Title: The Impact on Genetic Testing of Mutational Patterns of CFTR Gene in Different Clinical Macrocategories of Cystic Fibrosis
Abstract: More than 2000 sequence variations of the cystic fibrosis transmembrane conductance regulator gene are known. The marked genetic heterogeneity, poor functional characterization of the vast majority of sequence variations, and an uncertain genotype-phenotype relationship complicate the definition of mutational search strategies. We studied the effect of the marked genetic heterogeneity detected in a case series comprising 610 patients of cystic fibrosis (CF), grouped in different clinical macrocategories, on the operative characteristics of the genetic test designed to fully characterize CF patients. The detection rate in each clinical macrocategory and at each mutational step was found to be influenced by genetic heterogeneity. The definition of a single mutational panel that is suitable for all clinical macrocategories proved impossible. Only for classic CF with pancreas insufficiency did a reduced number of mutations yield a detection rate of diagnostic value. All other clinical macrocategories required an extensive genetic search. The search for specific mutational classes appears to be useful only in specific CF clinical forms. A flowchart defining a mutational search that may be adopted for different CF clinical forms, optimized in respect to those already available, is proposed. The findings also have consequences for carrier screening strategies. More than 2000 sequence variations of the cystic fibrosis transmembrane conductance regulator gene are known. The marked genetic heterogeneity, poor functional characterization of the vast majority of sequence variations, and an uncertain genotype-phenotype relationship complicate the definition of mutational search strategies. We studied the effect of the marked genetic heterogeneity detected in a case series comprising 610 patients of cystic fibrosis (CF), grouped in different clinical macrocategories, on the operative characteristics of the genetic test designed to fully characterize CF patients. The detection rate in each clinical macrocategory and at each mutational step was found to be influenced by genetic heterogeneity. The definition of a single mutational panel that is suitable for all clinical macrocategories proved impossible. Only for classic CF with pancreas insufficiency did a reduced number of mutations yield a detection rate of diagnostic value. All other clinical macrocategories required an extensive genetic search. The search for specific mutational classes appears to be useful only in specific CF clinical forms. A flowchart defining a mutational search that may be adopted for different CF clinical forms, optimized in respect to those already available, is proposed. The findings also have consequences for carrier screening strategies. Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF; Mendelian Inheritance in Man 219700), the most common lethal genetic disease in the caucasian population.1Lucarelli M. Pierandrei S. Bruno S.M. Strom R. The genetics of CFTR: genotype- phenotype relationship, diagnostic challenge and therapeutic implications. Cystic Fibrosis: Renewed Hopes Through Research. Edited by Sriramulu D. Intech - Open Access, Rijeka, Croatia2012: 91-122Google Scholar, 2Bombieri C. Seia M. Castellani C. Genotypes and phenotypes in cystic fibrosis and cystic fibrosis transmembrane regulator-related disorders.Semin Respir Crit Care Med. 2015; 36: 180-193Crossref PubMed Scopus (42) Google Scholar It is a multiorgan disease with highly variable clinical forms ranging from polysymptomatic severe forms to monosymptomatic forms limited to the male reproductive apparatus.3Elia J. Delfino M. 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Schwartz M. Witt M. Schwarz M. Girodon E. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders: updated European recommendations.Eur J Hum Genet. 