The Impact on Genetic Testing of Mutational Patterns of CFTR Gene in Different Clinical Macrocategories of Cystic Fibrosis  Marco Lucarelli, Sabina M. Bruno, Silvia Pierandrei, Giampiero Ferraguti, Giancarlo Testino, Gessica Truglio, Roberto Strom, Serena Quattrucci  The Journal of Molecular Diagnostics  Volume 18, Issue 4, Pages 554-565 (July 2016) DOI: 10.1016/j.jmoldx.2016.02.007 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Detection rates at each mutational step in the clinical macrocategories analyzed. Both the allelic (A) and genotypic (B) detection rates at each mutational step are shown (see Materials and Methods for definitions). Differences between mutational steps in the number of alleles (A) or genotypes (B) are statistically significant (for both, χ2, P < 0.0001). CBAVD, congenital bilateral absence of vas deferens; CF-OLA, analysis of the 32 most common mutations worldwide by the CF-OLA assay; CF (PI+PS), cystic fibrosis with either PI or PS; CF-PI, CF with pancreatic insufficiency; CF-PS, CF with pancreatic sufficiency; CF-SNAP+20, analysis of the 14 most frequent mutations in the specific geographical area by the primer extension assay; CFTR-RD, CFTR-related disorders and atypical CF forms; DEL, analysis of the most frequent macrodeletions worldwide by FC del assay; SEQ, analysis of the CFTR gene by extended sequencing; (TG)mTn, analysis of the (TG)mTn variant tracts by sequencing. See Materials and Methods for further details. The Journal of Molecular Diagnostics 2016 18, 554-565DOI: (10.1016/j.jmoldx.2016.02.007) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Cumulative frequencies of mutated alleles. The number of mutations included in an ideal genetic test is shown on the x axis, starting from the most frequent mutation and progressively adding the less frequent mutations according to their decreasing frequency. The order of mutations on the x axis of each graph varies according to the prevalence of mutations in each clinical macrocategory. In each graph, the vertical dotted line (the leftmost) indicates the number of nonindividual mutations found: the mutations added from the left up to the dotted line are those found in at least two unrelated patients, whereas those added to the right of the dotted line were found only once in a single patient or only in siblings. The vertical continuous line (the rightmost) indicates the overall number of mutations found in the clinical macrocategory. The x axis reflects the decreasing order of mutation frequency in the following populations: cystic fibrosis with pancreatic insufficiency (CF-PI; A), cystic fibrosis with pancreatic sufficiency (CF-PS; B), CFTR-related disorders and atypical CF forms (CFTR-RD; C), and congenital bilateral absence of vas deferens (CBAVD; D). See It Is Not Possible to Define Only One Mutational Panel That Is Suitable for All Populations (a Panel with High DRs Exists for the CF-PI Population Alone) for further explanations. CF (PI+PS), cystic fibrosis with either PI or PS. The Journal of Molecular Diagnostics 2016 18, 554-565DOI: (10.1016/j.jmoldx.2016.02.007) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Cumulative frequencies of complete mutated genotypes. The number of mutations included in an ideal genetic test is shown on the x axis, starting from the most frequent mutation and progressively adding the less frequent mutations according to their decreasing frequency. The order of mutations on the x axis of each graph varies according to the prevalence of mutations in each clinical macrocategory. In each graph, the vertical dotted line (the leftmost) indicates the number of nonindividual mutations found: the mutations added from the left up to the dotted line are those found in at least two unrelated patients, whereas those added to the right of the dotted line were found only once in a single patient or only in siblings. The vertical continuous line (the rightmost) indicates the overall number of mutations found in the clinical macrocategory. The x axis reflects the decreasing order of mutation frequency in the following populations: cystic fibrosis with pancreatic insufficiency (CF-PI; A), cystic fibrosis with pancreatic sufficiency (CF-PS; B), CFTR-related disorders and atypical CF forms (CFTR-RD; C), and congenital bilateral absence of vas deferens (CBAVD; D). See It Is Not Possible to Define Only One Mutational Panel That Is Suitable for All Populations (a Panel with High DRs Exists for the CF-PI Population Alone) for further explanations. CF (PI+PS), cystic fibrosis with either PI or PS. The Journal of Molecular Diagnostics 2016 18, 554-565DOI: (10.1016/j.jmoldx.2016.02.007) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A flowchart for mutational analysis of CFTR. If there is pancreatic insufficiency (PI), the right arm of algorithm should be followed: after search for a panel of mutations [including frequent macrodeletions but excluding (TG)mTn] and the finding of two mutated alleles, the search for complex alleles possibly already known to be based on CFTR variations in cis with the mutations found should be performed. Then, the evaluation of the genotype together with other laboratory and clinical findings should allow the diagnosis and the assignment to clinical macrocategory(ies). Alternatively, if there is pancreatic sufficiency (PS) or the pancreatic status is not known, the lower arm of the algorithm should be followed: after extended sequencing [including (TG)mTn and the search for complex alleles] and, possibly, the search for macrodeletions, if two mutated alleles are found the diagnosis and the assignment to a clinical macrocategory(ies) should be allowed. On the contrary, if at the end of the lower arm there is at least one unknown allele, further CFTR analyses could be performed. The lower arm of the algorithm should be followed also if, after the search for a panel of mutations and frequent macrodeletions (according to the right arm), at least one unknown allele remains. CF, cystic fibrosis; MLPA, multiplex ligation-dependent probe amplification; N, no; UTR, untranslated region; Y, yes. The Journal of Molecular Diagnostics 2016 18, 554-565DOI: (10.1016/j.jmoldx.2016.02.007) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions