small deletions or insertions
V	Reduced mRNA synthesis (reduction of the wild type mRNA)	Partial splicing mutations Promoter mutations	Restore wild-type mRNA levels by correctors, potentiators and/or antisense oligonucleotides	Under evaluation
VI	Decreased protein stability in membrane or reduced ability of other channel regulation	Missense mutations	Restore stability and regulation ability by potentiators, stabilizers and/or suppressors of overdue stop codons in-frame	Under evaluation
		Nonsense mutations
		Frameshift mutations (a common mechanism is the onset of overdue stop codons because of mutations of the protein C-terminus)

Table 2 Comparison of classic and next generation sequencing approaches for diagnostic mutation search in cystic fibrosis.

Feature	Classic approach	NGS approach
Analytical requirements for a full characterization of the CFTR gene	Multiple technical steps and different analytical platforms	Reduced number of technical steps and single analytical platform
Data elaboration	Multiple data elaboration steps handled by the laboratory itself	Reduced number of data elaboration steps often performed by internal dedicated personnel or external structures
Throughput	Low	High
Automation	Moderate	High
Timing	Time consuming	Rapid
Cost per sample	High	Low (if a reasonably high number of samples are processed in the same run)
No. of mutations analyzed	Progressively increasing from moderate (first steps) to high (last steps)	High
Detection rate	Progressively increasing from moderate (first steps) to high (last steps)	High
Possibility to analyze other genes involved in the modulation of CF clinical manifestations	Unlikely	Realistic
Feature	Classic approach	NGS approach

NGS: Next generation sequencing; CF: Cystic fibrosis; CFTR: Cystic fibrosis transmembrane conductance regulator