2009; 17: 51-65Crossref PubMed Scopus (190) Google Scholar, 8Chen H. Ruan Y.C. Xu W.M. Chen J. Chan H.C. Regulation of male fertility by CFTR and implications in male infertility.Hum Reprod Update. 2012; 18: 703-713Crossref PubMed Scopus (113) Google Scholar Genetic analysis, combined with a biochemical and clinical evaluation, is a fundamental step in the diagnosis of this disease.6Bombieri C. Claustres M. De B.K. Derichs N. Dodge J. Girodon E. Sermet I. Schwarz M. Tzetis M. Wilschanski M. Bareil C. Bilton D. Castellani C. Cuppens H. Cutting G.R. Drevinek P. Farrell P. Elborn J.S. Jarvi K. Kerem B. Kerem E. Knowles M. Macek Jr., M. Munck A. Radojkovic D. Seia M. Sheppard D.N. Southern K.W. Stuhrmann M. Tullis E. Zielenski J. Pignatti P.F. Ferec C. Recommendations for the classification of diseases as CFTR-related disorders.J Cyst Fibros. 2011; 10: S86-S102Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 7Dequeker E. Stuhrmann M. Morris M.A. Casals T. Castellani C. Claustres M. Cuppens H. Des G.M. Ferec C. Macek M. Pignatti P.F. Scheffer H. Schwartz M. Witt M. Schwarz M. Girodon E. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders: updated European recommendations.Eur J Hum Genet. 2009; 17: 51-65Crossref PubMed Scopus (190) Google Scholar The marked genetic heterogeneity and a multifaceted genotype-phenotype relationship9Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. A genotypic-oriented view of CFTR genetics highlights specific mutational patterns underlying clinical macrocategories of cystic fibrosis.Mol Med. 2015; 21: 257-275Crossref PubMed Scopus (31) Google Scholar complicate the definition of mutational search strategies. Although CF is a monogenic autosomal recessive disease, with what is, in theory, a relatively simple genetic makeup, only flowcharts for a general mutational search not optimized for the various clinical forms exist.7Dequeker E. Stuhrmann M. Morris M.A. Casals T. Castellani C. Claustres M. Cuppens H. Des G.M. Ferec C. Macek M. Pignatti P.F. Scheffer H. Schwartz M. Witt M. Schwarz M. Girodon E. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders: updated European recommendations.Eur J Hum Genet. 2009; 17: 51-65Crossref PubMed Scopus (190) Google Scholar This also severely hampers the definition of a strategy for carrier screening, thus limiting the possibility of primary prevention, which has been instead shown to reduce the disease incidence.10Picci L. Cameran M. Marangon O. Marzenta D. Ferrari S. Frigo A.C. Scarpa M. A 10-year large-scale cystic fibrosis carrier screening in the Italian population.J Cyst Fibros. 2010; 9: 29-35Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar With regard to the most suitable CFTR mutational search strategy for specific CF clinical forms, the issue of diagnostic sensitivity [detection rate (DR)] of mutational search approaches is crucial. Whether a single genetic test, which is also technically easy, rapid, and inexpensive, can be applied to perform a full mutational characterization is debated. The more suitable the next-generation sequencing platforms become, the more relevant this topic is. Alternatively, multistep genetic approaches that progressively increase the DR and diagnostic value in patients or subjects with suspected disease, and progressively reduce the carrier risk in subjects from the general population, may be applied. One limitation of these approaches is that the mutational search in patients or subjects with suspected disease is usually suspended when the first two mutations on different alleles are found. Because many CFTR mutations are not characterized from a functional point of view, especially with regard to the severity of the clinical form they can cause, the decision to suspend the mutational search is often arbitrary. Consequently, current mutational search algorithms often result in inaccurate mutated genotypes, even