Novel, rare and common pathogenic variants in the CFTR gene screened by high-throughput sequencing technology and predicted by in silico tools

Abstract

Cystic fibrosis (CF) is caused by ~300 pathogenic CFTR variants. The heterogeneity of which, challenges molecular diagnosis and precision medicine approaches in CF. Our objective was to identify CFTR variants through high-throughput sequencing (HTS) and to predict the pathogenicity of novel variants through in 8 silico tools. Two guidelines were followed to deduce the pathogenicity. A total of 169 CF patients had genomic DNA submitted to a Targeted Gene Sequencing and we identified 63 variants (three patients had three variants). The most frequent alleles were: F508del (n = 192), G542* (n = 26), N1303K (n = 11), R1162* and R334W (n = 9). The screened variants were classified as follows: 41 - pathogenic variants [classified as (I) n = 23, (II) n = 6, (III) n = 1, (IV) n = 6, (IV/V) n = 1 and (VI) n = 4]; 14 - variants of uncertain significance; and seven novel variants. To the novel variants we suggested the classification of 6b-16 exon duplication, G646* and 3557delA as Class I. There was concordance among the predictors as likely pathogenic for L935Q, cDNA.5808T>A and I1427I. Also, Y325F presented two discordant results among the predictors. HTS and in silico analysis can identify pathogenic CFTR variants and will open the door to integration of precision medicine into routine clinical practice in the near future.

Figure 1

Flowchart showing the steps to characterize the pathogenicity of variants in the CFTR gene with the use of predictors: (i) MutationTaster (http://www.mutationtaster.org/); (ii) SNPEffect 4.0 (http://snpeffect.switchlab.org); (iii) PolyPhen-2 (Polymorphism Phenotyping v2) (http://genetics.bwh.harvard.edu/pph2/); (iv) CADD – Combined Annotation Dependent Depletion) (https://cadd.gs.washington.edu/); (v) MutPred-2 (Mutation Prediction 2) (http://mutpred.mutdb.org/index.html); (vi) MutPred-LOF (Loss-of-function); (vii) MutPred Splice; and (viii) Human Splicing Finder version 3.1 (http://www.umd.be/HSF3/). CFTR, cystic fibrosis transmembrane conductance regulator.

Figure 2

Molecular visualization of WALTZ, LIMBO and TANGO. (A) Molecular visualization of WALTZ amyloid-forming regions showing the WALTZ aggregation-prone regions as blue-colored segments. (B) Molecular visualization of LIMBO chaperone-binding sites showing the LIMBO chaperone-binding sites as pink-colored segments. (C) Molecular visualization of TANGO aggregation-prone regions showing the TANGO aggregation-prone regions as red-colored segments. The structural location of the variant residue is colored in yellow. The data was achieved from SNPeffect 4.0 (http://snpeffect.switchlab.org/menu).

Figure 3

Molecular visualization of the wild-type (WT) (left – red color) and amino acid variant (right – red color) using the FoldX predictor. I, Ile – Isoleucine; T, Thr – Threonine; E, Glu – Glutamic Acid; D, Asp – Aspartate; N, Asn – Asparagine; S, Ser – Serine; R, Arg – Arginine; G, Gly – Glycine; A, Ala – Alanine; V, Val – Valine; L, Leu – Leucine; P, Pro – Proline; F, Phe – Phenylalanine; W, Trp – Tryptophan; Q, Gln – Glutamine; Y, Tyr – Tyrosine. The data was obtained from SNPeffect 4.0 (http://snpeffect.switchlab.org/menu).

Figure 4

Description of the variants in the CFTR gene according to functional classes. (A) Map graph (with squares) showing the prevalence of each class of variant in CFTR identified in the study sample and the drug, used in precision medicine, available for use with the approval of the FDA (US Food and Drug Administration) – 2018. The approval for use in cystic fibrosis patients is limited to some variants within each class. Therefore, the representation per class is a possibility of grouping the patients and showing, visually, the number of individuals who may have benefits for each class of variant. (B) Detailed description of the groups of variants that were not described due to the limitations of the study as belonging to the known classes of CFTR, and further studies are needed for the definitive classification. CFTR, cystic fibrosis transmembrane conductance regulator. Lumacaftor (CFTR chaperone, VX-809, C₂₄H₁₈F₂N₂O₅)/ivacaftor (CFTR potentiator, VX-770, C₂₄H₂₈N₂O₃) (Orkambi); Tezacaftor (VX-661, C₂₆H₂₇F₃N₂O₆) /Ivacaftor (Symdeko); Ivacaftor (Kalydeco).