because the mutational search is rarely designed to detect specific clinical forms. Even for carrier screening, because multistep approaches are inappropriate, prefixed mutational panels are used. In this case, however, general mutational panels are seldom optimized for the specific clinical form of CF for which the program is designed, which reduces the DR. Our aim was to improve the application of genetic tests used to diagnose different clinical forms of CF and to perform carrier screening. The details of the mutational pattern and of the clinical, microbiological, and biochemical characteristics of the case series analyzed in this study have already been published.9Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. A genotypic-oriented view of CFTR genetics highlights specific mutational patterns underlying clinical macrocategories of cystic fibrosis.Mol Med. 2015; 21: 257-275Crossref PubMed Scopus (31) Google Scholar Briefly, we enrolled 610 patients (1220 alleles), most of whom were from central Italy, and were examined and characterized on the basis of their clinical,11Borowitz D. Robinson K.A. Rosenfeld M. Davis S.D. Sabadosa K.A. Spear S.L. Michel S.H. Parad R.B. White T.B. Farrell P.M. Marshall B.C. Accurso F.J. Cystic Fibrosis Foundation evidence-based guidelines for management of infants with cystic fibrosis.J Pediatr. 2009; 155: S73-S93Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 12Canton R. Cobos N. de G.J. Baquero F. Honorato J. Gartner S. Alvarez A. Salcedo A. Oliver A. Garcia-Quetglas E. Antimicrobial therapy for pulmonary pathogenic colonisation and infection by Pseudomonas aeruginosa in cystic fibrosis patients.Clin Microbiol Infect. 2005; 11: 690-703Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar instrumental, laboratory,13Farrell P.M. Rosenstein B.J. White T.B. Accurso F.J. Castellani C. Cutting G.R. Durie P.R. Legrys V.A. Massie J. Parad R.B. Rock M.J. Campbell III, P.W. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report.J Pediatr. 2008; 153: S4-S14Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar microbiological,14Miller M.B. Gilligan P.H. Laboratory aspects of management of chronic pulmonary infections in patients with cystic fibrosis.J Clin Microbiol. 2003; 41: 4009-4015Crossref PubMed Scopus (66) Google Scholar, 15Zhou J. Garber E. Desai M. Saiman L. Compliance of clinical microbiology laboratories in the United States with current recommendations for processing respiratory tract specimens from patients with cystic fibrosis.J Clin Microbiol. 2006; 44: 1547-1549Crossref PubMed Scopus (33) Google Scholar biochemical (sweat test16Gibson L.E. Cooke R.E. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis.Pediatrics. 1959; 23: 545-549PubMed Google Scholar and fecal elastase dosage 117Gullo L. Graziano L. Babbini S. Battistini A. Lazzari R. Pezzilli R. Faecal elastase 1 in children with cystic fibrosis.Eur J Pediatr. 1997; 156: 770-772Crossref PubMed Scopus (66) Google Scholar), and genetic data (see below). According to recent CF guidelines and recommendations,6Bombieri C. Claustres M. De B.K. Derichs N. Dodge J. Girodon E. Sermet I. Schwarz M. Tzetis M. Wilschanski M. Bareil C. Bilton D. Castellani C. Cuppens H. Cutting G.R. Drevinek P. Farrell P. Elborn J.S. Jarvi K. Kerem B. Kerem E. Knowles M. Macek Jr., M. Munck A. Radojkovic D. Seia M. Sheppard D.N. Southern K.W. Stuhrmann M. Tullis E. Zielenski J. Pignatti P.F. Ferec C. Recommendations for the classification of diseases as CFTR-related disorders.J Cyst Fibros. 2011; 10: S86-S102Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 7Dequeker E. Stuhrmann M. Morris M.A. Casals T. Castellani C. Claustres M. Cuppens H. Des G.M. Ferec C. Macek M. Pignatti P.F. Scheffer H. Schwartz M. Witt M. Schwarz M. Girodon E. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders: updated European recommendations.Eur J Hum Genet. 2009; 17: 51-65Crossref PubMed Scopus (190) Google Scholar, 13Farrell P.M. Rosenstein B.J. White T.B. Accurso F.J. Castellani C. Cutting G.R. Durie P.R. Legrys V.A. Massie J. Parad R.B. Rock M.J. Campbell III, P.W. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report.J Pediatr. 2008; 153: S4-S14Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar, 18Castellani C. Southern K.W. Brownlee K. Dankert R.J. Duff A. Farrell M. Mehta A. Munck A. Pollitt R. Sermet-Gaudelus I. Wilcken B. Ballmann M. Corbetta C. de M.I. Farrell P. Feilcke M. Ferec C. Gartner S. Gaskin K. Hammermann J. Kashirskaya N. Loeber G. Macek Jr., M. Mehta G. Reiman A. Rizzotti P. Sammon A. Sands D. Smyth A. Sommerburg O. Torresani T. Travert G. Vernooij A. Elborn S. European best practice guidelines for cystic fibrosis neonatal screening.J Cyst Fibros. 2009; 8: 153-173Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar they were classified in the following clinical macrocategories: i) CF with pancreatic insufficiency (CF-PI, 354 patients, 708 alleles); ii) CF with pancreatic sufficiency (CF-PS, 138 patients, 276 alleles); iii) monosymptomatic or oligosymptomatic forms of CF, which for the purposes of this work included both CFTR-related disorders and atypical CF forms (herein all referred to as CFTR-RD, 71 patients, 142 alleles); iv) congenital bilateral absence of vas deferens, which for the purposes of this work was selected as the only clinical manifestation (ie, with no other CF symptoms; CBAVD, 47 patients, 94 alleles). When the diagnosis according to CF guidelines and recommendations was in contrast to clinical evidence, the latter prevailed. Informed consent was obtained from every patient (or parents) before enrollment. The study was approved by the institutional Ethics Committee and performed according to the Declaration of Helsinki. The mutational search on the CFTR gene (RefSeq NM_000492.3, NG_016465.3) was performed as already published.9Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. A genotypic-oriented view of CFTR genetics highlights specific mutational patterns underlying clinical macrocategories of cystic fibrosis.Mol Med. 2015; 21: 257-275Crossref PubMed Scopus (31) Google Scholar Briefly, DNA was extracted from peripheral blood using the QIAamp DNA blood midi kit (Qiagen, Hilden, Germany). The mutational search was conducted using a multistep approach, with the progressive application of five steps for the analysis of the following: i) the 32 most common mutations worldwide, by the CF-OLA assay (Abbott, Wiesbaden, Germany); ii) the 14 most frequent mutations in our geographical area, by our assay based on primer extension (CF-SNAP+20; see below); iii) the (TG)mTn variant tracts [specifically the (TG)13T5 (c.[1210-34TG[13];1210-12T[5]]), the (TG)12T5 (c.[1210-34TG[12];1210-12T[5]]) and the (TG)11T5 (c.[1210-34TG[11];1210-12T[5]]), by our assay19Lucarelli M. Grandoni F. Rossi T. Mazzilli F. Antonelli M. Strom R. Simultaneous cycle sequencing assessment of (TG)m and Tn tract length in CFTR gene.Biotechniques. 2002; 32: 540-547PubMed Google Scholar based on DNA sequencing; iv) the proximal 5′-flanking, all exons and adjacent intronic zones, by our assay based on DNA sequencing (SEQ),20Lucarelli M. Narzi L. Piergentili R. Ferraguti G. Grandoni F. Quattrucci S. Strom R. A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene.Anal Biochem. 2006; 353: 226-235Crossref PubMed Scopus (30) Google Scholar always applied to completion when included; v) the seven most frequent macrodeletions worldwide, by the FC del assay (DEL; Nuclear Laser Medicine, Milan, Italy). The mutational search was usually interrupted when the first two CFTR mutations already characterized as disease causing were found on different alleles.9Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. A genotypic-oriented view of CFTR genetics highlights specific mutational patterns underlying clinical macrocategories of cystic fibrosis.Mol Med. 2015; 21: 257-275Crossref PubMed Scopus (31) Google Scholar As previously described in more detail,9Lucarelli M. Bruno S.M. Pierandrei S. Ferraguti G. Stamato A. Narzi F. Amato A. Cimino G. Bertasi S. Quattrucci S. Strom R. A genotypic-oriented view of CFTR genetics highlights specific mutational patterns underlying clinical macrocategories of cystic fibrosis.Mol Med. 2015; 21: 257-275Crossref PubMed Scopus (31) Google Scholar the mutational search from step i) to step iv) was performed, in a 96-well format, using a semiautomated platform made up of a robotic system (Microlab Starlet; Hamilton, Bonaduz, Switzerland) and two genetic analyzers (ABI PRISM 3100 Avant and ABI PRISM 3130 xl; Applied Biosystems, Foster City, CA). For data analysis, specific approaches and/or templates for Genotyper version 3.7, GeneMapper version 4.1, and Seqscape software version 2.7 (Applied Biosystems) were used19Lucarelli M. Grandoni F. Rossi T. Mazzilli F. Antonelli M. Strom R. Simultaneous cycle sequencing assessment of (TG)m and Tn tract length in CFTR gene.Biotechniques. 2002; 32: 540-547PubMed Google Scholar, 21Ferraguti G. Pierandrei S. Bruno S.M. Ceci F. Strom R. Lucarelli M. A template for mutational data analysis of the CFTR gene.Clin Chem Lab Med. 2011; 49: 1447-1451Crossref PubMed Scopus (12) Google Scholar (see also next paragraph). The segregation of all mutated alleles was ascertained by analysis of parents and/or relatives. From the data obtained by the mutational search, both the allelic and genotypic DRs were calculated. The allelic DR is defined as the proportion of mutated alleles selected by the genetic test; the genotypic DR is defined as the proportion of full mutated genotypes (with both mutated alleles) detected by the genetic test. They can be seen as the genetic counterparts of diagnostic sensitivity. In particular, the genotypic DR defines the ability of a genetic test to characterize both mutated alleles, thereby allowing the full genetic characterization of a patient. A search was performed on 20 CFTR mutations by an original assay called CF-SNAP+20. Fourteen of these were preliminarily selected from a survey of frequent CFTR mutations not included in the first step of the mutational search (CF-OLA assay). These 14 mutations represented a preliminary best panel of the most frequent CFTR mutations in our geographical area, which were to be added to the CF-OLA assay. As a consequence, this panel was added as a second step of the mutational search, without ruling out the possibility that it might be changed after the analysis of the entire case series. By using the CF-SNAP+20 assay, it proved possible to detect the presence of an additional six mutations, which were not represented in our case series, only because they overlapped the mutations represented herein. The list of CFTR mutations, including both those that were and were not represented in our case series, is reported in Supplemental Table S1. A typical protocol of CF-SNAP+20 includes the following steps: genomic DNA PCR amplification and purification, minisequencing (single-nucleotide primer extension) and purification, capillary electrophoresis, and data analysis. Each step is described below. The genomic DNA was amplified by four PCRs, spanning seven CFTR exons (Table 1). Each singlex and multiplex PCR was performed in a total volume of 15 μL with 50 ng of genomic DNA, 6 pmol of each primer, 0.5 U of GoTaq hot start polymerase (Promega, Madison, WI), 175 μmol/L of each dNTP (Fermentas, Thermofisher, Waltham, MA), 1.5 mmol/L MgCl2, and 1× manufacturer's buffer, on a PTC100 thermal cycler (MJ Research, St. Bruno, QC, Canada). After an initial activation step of 2 minutes at 95°C, 35 cycles were performed as follows: 45 seconds at 94°C, 1.5 minutes at 54°C (Ta common to all PCRs), 4 minutes at 72°C; this PCR cycle was followed by an extension step of 7 minutes at 72°C. PCR products were checked by 1% agarose gel electrophoresis and purified by a common exonuclease I (Fermentas)–alkaline phosphatase (Fermentas) method.Table 1Characteristics of the Multiplexed PCR Step of the CF-SNAP+20 AssayPCRExon (legacy name)Exon (HGVS name)Forward primerReverse primerSize (bp)Multiplex A445′-CTCCCACTGTTGCTATAACAAATCCC-3′5′-AGCATTTATCCCTTACTTGTACCAGC-3′52720235′-GGTCAGGATTGAAAGTGTGCAACAAGG-3′5′-TCCCAAACTTTTAGAGACATCTTTTCTGCC-3′502Singlex B785′-ATAACATGCCCAAGGTCACACAGG-3′5′-GGTGAACATTCCTAGTATTAGCTGGC-3′564Multiplex C17a195′-AAAAAGTTTGAGGTGTTTAAAGTATGC-3′5′-CACCAACTGTGGTAAGATTCTATATACC-3′52717b205′-TCAAAGAATGGCACCAGTGTG-3′5′-TGCAGCATTTTATTCATTGA-3′449Multiplex D11125′-AAATTGCATTTGAAATAATGGAGATGC-3′5′-AAGATACGGGCACAGATTCTGAGTAACC-3′47514a155′-GGTGGCATGAAACTGTACTG-3′5′-TGTATACATCCCCAAACTATCT-3′251Primers and characteristics of the PCRs used to amplify the seven CFTR exons.HGVS, Human Genome Variation Society. Open table in a new tab Primers and characteristics of the PCRs used to amplify the seven CFTR exons. HGVS, Human Genome Variation Society. For the primer extension, the 14 oligonucleotides (high-performance liquid chromatography purified) listed in Table 2 were used in a single multiplex reaction based on the SNaPshot Multiplex Kit (Applied Biosystems). To optimize electrophoretic mobility, a nonhybridizing mobility tail was added to 11 oligonucleotides. Each mutation and corresponding wild-type proved to be univocally recognized by a unique combination of fluorescence (dependent on the specific ddNTP incorporated) and electrophoretic mobility (dependent on the overall length of extended specific oligonucleotide). To equilibrate the oligonucleotide peak heights, a different concentration of each oligonucleotide was used (Table 2). For practical purposes, a master mix with all oligonucleotides, each at an appropriate concentration, was prepared. The sample mix was then prepared as follows: 2 μL of the purified PCR, 0.5 μL of the oligonucleotide master mix, 2.5 μL of the Multiplex Ready Reaction Mix (of the SNaPshot Multiplex kit), in a final volume of 5 μL. Using a PTC100 thermal cycler (MJ Research), 30 cycles were performed as follows: 10 seconds 96°C, 5 seconds 50°C, 30 seconds 60°C. Minisequencing products were purified by a common alkaline phosphatase (Fermentas) method and underwent capillary electrophoresis on an ABI PRISM 3130 xl using POP6 polymer.Table 2Characteristics of the Minisequencing Step of the CF-SNAP+20 AssayNameSequenceLen (b)Concentration (μmol/L)mD110HYr5′-CCTCCTTGTTATCCGGGT-3′181mT338I5′-ACAATGCAGAATGAGATGGTG-3′218mS549R(A>C)5′-TCTTGGAGAAGGTGGAATCACACTG-3′250.25mL1065PRr5′-AAGGGAAGGAGCTGCCGTCCGAAGGCACG-3′302mY849X5′-GAAGAGCCAGCAGTGACTACATGGAACACATA-3′322mG1244R5′-AGAAAAGAAGCTTTTACCTTATAGGTGGGCCTCTTG-3′361mR117C5′-GGAAGGGGAAAAAAGGAAGGGGAAAACTATGCCTAGATAAATCGCGATAGAGC-3′408mR334LQ5′-AAAAAAGAAAAAAAAAAACTATGCACTAATCAAAGGAATCATCCTCC-3′471mH139RL5′-GAAAGAGAAAAAGAAAAAACTCTTTATTGTGAGGACACTGCTCCTAC-3′470.50mA349V5′-AGGAGAGAAAGAGAAGAAAAAAAGAGAACCAGGGAAAATTGCCGAGTGACC-3′502mR1066CS5′-AAGAAAAGGAAAAAGAAAGAAATGTTACAAGCTTAAAAGGACTATGGACACTT-3′531mL1077Pr5′-AAAAAAGAGAAAGAGGAGGAAGAAAGCAGTATGTAAATTCAGAGCTTTGTGGAAC-3′561mG1244EVr5′-AGAAAAAAGAAGAAAAAGGGGGGAAAAATAACAAAGTACTCTTCCCTGATCCAGTTCTT-3′592mL997F5′-GAAAAAAAAGAAAAAAGGAAAAGGGGAAAATACTCACCAACATGTTTTCTTTGATCTTACAGTT-3′648Sequence and characteristics of oligonucleotides used for minisequencing. The underlined portion of the sequence is the nonhybridizing mobility tail. The length (Len) of the oligonucleotides is reported as number of bases (b), mobility tail included. Final concentration of each oligonucleotide in the total volume of minisequencing oligonucleotide master mix is shown in the last column (Materials and Methods). Oligonucleotides whose name ends in "r" are designed on the reverse strand. Open table in a new tab Sequence and characteristics of oligonucleotides used for minisequencing. The underlined portion of the sequence is the nonhybridizing mobility tail. The length (Len) of the oligonucleotides is reported as number of bases (b), mobility tail included. Final concentration of each oligonucleotide in the total volume of minisequencing oligonucleotide master mix is shown in the last column (Materials and Methods). Oligonucleotides whose name ends in "r" are designed on the reverse strand. Data analysis was performed by originally set up templates for GeneMapper software (Applied Biosystems). These templates allow the automatic analysis of the results obtained by our primer extension protocol. The corresponding supplemental files contain the scripts for the analysis methods (Supplemental Code S1), plot settings (Supplemental Code S2), and table settings (Supplemental Code S3) templates. Template files that can be directly uploaded in the GeneMapper software may be requested from the corresponding author. Supplemental Figure S1 is an example of electropherograms of the CF-SNAP+20 assay. Contingency tables and χ2 test were used for the statistical analysis of experimental data, by using the SPSS software version 22 (SPSS-IBM, Armonk, NY). In the test phase of the CF-SNAP+20 assay, 170 mutated alleles were analyzed. For each mutation, the following number of alleles were analyzed: 12 D110H (p.Asp110His), eight R117C (p.Arg117Cys), nine H139R (p.His139Arg), 11 R334L (p.Arg334Leu), 13 T338I (p.Thr338Ile), 24 S549R(A>C) (p.Ser549Arg), five Y849X (p.Tyr849*), 35 L997F (p.Leu997Phe), 12 L1065P (p.Leu1065Pro), three L1065R (p.Leu1065Arg), 11 R1066C (p.Arg1066Cys), 20 L1077P (p.Leu1077Pro), three G1244R (p.Gly1244Arg), and four G1244E (p.Gly1244Glu). In addition, 220 wild-type alleles were analyzed. In the test phase, each sample was analyzed at least twice and results confirmed by sequencing. No discrepancies between replicates or between the CF-SNAP+20 assay and sequencing arose (100% of analytical sensitivity and specificity). The CF-SNAP+20 assay revealed the following advantageous characteristics. The minisequencing oligonucleotide design, with nonhybridizing mobility tail, allows the most suitable electrophoretic mobility to be chosen easily. The high-performance liquid chromatography purification of the minisequencing oligonucleotide reduces the electrophoresis background. The multiplex format of both the PCR and the minisequencing steps, the speed of enzymatic purification steps, and the possibility of reaction setup automation in 96- or 384-well format greatly enhance the throughput. In addition, the possibility of using this assay on a common analytical platform, already in use in most molecular genetics laboratories, greatly facilitates the implementation of this assay. Finally, another suitable characteristic of this assay is the marked flexibility of the oligonucleotide design when adding or removing mutations, according to the geographical areas and possible experimental refinement of mutation prevalence. The genetic heterogeneity was found to influence the allelic and the genotypic DR (Materials and Methods). Both the allelic and the genotypic DRs were found to vary greatly (particularly the genotypic DR) between the populations analyzed and at each step of the mutational search (Figure 1 and Table 3). The differences detected between mutational steps in the number of alleles or genotypes were statistically significant (for both allelic and genotypic DRs, χ2, P < 0.0001).Table 3Cumulative DRs Depending on the Mutational Search StepAssayCF (PI+PS)CF-PICF-PSCFTR-RDCBAVDCumulative allelic DRs CF-OLA0.7700.8390.5940.4510.181 + CF-SNAP+200.8430.8900.7240.5360.255 + (TG)mTn0.8630.8900.7960.6840.446 + SEQ0.9660.9690.9590.9660.563 + DEL0.9850.9930.9660.9660.563 Unknown∗Alleles with no mutation detected.0.0150.0070.0340.0340.437Cumulative genotypic DRs CF-OLA0.6000.7120.3120.0280 + CF-SNAP+200.7140.7910.5150.1410.043 + (TG)mTn0.7440.7910.6240.4230.341 + SEQ0.9370.9440.